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THE EFFECTS OF ZINC DEFICIENCY ON CARBONIC ANHYDRASE ACTIVITY AND RENAL FUNCTION By Penny Lee Kasch A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1978 ABSTRACT THE EFFECTS OF ZINC DEFICIENCY 0N CARBONIC ANHYDRASE ACTIVITY AND RENAL FUNCTION By Penny Lee Kasch This study was designed to determine the effects of zinc deficiency on carbonic anhydrase activity and renal function in the rat. Body and kidney growth were severely compromised after three and six weeks of feeding a zinc— deficient diet. Plasma and kidney zinc concentration were significantly decreased in zinc-deficient animals fed six weeks. While whole blood carbonic anhydrase activity of zinc-deficient rats was significantly greater than zinc- supplemented animals, no differences in renal carbonic anhydrase activity were found between zinc-deficient animals and pair-fed controls. Sodium excretion in untreated zinc-deficient animals was significantly greater than in animals fed zinc-supplemented diets, suggesting a renal defect in the reabsorptive capacity for sodium. Renal function, quantified as sodium, potassium, chloride, and bicarbonate excretion and glomerular filtration rate, was differentially affected by administration of three prototype diuretics: acetazolamide, furosemide and hydrochlorothiazide. ACKNOWLEDGMENTS I would like to express my sincere appreCiation to my advisor, Dr. Jenny Bond, for continued support and guidance during this study, and for her genuine concern throughout my graduate program. I also wish to thank Dr. Jerry Hook, Dr. Richard Luecke and Dr. Michael Bailie for their assistance in the preparation of this thesis. Special thanks are extended to Dr. Byron Noordewier for his infinite wisdom and never-ending patience, and to Nancy Lumbert for her photographic expertise. My fellow graduate students, Bill Evers, Ellen Rolig, Robin Gold- stein and Sue Ford deserve worthy mention for their con- structive criticisms, unselfish help, and for making life in the lab bearable. A special tribute is paid to my parents, Bud and Dorothy Kasch, for their confidence in me, patience and understanding, and to my fiancee, Doug, for giving me the incentive to complete this project. The financial support provided by Dr. Jenny Bond and the Department of Food Science and Human Nutrition, making this research project a reality, is gratefully acknow- ledged. ii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . vii INTRODUCTION. . l Zinc Metalloenzymes. . . 3 Carbonic Anhydrase and the Mechanism of. Urine Acidification. 4 Renal Zinc and the Functional Significance of Metallothionein. 9 Diuretics. . l2 Acetazolamide. . . . . . . . . . . . . . . l3 Furosemide . . . . . . . . . . . . . . . . l4 Hydrochlorothiazide. l6 Rationale. l7 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 2l Animals and Diets. . . . . . . . . . . . 21 Determination of Zinc. Status . . . . . . . . . . 23 Determination of Renal Function. . . . . . . . . 26 Statistical Analyses . . . . . . . . . . . . . . 28 RESULTS . . . . . . . . . . . . . . . . . . . . . 29 Food Intake. . . . . . . . . . . . . . . . . . . 29 Body Weight. . . 29 Kidney Weight and Kidney Weight to Body ONeight Ratio. . . . 29 Plasma Zinc Concentration. . . . . . . . . . . . 38 Whole Blood Zinc Concentration . . . . . . . . . 43 Kidney Zinc Concentration. . . . . . . 43 Whole Blood Carbonic Anhydrase Activity. . . . . 43 Kidney Carbonic Anhydrase Activity . . . . . . . 49 Acetazolamide. . . . . . . . . . . . 49 Blood pH . . . . . . . . . . . . . . . . . . 49 Blood pCOZ . . . . . . . . . . 49 Plasma Sodium Concentration. . . . . . . . . 53 Plasma Potassium Concentration . . . . . . . 53 Plasma Chloride Concentration. . . . . . . . 53 Urinary Sodium Excretion . . . . . . . . . . 53 Urinary Potassium Excretion. . . . . . . . . 57 TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . vii INTRODUCTION. . l Zinc Metalloenzymes. 3 Carbonic Anhydrase and the MeChanism of Urine Acidification. . 4 Renal Zinc and the Functional Significance of Metallothionein. 9 Diuretics. l2 Acetazolamide. l3 Furosemide l4 Hydrochlorothiazide. l6 Rationale. l7 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 2l Animals and Diets. . . . . . . . . . . . . 21 Determination of Zinc Status . . . . . . . . . . 23 Determination of Renal Function. . . . . . . . . 26 Statistical Analyses . . . . . . . . . . . . . . 28 RESULTS . . . . . . . . . . . . . . . . . . . . . 29 Food Intake. . . . . . . . . . . . . . . . . . . 29 Body Weight. . . 29 Kidney Weight and Kidney Weight to Body Weight Ratio. . . . . . 29 Plasma Zinc ConCentration. . . . . . . . . . . . 38 Whole Blood Zinc Concentration . . . . . . . . . 43 Kidney Zinc Concentration. . . . . . . 43 Whole Blood Carbonic Anhydrase Activity. . . . . 43 Kidney Carbonic Anhydrase Activity . . . . . . . 49 Acetazolamide. . . . . . . . . . . . 49 Blood pH . . . . . . . . . . . . . . . . . . 49 Blood pCOZ . . . . . . . . . . . 49 Plasma Sodium ConCentration. . . . . . . . . 53 Plasma Potassium Concentration . . . . . . . 53 Plasma Chloride Concentration. . . . . . . . 53 Urinary Sodium Excretion . . . . . . . . . . 53 Urinary Potassium Excretion. . . . . . . . . 57 111' TABLE 10 LIST OF TABLES Diet composition. Kidney weight of rats fed zinc-deficient or zinc- supplemented diets for three or six weeks Kidney weight to body weight ratio (KW/BW) of rats fed zinc-deficient or zinc-supplemented diets for three or six weeks. . . . . Concentration of zinc in whole blood of rats fed zinc-deficient or zinc-supplemented diets for three or six weeks. . . . . . . . . . . Blood pH and blood pCO of control and acetazo- lamide treated rats fe zinc-deficient or zinc- supplemented diets for six weeks. Plasma concentrations of sodium, potassium and chloride in control and acetazolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks . . . . Urinary bicarbonate excretion of control and acetazolamide treated rats fed zinc-deficient and zinc-supplemented diets for six weeks Glomerular filtration rate in control and aceta- zolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks Blood pH and blood pCO of control and furosemide treated rats fed zinc- eficient or zinc-supple- mented diets for six weeks. . . . . . . . Plasma concentrations of sodium, potassium and chloride in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks PAGE 22 39 4O 44 52 54 63 65 66 68 LIST OF TABLES TABLE PAGE l Diet composition. . . . . . . . . . . . . . . . . 22 2 Kidney weight of rats fed zinc-deficient or zinc- supplemented diets for three or six weeks . . . . 39 3 Kidney weight to body weight ratio (KW/8W) of rats fed zinc-deficient or zinc-supplemented diets for three or six weeks. . . . . . . . . . . 40 4 Concentration of zinc in whole blood of rats fed zinc-deficient or zinc-supplemented diets for three or six weeks. . . . . . . . . . . . . . . . 44 5 Blood pH and blood pC02 of control and acetazo- lamide treated rats fed zinc-deficient or zinc- supplemented diets for six weeks. . . . . . . . . 52 6 Plasma concentrations of sodium, potassium and chloride in control and acetazolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks . . . . . . . . . . . . . . . 54 7 Urinary bicarbonate excretion of control and acetazolamide treated rats fed zinc-deficient and zinc-supplemented diets for six weeks . . . . 63 8 Glomerular filtration rate in control and aceta- zolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks . . . . . . 65 9 Blood pH and blood pCO of control and furosemide treated rats fed zinc-Eeficient or zinc-supple- mented diets for six weeks. . . . . . . . . . . . 66 IO Plasma concentrations of sodium, potassium and Chloride in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks . . . . . . . . . . . . . . . . . . . . 68 Table II 12 l3 I4 IS l6 l7 Urinary bicarbonate excretion of control and furosemide treated rats fed zinc—deficient or zinc-supplemented diets for six weeks. Glomerular filtration rate in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks Blood pH and blood pCOZ of control and hydro- chlorothiazide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks Plasma concentrations of sodium, potassium and Chloride in control and hydrochlorothiazide treated rats fed zinc-deficient or zinc- supplemented diets for six weeks Urinary bicarbonate excretion of control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. . . . . . . . . . Glomerular filtration rate in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. Fractional sodium excretion of control and drug-treated rats fed zinc-deficient or zinc- supplemented diets for six weeks vi Page 77 78 80 BI 9O 91 lO9 LIST OF FIGURES Figure I 10 ll Food intake of rats fed zinc-deficient or supplemented diets for three weeks. Food intake of rats fed zinc-deficient or supplemented diets for six weeks. Body weight of rats fed zinc-deficient or supplemented diets for three weeks. Body weight of rats fed zinc-deficient or supplemented diets for six weeks. zinc- zinc- zinc- zinc— Plasma zinc concentration of animals sacrificed prior to dietary treatment or fed zinc— deficient or zinc-supplemented diets. Kidney zinc concentration of animals sacrificed prior to dietary treatment or fed zinc- deficient or zinc-supplemented diets. Carbonic anhydrase activity in the blood of zinc-deficient or zinc-supplemented rats at three and six weeks Carbonic anhydrase activity in the kidney of zinc-deficient or zinc-supplemented rats at three and six weeks Urinary sodium excretion in control and aceta- zolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks Urinary potassium excretion in control and acetazolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks. Urinary chloride excretion in control and acetazolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks. vii Page BI 33 35 37 42 46 48 51 56 59 61 Figure 12 l3 T4 15 l6 l7 Urinary sodium excretion in control and furo- semide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks. Urinary potassium excretion in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks. Urinary chloride excretion in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks Urinary sodium excretion in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. . . . . . . . . . Urinary potassium excretion in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. . . . . . . . Urinary chloride excretion in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. . . . . . . . . . viii Page 70 73 75 84 86 89 INTRODUCTION Over a century ago Raulin demonstrated that zinc was essential for the growth of the mold, Aspergillus niger (Underwood, l97l). Attempts to demonstrate a similar need for zinc in higher animals met with limited success, until Todd £3.1l- (1934) provided the first conclusive evidence that zinc was an essential component of the rat's diet. These findings were confirmed by Hove gt l. (l937). Sub- sequent studies have shown a similar need in pigs (Miller (D t l., l968), chickens (O'Dell t l., l958), and cattle (Miller and Miller, l962). Growth retardation, testicular atrophy, hyperkeratotic skin lesions and anorexia are some of the clinical manifestations of zinc deficiency in these Species. In man, the existence of zinc deficiency was first suspected when Prasad 33 31. (l96l) described a group of l8-20 year old Iranian males with iron deficiency anemia, hepatosplenomegaly, geophagia, hypogonadism and dwarfism. Similar clinical features were seen in Egyptian male pa- tients (Prasad gt 31., l963a). The diet of these people consisted only of bread made from wheat flour, and the intake of animal protein was negligible. After extensive studies, it was established that these dwarfs were indeed zinc-deficient. Diagnosis was based on decreased zinc concentration in plasma, red cells and hair and an increase in plasma zinc turnover rate in these subjects (Prasad gg gl., l963b). Further studies showed that the rate of growth was greater in patients receiving supplemental zinc compared to those who received iron alone or an animal- protein supplemented diet (Sandstead gg gl., 1967). Growth retardation, so commonly seen as a result of zinc deficiency, is most likely due to its effect on nucleic acid metabolism and decreased protein synthesis. Direct evidence that zinc plays an essential role in replication and transcription comes from the demonstration that a number of DNA-dependent DNA polymerases, and DNA-dependent RNA polymerases are zinc metalloenzymes (Vallee, 1976). Several RNA-dependent DNA polymerases are also zinc metal- loenzymes (Auld g_ gl., 1974, 1975; Poiesz _g.gl., 1974). Nucleic acid and protein synthesis have been shown to be impaired in zinc-deficient microorganisms (Nason gg gl., 1953), plants (Underwood, 1971), and rats (Buchanan and Hsu, 1968; Fujioka and Lieberman, 1964). These defects in protein and nucleic acid metabolism respond rapidly to supplemental zinc (Underwood, 1971). Zinc- and iron-deficient hypogonadal dwarfism is likely to be prevalent in countries where cereal foods are the primary source of protein consumed by the population (Prasad, 1976). Such disturbances develop despite the fact that village diets, consisting mainly of bread and beans, contain more than enough zinc and iron to fulfill daily requirements for these metals (Maleki, 1973). This discrepancy has been attributed to the high phytate content of unleavened wheat bread, which inhibits intestinal absorption of metals (Reinhold, 1972; Ismail-Beigi g; _l., 1977). Thus it appears that zinc, in addition to other essential nutrients, may be a limiting factor in normal growth of certain popu- lations in underdeveloped regions of the world. Zinc Metalloenzymes The isolation and purification of the enzyme carbonic anhydrase by Keilin and Mann (1940) offered the first clue to the mode of action of zinc. This metal ion was shown to be essential for the activity of the enzyme which catalizes the reversible hydration of carbon dioxide (Bundy, 1977). In the last 15 years over two dozen zinc- containing metalloenzymes have been isolated and purified from various tissues of diverse species (Parisi and Vallee, 1969), thereby indicating the metabolic importance of the element. Induction of a zinc-deficient state in an animal often produces changes in the activity of many of these enzymes. Prasad gg g1. (1967) found reduced activities of certain enzymes accompanied by reduced concentrations of zinc in the bones, testes, esophagus, and kidneys of zinc—deficient rats. Similar findings were obtained in baby pigs (Prasad gg gl., 1969). Macapinlac g3 g1. (1966) observed zinc concentrations in bone to be decreased by 66% in rats main- tained on a low-zinc diet for seven weeks. Roth and Kirch- gessner (1974) reported significantly decreased blood carbonic anhydrase activity in rats after only four days of zinc depletion. Attempts to relate the effects of low zinc intake to enzyme changes in the kidney have yielded conflicting re- sults. Several researchers (Hsu and Anilane, 1966; Prasad gt _1., 1967; Iqbal, 1971) have reported the activity of alkaline phosphatase in the kidney of zinc-deficient rats to be approximately 50-60% below normal. Other workers, however, found no significant differences in enzyme acti- vity between deficient and control groups (Day and McCollum, 1940; Kfoury g; _l., 1968). Iqbal (1971) found decreased carbonic anhydrase activity in kidneys from zinc-deficient rats. More recently Huber and Gershoff (1973) indicated no detectable Change. Prasad and Oberleas (1971) demonstrated that the zinc content of kidney tissue was reduced in zinc-deficient pigs, in contrast to a previous study which showed no significant reductions (Prasad gg gl., 1969). Carbonic Anhydrase and the Mechanism of Urine Acidification Carbonic anhydrase is among the most widely occurring enzymes, having been found in bacteria, lower and higher plants, invertebrates, and vertebrates (Maren, 1967; Lindskog gg gl., 1971). The enzyme from mammalian sources consists of a single polypeptide chain, has a molecular weight of 30,000, and contains one zinc atom per enzyme molecule (Bernhard, 1968). The zinc atom is tightly bound and is an essential cofactor for this enzyme (Keilin and Mann, 1940; Maren, 1967). Carbonic anhydrase is present in high concentration in the kidney and other tissues where rapid exchanges between carbon dioxide and bicarbonate occur.’ Lonnerholm (1970; 1973) utilizing histochemical staining techniques, demonstrated the presence of carbonic anhydrase in the proximal convoluted tubules, including the luminal brush border, and along straight collecting tubules of human and rat kidney. In the distal nephron, enzyme activity is confined to the basal 1/3 to 1/2 of the epithelial cells. Functionally, carbonic anhydrase plays an important role in the process of urinary acidification and bicarbonate reab- sorption in this organ (Rector, 1976). Thus, this enzyme is a potential regulator of available concentrations of carbon dioxide, hydrogen ions, and bicarbonate; and conse- quently of such processes as gas exchange and acid-base balance. The renal mechanisms involved in acidification of the urine and the role of carbonic anhydrase in this process have been an area of intense research in the past few decades. Pioneering work in this area was that of Pitts and Alexander in 1945. They showed that the reabsorption of filtered bicarbonate by the kidney is mediated by a single mechanism, operative in both the proximal and distal portions of the nephron, which involves the secretion of hydrogen ion into tubular urine in exchange for luminal sodium. The secreted hydrogen ion reacts with filtered bicarbonate to form carbonic acid, which then decomposes to carbon dioxide and water (Pitts and Alexander, 1945). The development and use of acetazolamide and other powerful carbonic anhydrase inhibitors in the early 1950's con- firmed Pitts' hypothesis in all respects (Maren, 1974). Difficulties arose when the details of the urine acidification process were examined. In a steady state, the rate at which the carbonic acid is removed from luminal fluid must equal the rate at which the hydrogen ion is secreted. Walser and Mudge (1960) estimated that in the absence of carbonic anhydrase, the concentration of carbonic acid in luminal fluid must be at least ten times greater than it would be if it were in equilibrium with the carbon dioxide tension of plasma, to account for observed rates of bicarbonate reabsorption. As a result of the excess carbonic acid, the pH of the fluid flowing along the tubule would be approximately one pH unit lower. They used the term ”disequilibrium pH” to describe the observation that the ratio for carbonic acidzcarbon dioxide was increased beyond the chemical equilibrium value of 1:400. Studies based on these theoretical considerations were performed by Rector gg _l. (1965). In a series of micropuncture studies using rats, these investigators found that the distal intraluminal pH was more acid than the calculated equilibrium pH during sodium bicarbonate diure- sis. The disequilibrium was obliterated by injection of carbonic anhydrase intravenously in amounts sufficient to give measured activity in the urine, indicating that the discrepancy was due to excess carbonic acid. Similar studies by Vieira and Malnic (1968) confirmed these find- ings. These results indicate that bicarbonate reabsorption in the distal tubule is mediated by hydrogen ion secretion. In the proximal tubule different results were obtained (Rector ggigl., 1965). In normal hydropenic rats, rats infused with sodium phosphate, and in rats undergoing sodium bicarbonate diuresis, the measured intratubular pH was equal to the calculated equilibrium pH. After inhibition of carbonic anhydrase with benzolamide, the i vivo intratubular pH became 0.8 pH units more acid than the calculated equilibrium pH. This was interpreted to mean that hydrogen ion secretion mediated bicarbonate reabsorp- tion in the proximal, as well as the distal tubule (Rector _g gl., 1965). However, due to differences in enzyme location, it is apparent that hydrogen ion secretory systems in the proximal and distal tubule will differ markedly with respect to the role of carbonic anhydrase. Inboth areas of the nephron, carbonic anhydrase probably plays a cellular role in main- taining an intracellular supply of carbonic acid and hydro- gen ion by catalyzing the hydration of carbon dioxide (Rector gt 31., 1965). In addition, carbonic anhydrase serves a second function in the proximal tubule. The enzyme appears to be in contact with proximal tubule fluid so that as carbonic acid is formed it is rapidly broken down to carbon dioxide and water. Carbonic anhydrase then, prevents the accumulation of excess carbonic acid and as a result of this action the steady state intratubular pH is approximately one pH unit higher than it would be if carbonic anhydrase were not present in the luminal membrane (Rector gg gl., 1965). In the proximal tubule, therefore, carbonic anhydrase facilitates the transport of large quan- tities of hydrogen ion, by both furnishing an intracellular supply of hydrogen ion and preventing the generation of steep pH gradients. In contrast, the distal tubule does not contain carbonic anhydrase in its luminal membrane; tubular fluid remains relatively acidic even in the presence of high bicarbonate concentrations and hydrogen ions are always secreted against a concentration gradient (Rector t 1., 1965). Renal Zinc and the Functional Significance of Metallothi- 9_n_e_1'_r_w_ Although intrarenal mechanisms controlling urinary zinc excretion remain poorly defined and are dissociated from factors controlling the urinary excretion of other electrolytes, serum zinc concentrations are maintained within a narrow range implying the presence of effective homeostatic mechanisms (Lindeman gg,_l., 1977). Early studies of the fate of ingested and injected zinc led investigators to speculate that the kidney can take little or no part in regulating the amount of this metal in the body; nor does the kidney vary its excretion of zinc in accordance with the amount in the plasma or the amount absorbed (McCance and Widdowson, 1942). Steele (1973) showed that diuretic therapy designed to increase urinary excretion of sodium, potassium, calcium, and magnesium had little effect on zinc excretion, suggesting that the renal handling of zinc differs markedly from other cations. In contrast to most organs where the zinc level has been found to be practically constant throughout life (Schroeder gg gl., 1967), renal zinc concentration increases between infancy and adulthood (Piscator and Lind, 1972; Livingston, 1972; Elinder gg _l., 1977). In the infant the total renal zinc concentration is not only lower, but scarcely any concentration gradient exists between cortex and medulla (Livingston, 1972). Adult kidneys have both an 10 elevated zinc concentration and a steeper concentration gradient between cortex and medulla (Millar _g _l., 1961; Livingston, 1972). Piscator (1966) has postulated the build-up is due to the metalloprotein, metallothionein, which binds zinc and other heavy metals, and transports them to the renal cortex as a detoxification mechanism. Metallothioneins constitute a family of cytoplasmic, low molecular weight, metal-binding proteins, synthesized in liver and other tissues of animals exposed to heavy metals such as cadmium and zinc (Nomiyama and Foulkes, 1977). Originally, metallothionein was isolated in equine renal cortex (Margoshes and Vallee, 1957), and later in liver or kidney of rats (Winge and Rajagoplan, 1972), rabbits (Nordberg gg gl., 1972), calves (Bremner and Marshall, 1974), and humans (Pulido g; gl., 1966). The initial work on metallothionein originated from interest in the physiological function of cadmium; however, the pro- tein has repeatedly been shown to contain a large amount of zinc as well (Kagi and Vallee, 1961; Kagi g3 g1., 1974; Weser g3 g1., 1973; Bremner and Marshall, 1974). The metal composition of metallothionein is dependent on the tissue of origin; cadmiUm and zinc are often of nearly equal abundance in the protein from kidney, while zinc is the principle metallic constituent in the one from liver (Kojima and Kagi, 1978). 11 Although this protein was isolated two decades ago and numerous studies have been subsequently conducted, the biological role of metallothionein is still speculative. The ability of this protein to bind large quantities of metals may imply an intracellular reservoir function for essential elements such as zinc and copper (Cherian, 1977; Kojima and Kagi, 1978). Since these ions must be supplied continually for the biosynthesis of metalloenzymes, pro- teins like metallothionein could maintain metal homeostasis by fulfilling an intracellular metal sequestering and dis- pensing task (Kojima and Kagi, 1978). Richards and Cousins (1975) propose a dual role for metallothionein: when plasma zinc levels are high, metallothionein serves an uptake and storage function in hepatocytes and a sequestration func- tion to control zinc absorption in intestinal cells. In contrast, Chen gg _l. (1977) demonstrated that zinc from metallothionein was excreted from the body instead of being reutilized. This led them to suggest that metallothionein plays a key role in heavy metal metabolism by temporarily holding excessive levels of the metal to prevent its attack on certain critical targets, and subsequently releasing it for excretion when high dietary levels are discontinued. This probable role of metallothionein in heavy metal detoxi- fication is supported by other investigators (Winge and Rajagoplan, 1972; Piotrowski gt 1., 1974; Webb and Magos, 1976). 12 Discrepancies have risen over the site of biosynthesis of metallothionein. According to the hypothesis proposed by Piscator (1966), and later supported by Nordberg gg _l. (1972), metallothionein is synthesized in the liver, slowly released into the circulation, and accumulated by the kidney. This theory has been recently challenged by Shaikh and Smith (1976) who showed metallothionein is not only synthesized in the liver but is also actively produced in the kidney. Webb and Daniel (1975) confirmed the production of metallo- thionein by kidney cells. Control for the biosynthesis of this protein appears to be regulated at the transcriptional level in the liver, and in the kidney, at the translational level (Squibb and Cousins, 1974; Squibb gg al., 1977; Richards t l., 1975; Shaikh and Smith, 1976, 1977). ——-———— Diuretics Diuretics are clinically useful pharmacological agents which induce a net lossof water and sodium in the urine (Suki _g,gl., 1973). There is general agreement that most diuretics act directly on the kidneys to inhibit solute reabsorption by the epithelial cells of the renal tubules, so that a greater fraction of the glomerular filtrate is excreted in the urine. Since sodium, the major solute of the tubular fluid, is reabsorbed throughout most portions of the nephron, many of the drugs inhibiting its reabsorp- tion can act at more than one site. The mechanism and 13 localization of drug action to a particular site has been studied extensively with multiple experimental techniques: clearance studies, stop-flow analysis, micropuncture, toad bladder studies, and most recently, ig_giggg microperfusion of isolated tubules (Mudge, 1975). Acetazolamide Acetazolamide, one of a class of sulfonamide compounds used as diuretics, acts in the renal tubule by means of its inhibition of carbonic anhydrase (Mudge, 1975). The pre- sence of carbonic anhydrase in a number of intraocular structures makes acetazolamide clinically useful in the treatment of glaucoma, where it reduces the rate of aqueous humor formation and thus reduces intraocular pressure. Therapeutic useage has also been indicated in the event of epileptic seizures where acetazolamide has a tran- quilizing effect on the central nervous system (Mudge, 1975). Inhibitors of carbonic anhydrase then, reduce the rate of formation of hydrogen ions for secretion within tubular cells and reduce their availability to luminal exchange mechanisms, thereby depressing bicarbonate reabsorption (Maren, 1967). Micropuncture studies in dogs and rats have identified the proximal tubule as the principle site of action of acetazolamide (Dirks g3 g1., 1966; Weinstein, 1968). Clapp t al. (1963) found a rise in proximal 14 tubular bicarbonate tubular fluid: plasma (TF/P) ratios in the rat, to values approaching 3.0 during acetazola- mide diuresis. Weinstein (1968) and Rector gg‘gl. (1965) reported that a maximum of 50% of the filtered bicarbonate escapes reabsorption in the proximal tubule of acetazola- mide treated animals. A recent study by McKinney and Burg (1977) showed acetazolamide inhibited bicarbonate reabsorption almost completely, in contrast to the previous results where only partial inhibition was noted. Furosemide Furosemide is a carbonic anhydrase inhibitory analogue of sulfanilamide, clinically useful in the acute and chro- nic treatment of edema of cardiac, hepatic, or renal origin (Burg, 1976). The drug has a rapid and short duration of action and relatively steep dose-response curves, proving itself useful in those patients with mild to severe conges- 1., 1971; Suki (11;ng 1973). tive heart failure (Kim gg Renal clearance studies in the dog have shown that furosemide decreases the urinary concentrating capacity (Seldin g3 g1., 1966), and decreases the renal medullary sodium gradient (Hook and Williamson, 1965a), implying interference with electrolyte transport systems in the ascending limb of the loop of Henle. Micropuncture studies in rats confirm the action of furosemide at this site since early distal sodium (Deetjen, 1966), and chloride (Malnic 15 _g _l., 1965) concentrations were considerably elevated and approached plasma concentrations under the influence of the drug. Reabsorption of sodium chloride in the ascen- ding limb of the loop of Henle occurs without an equivalent movement of water, and results in the production of the hypotonic fluid usually found in the first part of the distal tube (Mudge, 1975). More recently, Burg and Stoner (1976) demonstrated a primary effect of furosemide on active chloride transport in the isolated perfused ascen- ding limb of rabbit kidneys. It is now apparent that furosemide inhibits active chloride transport, rather than sodium transport in this segment of the nephron. Furosemide has been demonstrated to exert an inhibi- tory effect on the proximal tubular reabsorption of sodium and chloride, possibly due to its action as a weak carbonic anhydrase inhibitor (Baer and Beyer, 1966; Kim _g gl., 1971). In addition the effects of furosemide on free water clearance and bicarbonate excretion provide further evidence for a proximal tubule effect (Stein et 1., 0) ° ’ m _.a 1968; Puschett and Fernandez, 1968; Alguire g; 1974). The exact mechanism by which furosemide inhibits active chloride transport in the loop of Henle is unknown. Hook and Williamson (1965b) speculated that the drug inhibits the Na+ - K+ -dependent ATPase in the kidneys of rats. More recently, Schmidt and Dubach (1970) demonstrated 16 that a large dose of furosemide inhibited this enzyme in the diluting segments of rat kidney. However, the rela- tionship between enzyme activity and active chloride trans- port in this segment of the tubule remains unclear. Hydrochlorothiazide Benzothiadiazides, or thiazide diuretics, are synthetic drugs Chemically related to the sulfonamides, which stemmed from research on carbonic anhydrase inhibitors. Thiazides have their greatest usefulness as diuretics in the manage- ment of edema of chronic cardiac decompensation and hypertension (Mudge, 1975). The dominant action of the thiazides is to increase the renal excretion of sodium and chloride, but unlike the carbonic anhydrase inhibitors, the action of the thiazides is virtually independent of acid-base balance (Mudge, 1975). The inhibition of carbonic anhydrase that they do cause in the proximal tubule results in decreased reab- sorption of sodium, chloride and water in this segment. However, this action is relatively unimportant for their overall diuretic action. The primary diuretic effect of these drugs is the inhibition of sodium and chloride reab- sorption in the distal nephron (Earleyahd Orloff, 1962; Mudge, 1975; Burg, 1976). 17 Rationale It is now becoming clear that certain factors other than a primary lack of dietary zinc may compromise zinc status in a number of disease states. It has been observed that serum zinc concentrations are decreased and marked zincuria occurs in patients with a variety of clinical dis- orders including cirrhosis, nephrotic syndrome, and renal insufficiency (Lindeman gghgl., 1978). Many of the acute and chronic manifestations of zinc deficiency are common in these patients, suggesting the possibility of a true clinical deficiency state. Similar increases in zinc excretion are seen in patients with renal failure (Condon and Freeman, 1970), and following renal transplant opera- tions (Ellis, 1978). Certain amino acids have been shown to exert a tre- mendous influence on urinary zinc excretion. Lindeman _g _l. (1977) have proposed that variations in serum con- centrations of these amino acids may explain the hyper- zincuria observed in many pathological conditions. Furthermore, excessive urinary zinc losses have been seen during total parenteral nutrition. This may be due to the chelating effect of the amino acid preparation used, since aminoaciduria has also been observed (Van Rij g3 g1., 1975). Recently, the use of thiazide diuretics in the treat- ment of calcium stones of renal origin has been advocated (Yendt gg gl., 1966; 1970). The efficacy of this form of 18 treatment is related to a reduction in urinary calcium excretion and increased urinary magnesium excretion in- duced by thiazides. However, it is now clear that these diuretics profoundly effect zinc excretion. In patients with renal calculi receiving hydrochlorothiazide therapy, Cohanim and Yendt (1975) report a striking and sustained increase in urinary zinc excretion. Occasional findings of subnormal serum zinc levels in these patients suggest that long term thiazide therapy carries with it the hazard of zinc deficiency. Pak g£_gl, (1972) demonstrated that treatment with hydrochlorothiazide significantly increased zinc excretion in normal volunteers, and in patients with hypercalcuria, osteoporosis, and hyperparathyroidism. Furthermore, stimulation of the renal excretion of zinc persisted after one month of therapy. The ability to increase urinary zinc excretion may not be confined to the thiazide group of diuretics. Steele (1973) showed that during the first hour after intravenous administration of furosemide or ethacrynic acid to humans, zinc excretion increased significantly from 51.6 mEq/ minute to 95.0 mEq/minute. The high zinc content of human renal cortex suggests that significant urinary zinc could be derived from renal tissue sources. Zinc deficiency may not be uncommon in children less than four years of age in this country (Cordas and Holland, 1977). Studies where zinc was added to baby formula showed 19 that the zinc-supplemented children gained more weight and experienced an increase in height faster than controls, and that these gains were permanent (Med. World News, 1976). A recent study seems to lend support to the ori- ginal observations relating zinc deficiency to poor growth in humans. Hambridge g3 g1. (1972) demonstrated low con- centrations of zinc in the hair of children from middle- and upper-income families in Denver. These children had poor growth, and many had diminished taste acuity. Zinc supplementation resulted in improved appetite, growth and taste. The occurrence of zinc in virtually all biological systems, and definitive knowledge that zinc is indispen- sible to living matter, has given direction to critical experiments in biochemistry, physiology, medicine and nutrition. As a result, it has become clear that zinc participates in a wide variety of processes including pro- tein, nucleic acid, carbohydrate, and lipid metabolism. Since an increasing number of diseases have proved to produce a zinc deficiency or imbalance, the present study was undertaken to examine the effects such a deficiency has on renal function in the rat. As a zinc metalloenzyme, carbonic anhydrase plays an important role in regulating acid-base balance in the body; hence a zinc-deficient state may alter enzyme activity and upset regulatory processes. To further ascertain possible alterations in renal 20 function, three diuretic drugs with minimal to maximal carbonic anhydrase inhibitory activity were administered. Changes in urinary constituents, as a result of diuretic therapy, were assessed and compared to control values. MATERIALS AND METHODS Animals and Diets Weanling, male Sprague-Dawley strain rats, obtained from Spartan Research, Inc., Haslett, Ml, were used for the development of a zinc-deficient model. Upon arrival the animals were randomly assigned to one of four treat- ment groups: Group I: Day zero controls: Animals were killed for determination of plasma and kidney zinc con- centration prior to dietary treatment. Group II: Zinc- deficient: Animals were fed a commercially prepared zinc- deficient diet ad libitum. Group III: Pair-fed control: Animals were pair-fed a zinc-supplemented diet so that the food intake of Group III was equivalent to that of Group II. Group IV: Zinc-supplemented: Animals were fed a zinc supplemented diet ad libitum. Groups II, III and IV were fed for three or six weeks. Diets used during this study were purchased from Teklad Test Diets, Madison, WI (Table l). The zinc-deficient diet contains less than 1 ppm zinc. The zinc control diet is identical to the deficient diet except that zinc carbonate has been added at a level of 50 ppm. Diets were isocaloric and complete with respect to fat, protein, energy, vitamin and mineral content. 21 22 Table 1. Zinc Deficient Rat Test Diet* Ingredient 9/k9 Egg White Solids, Spray Dried 200.0 Dextrose 636.498 Corn Oil 100.0 Non—Nutritive Fiber (Cellulose) 30.0 Sodium Chloride 5.551 Potassium Phosphate 10.687 Calcium Phosphate 2.489 Magnesium Sulfate 1.651 Calcium Carbonate 9.944 Ferric Citrate 0.911 Potassium Iodide 0.026 Manganese Sulfate 0.008 cupric Sulfate 0.009 Cobalt Chloride 0.001 Biotin 0.004 Vitamin 812 0.02 Calcium Pantothenate 0.016 Choline Chloride 1.5 Folic Acid 0.000 Niacin 0.025 Pyridoxine HCl 0.004 Riboflavin 0.006 Thiamin HCl 0.01 Vitamin A Palmitate 0.02 Vitamin 02 0.002 Vitamin E Acetate 0.22 Menadione 0.001 Chlortetracycline HCl 0.39 *In the Control Diet, Zinc Carbonate (ZnCO3) is added at a level of 0.0892857 g/kg of diet (adds 50 ppm zinc). 23 Animals were housed individually in stainless steel cages held on a stainless steel rack, in a room with limited access specifically designated for this trace element research. All animals received double distilled water ad libitum from glass water bottles specially fitted with plastic stoppers and stainless steel water spouts (Swenerton and Hurley, 1968). Special precautions were taken throughout this study to eliminate all sources of environmental contamination which may have interfered with analytical techniques. Cages were rinsed thoroughly with double distilled water before housing animals. All glassware, including water bottles and glass food cups, were washed in a 4 N hydrochloric acid solution and rinsed repeatedly with doubled distilled water prior to use. Animals were weighed three times weekly and food intake recorded daily. Determination of Zinc Status Animals in Group I were killed prior to any dietary treatment following anesthesia with sodium pentobarbital. A large blood sample was drawn from the abdominal aorta for determination of plasma zinc concentration. The kidneys of the animal were perfused and wet-ashed, in the manner described below and the digest was used to determine kidney zinc content. After the remaining animals were on the three feeding regimens for three or six weeks they were weighed and 24 anesthetized with sodium pentobarbital (40 mg/kg I.P.). A 50 ml blood sample was drawn from the abdominal aorta and 4 m1 used for the determination of plasma zinc concentra- tion. A one-tenth m1 aliquot of the sample was diluted 1:100 with double distilled water and used to determine whole blood carbonic anhydrase activity according to the changing pH principle of Philpot and Philpot (1936), as described below. Three-tenths m1 of the sample was diluted 1:4 with .l N hydrochloric acid for determination of whole blood zinc concentration (Dawson and Walker, 1969). The kidneys of the animal were perfused through the abdominal aorta with .25 M sucrose until the organ was light tan in color and the effluent from the renal vein colorless. The kidneys were removed, decapsulated, and weighed separately. The left kidney was used for deter- mination of tissue zinc concentration. A known amount of tissue (wet weight) was wet-digested with a mixture of nitric acid and perchloric acid (5:1) on a hot plate and then diluted with .1 N hydrochloric acid to 10 ml for metal analysis (Sugawara, 1977). The right kidney was thoroughly minced and homogenized in 20 volumes of .25 M sucrose using a Potter-Elvehjem glass homogenizer with a motor driven pestle. The homogenate was centrifuged for one hour at 100,000 g in a refrigerated ultracentrifuge. The supernatant, following careful separation from the pellet, was diluted 1:10 with double distilled water and 25 used for determination of renal carbonic anhydrase activity. The changing pH principle of Philpot and Philpot is as follows: Carbon dioxide is bubbled at a constant rate through a phenol red indicator solution which changes color near pH 7. A standard amount of alkaline buffer (pH 9) is added, causing the indicator to change color as the reac- tion starts. The hydration of carbon dioxide to form car- bonic acid neutralizes the added buffer base and the indicator turns back to the original acid color. The time of hydration (measured from the addition of buffer) is 62-72 seconds in the absence of carbonic anhydrase. When enzyme is added, the reaction rate is increased and a reproducible relationship exists between time and enzyme activity. One enzyme unit is defined as the quantity which will double the rate of the uncatalyzed reaction under the specified conditions. For each determination of carbonic anhydrase activity carbon dioxide was bubbled through water in a bottle im- mersed in a constant temperature ice bath, and led to the reaction vessel (15 ml glass test tube) at a constant rate as monitored using a flowmeter. Five ml of phenol red indicator solution (12.5 mg phenol red dissolved in 1 liter of 0.0026 M NaHCO were added to the reaction 3) vessel. Diluted preparations of whole blood or kidney supernatant were added at concentrations of .05 m1, .1 m1, .2 ml, .4 ml, or .6 m1. Distilled water was used to make 26 a final volume of 6 ml in the reaction vessel. A stop- watch was started upon addition of the buffer (300 ml of l M Na2C03 was added to 206 m1 of l M NaHC03 and made up to 1 liter. 1 ml was used.) The timer was stopped when the solution turned yellow, giving the time of hydration of carbon dioxide over the pH range of phenol red. The standard deviation of a single determination was on the average i 10% of the concentration of enzyme. Carbonic anhydrase activity of whole blood and kidney was determined the day an experiment was carried out. Plasma and kidney samples were frozen for future zinc analysis. Zinc content of all tissues was determined by atomic absorption spectrophotometry using a Varian 375 Atomic Absorption Spectrophotometer. Determination of Renal Function To quantify the effects of zinc deficiency on renal function, clearance experiments were carried out on animals maintained on each dietary treatment for six weeks. Animals were anesthetized with 40 mg/kg sodium pentobar- bital, intraperitoneally. Body temperature was monitored with a rectal temperature probe and maintained at 37°C using heat lamps. A PE 50 cannula was inserted into the bladder and urine collected under mineral oil in pre-weighed glass vials. The left jugular vein was cannulated for infusion of a solution containing 2.5% inulirT and 25% rat 27 plasma in normal saline. 3H inulin (0.5 uCi/ml) was added to this solution which was infused at a constant rate using a Harvard infusion pump. Animals were volume expan- ded at 4% of body weight for 20 minutes with this solution. Following the volume expansion period, the animals were given one of three diuretics intravenously - acetazolamide, furosemide or hydrochlorothiazide. Two doses of each drug were used: acetazolamide - 5 mg/kg, 20 mg/kg; furosemide - 2 mg/kg, 20 mg/kg; hydrochlorothiazide - 5 mg/kg, 20 mg/ kg. Urine collections for a one hour period were initiated following drug injection. During the drug period, a saline infusion solution containing 1.4% inulin, and 3H inulin (0.5 uCi/ml) was infused to compensate for fluid losses occurring with diuresis. At the end of the clearance, a terminal blood sample was taken. Blood pH and pCO2 were measured immediately with an Instrumentation Laboratory pH - gas analyzer. Urine collection vials were reweighed to determine urine volume. Urinary bicarbonate concentration was calculated from the Henderson-Hasselbach equation using pH and pCO2 measurements. Sodium and potassium was measured in plasma and urine samples by lithium standard flame photometry (Instrumentation Laboratory flame photo- meter Model 143). Chloride was measured using a Buchler- Cotlove automatic titration chloridometer. 3H inulin in the plasma and urine was determined using a Beckman LS-250 Liquid Scintillation counter and employing internal 28 standards. The clearance of inulin, calculated from urine flow rate and the concentrations of inulin (dpm/ml) in urine and plasma, was used to estimate glomerular filtration rate. Statistical Analyses All data were reported as means i S.E.M. Differences between means were analyzed statistically using the Student- Newman-Keuls test following analysis of variance. The 0.05 level of probability was used as the criterion for signi- ficance. RESULTS Food Intake Food consumption of animals fed the zinc-deficient diet was significantly less than animals fed the zinc- supplemented diet. Significant differences in food intake between the two groups were noted after six days of feeding and continued throughout the three week (Figure l) and six week (Figure 2) study periods. Body Weight Following the three week feeding period body weight of the zinc-deficient animals (62.02 E 2.9 g) and pair-fed controls (71.77 E 3.2 g) was significantly less than animals fed the zinc-supplemented diet ad libitum (168.11 E 4.3 9) (Figure 3). After six weeks on the respective diets zinc- deficient animals weighed 68.83 E 4.0 g and pair-fed con- trols weighed 90.66 E 3.7 9. These values are significantly less than those of animals fed the zinc-supplemented diet ad libitum (300.66 E 7.2 g) (Figure 4). Kidney Weight and Kidney Weight to Body Weight Ratio No differences were noted in kidney weight of zinc- deficient animals after three or six weeks (.804 E .05 g and .818 E .03 g respectively) when compared to animals 29 Figure l. 30 Food intake of rats fed zinc-deficient or zinc-supplemented diets for three weeks. Each value represents mean E S.E.M. of nine animals. Absence of a vertical bar indicates that S.E.M. is within the radius of the point. Asterisks denote a statistical dif- ference from zinc-supplemented animals (p < .05). FOOD INTAKE (g) 24 31 £0 ZINC'DEFICIENT OI— ZINC'SUPPLEMENTED DAYS Figure 2. 32 Food intake of rats fed zinc-deficient or zinc-supplemented diets for six weeks. Each value represents mean E S.E.M. of nine animals. Absence of a vertical bar indicates that S.E.M. is within the radius of the point. Asterisks denote a statistical dif- ference from zinc—supplemented animals (p < .05). FOOD INTAKE (g) 28 I 24 20- 16- 12 .E‘ ZlNC-DEFICIENT .— ZlNC" SUPPLEMENTED /\/\ / \ flail/r” LH 6 12 L# 18 DAYS 24 3O Figure 3. 34 Body weight of rats fed zinc-deficient or zinc-supplemented diets for three weeks. Each value represents mean E S.E.M. of nine animals. Absence of a vertical bar indicates that S.E.M. is within the radius of the point. Asterisks denote a statis- tical difference from zinc-supplemented animals (p< .05). aoov WEIGHT (9) 180 160 5’ '8' 2O 35 — ZlNC-DEFICIENT m PAIR-FED CONTROL O--o ZINC'SUPPLEMENTED . 0“”. DAYS Figure 4. 36 Body weight of rats fed zinc-deficient or zinc-supplemented diets for six weeks. Each value represents mean E S.E.M. of six animals. Absence of a vertical bar indi- cates that S.E.M. is within the radius of the point. Asterisks denote a statistical difference from zinc-supplemented animals (p < .05). BODY WEIGHT (9) 350 300 250 200 150 100 37 —0 ZINC-DEFICIENT 0mm. PAIR-FED CONTROL o—-o ZINC-SUPPLEMENTED I “In”... “.0uuuuun0uunuuun I DAYS “quIIIIOI.IIIIIIIIIIMI. *- 'X- 38 pair-fed the zinc-supplemented diet for the same length of time (.800 E .04 g and .863 E .03 g respectively). In contrast, the kidney weight of zinc-supplemented animals fed ad libitum was significantly increased after three and six weeks (Table 2). Kidney weight, relative to body weight, KW/BW, was significantly reduced in animals fed the zinc-supplemented diet ad libitum after three weeks (1.06 E .04) and six weeks (.88 E .02) when compared with animals on the zinc-deficient diet for these times (1.24 E .02 and 1.20 E .06 respectively). These differences were also observed after six weeks in pair-fed controls but not after three weeks of feeding (Table 3). Plasma Zinc Concentration Plasma zinc concentration of all animals maintained on the zinc-deficient diet was significantly less than day zero controls, pair-fed controls or zinc-supplemented animals fed ad libitum (Figure 5). Pair-fed controls had plasma zinc concentrations of 1.4 E .10 ug/ml at the end of three weeks: not significantly different than zinc- supplemented animals fed ad libitum for three weeks (1.35 E .13 ug/ml) or six weeks (1.44 E .05 ug/ml). However, by six weeks, plasma zinc concentration of pair-fed controls had fallen significantly below values obtained at three weeks (Figure 5). Plasma zinc concentration of animals sacrificed immediately upon arrival (.93 E .03 ug/ml) was 39 Table 2. Kidney weight (g) of rats fed zinc-deficient or zinc-supplemented diets for three or six weeks Diet Weeks on Diet Feeding Pattern 3 6 Zinc-deficient a Ad libitum .804 i .05 .818 i .03 Zinc-supplemented Pair-fed .800 i .04 .863 i .03 Zinc-supplemented Ad libitum 1.731 i .Tob’C 2.586 t .69b’C 1+ aValues represent mean S.E.M. of five animals bSignificantly different from zinc-deficient animals cSignificantly different from animals pair-fed the zinc-supplemented diet 40 Table 3. Kidney weight to body weight ratio (KW/BW x 100) of rats fed zinc-deficient or zinc- supplemented diets for three or six weeks Diet _fi Weeks on Diet Feeding Pattern 3 5 Zinc-deficient a Ad libitum 1.24 E .02 1.20 E .06 Zinc-supplemented b Pair-fed 1.20 E .03 .98 E .02 Zinc-supplemented b c b c Ad libitum 1.06 E .04 ’ .88 E .02 ’ aValues represent mean i S.E.M. of five animals bSignificantly different from zinc-deficient rats CSignificantly different from animals pair-fed the zinc- supplemented diet Figure 5. 41 Plasma zinc concentration of animals sac- rificed prior to dietary treatment or fed zinc-deficient or zinc-supplemented diets. Each value represents mean E S.E.M. of five animals. Absence of a vertical bar indicates that S.E.M. is within the radius of the point. Asterisks denote a statis- tical difference from zinc-supplemented animals fed for three or six weeks (p < .05). The star represents a statistical difference between day zero controls and animals fed the zinc-supplemented diet ad libitum (p < .05). (pg/ml) PLASMA ZINC CONCENTRATION 1.60 1.40 1.20 1.00 .80 .60 - .40 - .20 - 42 _ ZINC'DEFICIENT .uuu. PAIR'FED CONTROL o——o ZINC'SUPPLEMENTED' h 43 significantly less than animals fed the zinc-supplemented diet ad libitum for three and six weeks. Whole Blood Zinc Concentration No differences in whole blood zinc concentration were observed among the three dietary groups at any time through- out this study (Table 4). Kidney Zinc Concentration Kidney zinc concentration was significantly reduced in zinc—deficient animals after six weeks (12.62 i .92 ug/g) when compared to zinc-deficient animals at three weeks (18.27 E .62 ug/g), pair-fed controls at three and six weeks (21.28 E 1.08 ug/g and 22.63 E .78 ug/g respectively), and animals fed the zinc-supplemented diet ad libitum for three and six weeks (20.26 E .82 pg/g and 23.68 E 1.03 ug/g respectively). Kidney zinc concentration of day zero controls did not vary from values obtained for animals fed the zinc-supplemented diet for three and six weeks (Figure 6). Whole Blood Carbonic Anhydrase Activity Carbonic anhydrase units of activity in the blood of zinc-deficient animals and pair-fed controls after three weeks were significantly greater than in zinc-supplemented animals fed ad libitum (Figure 7). After six weeks of feeding, carbonic anhydrase activity in the blood of 44 Table 4. Concentration of zinc (ug/ml) in whole blood of rats fed zinc-deficient or zinc-supplemented diets for three or six weeks Diet Weeks on Diet Feeding Pattern 3 6 Zinc-deficient a Ad libitum 2.97 E .26 3.12 E .18 Zinc-supplemented Pair-fed 3.47 i .27 3.45 E .13 Zinc-supplemented Ad libitum 3.31 E .10 3.31 + .15 aValues represent mean i S.E.M. of five animals Figure 6. 45 Kidney zinc concentration of animals sacri- ficed prior to dietary treatment or fed zinc-deficient or zinc-supplemented diets. Each value represents mean E S.E.M. of five animals. Absence of a vertical bar indi- cates that S.E.M. is within the radius of the point. Asterisks denote a statistical difference from zinc-supplemented animals and)pair-fed controls at six weeks (p < .05 . KIDNEY ZINC CONCENTRATION (pg/gm) 24 20 16 12 46 "‘I’IIIOOIIIIIOIIII IIIIIIIIIIIIIII. — ZINC-DEFICIENT ."m'. PAIR-FED CONTROL .— ZINC‘ SUPPLEMENTED WEEKS Figure 7. 47 Carbonic anhydrase activity in the blood of zinc-deficient or zinc-supplemented rats at three and six weeks. Each bar represents the mean E S.E.M. of five animals. Stars denote a statistical difference from all other dietary groups at that time period (p < .05). C. A. UNITS /ml BLOOD 2000 1000 48 ZlNC-DEFICIENT PAIR-FED CONTROL 1:1 ZlNC-SUPPLEMENTED 3 WEEKS 6 WEEKS 49 zinc-deficient animals was significantly greater than in pair-fed controls and zinc-supplemented animals (Figure 7). Kidney Carbonic Anhydrase Activity No differences were found in renal carbonic anhy- drase activity, regardless of dietary treatment, when animals were fed for three weeks (Figure 8). After six weeks of feeding, carbonic anhydrase activity in kidney supernatant of zinc-deficient animals and pair-fed controls was significantly greater than in zinc-supplemented animals (Figure 8). ACETAZOLAMIDE Blood pH Blood pH of control animals was significantly greater than animals receiving a low or high dose of acetazolamide for any dietary treatment. No differences were noted in blood pH among the three dietary groups in control or drug- treated animals (Table 5). Blood pCOz Blood pCO2 did not differ among dietary groups in drug treated or control animals. No differences were noted between similar dietary groups in control animals or those given a low drug dose. Blood pCOZ was significantly greater in zinc-supplemented animals given acetazolamide when Figure 8. 50 Carbonic anhydrase activity in the kidney of zinc-deficient or zinc-supplemented rats at three and six weeks. Each bar re- presents the mean E S.E.M. of five animals. Stars denote a statistical difference from all other dietary groups at that time period (p < .05). 400 300 200 RENAL C.A. UNITS/9 100 51 ZINC-DEFICIENT PAIR-FED CONTROL CZ] ZlNC-SUPPLEMENTED 3 E w # ’ fl —_ —l * fl — * ’ fl * '- fl *- - ’ —-— * fl # fl — ’ # fl *- * ’ fl —- fl ’ # fl #- * ——— fl ” EEKS _ * E 111111111111111111111111111111 52 Amx\me cv pogucou EOCC a:msm&u.u spueuu¢5_=m.ma Acv .:.m.m A some »:Omoggmg mmapa>a any Anv Amy any AmC Am. e:»_a__ e< nme..«~o.me num.—«om.ee we.pwmw.~m amo.wen.~ a—o.fimm.n mc.Ame.~ cmucosm—aasm-uc_~ .mv .mV Am. An. AMV AGE eoe-t_aa mu.«~p.mm nu.mu~o.am ce.~«~o.~m cec.fimm.u apc.Amm.u mo.Awe.~ umucwsm_aa=m-u:_N Any AME Ase . AME . Amv EEE , EEA.E__ EE mm.umm.mm wo.~«~—.um mp.~flcm.om awc.fiem.~ a.o.«wm.n umO.ame.m u:m.u.uoo-o:_N cu m o cw m c :Cmuuma m:_ummu Amx\uav Omen ov—Eapouauoo< Aozssv Neva coo—m to u=o_o_woqu=_~ to» mac; vmuaogu oo_su_o~nuoum v Amx\mev Once oufisapo~uuoo< Ea uoopm aw.o mxmmz xym EOC mummv voucoEO—aaamio=_~ cu pogucoo Co c on Eco—E tea Ea woo—m .m space 53 compared to untreated animals in this dietary group (Table 5). Plasma Sodium Concentration No differences were found in plasma sodium concentra- tion between drug-treated and control animals, or among the three dietary groups for any drug treatment (Table 6). Plasma Potassium Concentration Plasma potassium concentration did not vary among drug- treated or control animals. No differences were seen between dietary groups for any treatment (Table 6). Plasma Chloride Concentration Animals treated with low and high doses of acetazo- lamide had significantly greater plasma Chloride concentra- tions than control animals (Table 6). No differences were seen among dietary groups for any treatment. Urinary Sodium Excretion Urinary sodium excretion was significantly enhanced in drug-treated animals when compared to control animals for any dietary treatment (Figure 9). Animals fed the zinc-supplemented diet ad libitum excreted a significantly greater amount of sodium than zinc-deficient animals or pair-fed controls when given a low or high dose of aceta- zolamide. Sodium excretion was significantly greater in untreated zinc-deficient animals (1.49 E .01 qu/minute) 54 Auxxms c. pogucou Seem «cogu»upu a—ucau_u—=m.m a Acv .:.m.m A com: acumosaos mos—E>E An. “EV- .E. “Ev .Ev AE. A”. An. RE. Em.~a En... EE.A EE.A E_.A EN.“ EE._A ~m._w EA.“ EEE—E.. EE EEE.._. EEE.E__ E...E. EA.” EE.E E... A..m.. EE.EE_ EA..E_ EEEEEEE.EEEA-EEEN A". An. AA. .mE Am. .E. .m. .E. Am. EA.~. E...H EE.A E_.A EE.A En.“ EE.A EE.~A EE.~A EEE-E..E EA~.~_. EAE.E._ nE.EE_ EE.E EE.E n~.. EE.EE_ mm.EE_ EE.AE_ EEEEEEE.EEEa-EE.N an. .n. 2A.- .EE An. “E. An. .n. .EE mm.mu EE... E_._. E~.. E~.. AN.“ E..n« mm.“ EE.EA EEE.E._ EE EEE.A_. EEE.E__ EE.EE_ n~.E a... EE.E A..EE, EE.EE. EE~.EE. EEE.E.CEE-EE.N EN E E EN E E EN m E aEJNmsq omov uudamdedmmmml Admwwsv one: ov.eE—o~ouoo< waxwev «moo mopeE—o~auou< :cmuuua EE'EEEE oo_Co—gu e=*mmeuoa EE—vom uc_a mama: x.m so» muo.e voucoso—Eaam-u=_~ so u:m.u.uoo-o=.~ to» was; toauoga av.sE—o~ououa ecu —osu:oo Ev av.gopgo ecu Ez—munuoa .22.OOm Co Eco—ungucoueou mama—E .o o—EEH Figure 9. 55 Urinary sodium excretion in control and. acetazolamide treated rats fed Zinc-defi- cient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). Stars denote a statistical difference from all other dietary groups at that drug dose (p E .05). URINARY SODIUM EXCRETION (qu/min) 12 10 56 - ZlNC-DEFICIENT E PAIR-FED CONTROL E ZINC-SUPPLEMENTED Ill|1|llllllllllllllllllllllllllllllllllllllllll lllllllllllllllllllllllllllllllllllHllllllllll O 5 ACETAZOLAMIDE DOSE (mg/k9) 57 than in untreated pair-fed or zinc-supplemented animals (.46 E .08 qu/minute and .69 E .04 qu/minute respectively) (Figure 9). Urinary Potassium Excretion Pair-fed controls and zinc-supplemented animals fed ad libitum excreted a significantly greater amount of potassium when given a low or high dose of acetazolamide than untreated animals in these dietary groups. Potassium excretion in zinc-deficient animals given a high drug dose (1.90 E .20 qu/minute) was significantly greater than in untreated zinc-deficient animals (.53 E .04 qu/minute) or those given a low dose of acetazolamide (.85 E .09 qu/ minute). No differences were found among dietary groups in control animals or when a high dose of acetazolamide was administered (Figure 10). Potassium excretion was significantly less in zinc-deficient animals and pair-fed controls given a low dose of drug when compared to animals fed the zinc-supplemented diet ad libitum. Urinary Chloride Excretion Urinary chloride excretion did not differ among con- trol animals or those given a low dose of acetazolamide for any dietary treatment, or between dietary groups at these doses (Figure 11). When a high dose of acetazolamide was given to zinc-supplemented animals, chloride excretion was significantly greater (3.87 E .49 qu/minute) than in Figure 10. 58 Urinary potassium excretion in control and acetazolamide treated rats fed zinc-defi— cient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). Stars denote a statistical difference from all other dietary groups at that drug dose (p < .05). URINARY POTASSIUM EXCRETION (qu/min) 59 - ZlNC-DEFICIENT E PAIR-FED CONTROL E ZlNC-SUPPLEMENTED 5 ACETAZOLAMIDE DOSE (mg/kg) Figure 11. 60 Urinary chloride excretion in control and acetazolamide treated rats fed zinc-defi- cient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). Stars denote a statisti- cal difference from all other dietary groups at that drug dose (p < .05). URINARY CHLORIDE EXCRETION (qu/min) 61 - ZlNC-DEFICIENT _=-.=_ PAIR-FED CONTROL 1: ZlNC-SUPPLEMENTED ° HHIHHHUHHHHH l- .. HUHHHHHHHIHH ACETAZOLAMIDE DOSE (mg/k9) k: *— § —— _— *— ‘ § —— _ —. _—_ ‘ § ‘ ~ __ ——-— - *- N O 62 pair-fed controls (1.85 E .02 qu/minute) or zinc-defi- cient animals (1.23 E .10 qu/minute). Chloride excretion was greatest in zinc—supplemented animals given a high drug dose when compared to untreated animals or those given a low drug dose. Urinary Bicarbonate Excretion Urinary bicarbonate excretion of animals in each die- tary group was significantly enhanced by treatment with acetazolamide when compared to animals not receiving the drug (Table 7). In zinc-supplemented animals given a high dose of acetazolamide, the urinary excretion of bicarbonate was significantly greater (9.06 E1.06 qu/minute) than in zinc-deficient animals (4.16 E .62 qu/minute) or pair-fed controls (3.45 E .22 qu/minute). No differences were noted among dietary groups in untreated animals or those given a low drug dose. Glomerular Filtration Rate Glomerular filtration rate (GFR) was significantly depressed in zinc-deficient animals (.27 E .06 ml/minute) and pair-fed controls (.22 i .05 ml/minute) when compared to zinc-supplemented animals (.82 E .06 ml/minute) in the control group. GFR did not differ among zinc-deficient animals or pair-fed controls when given a low dose of acetazolamide; however, GFR's were significantly less for both groups than the GFR for zinc-supplemented animals 63 Table 7. Urinary bicarbonate excretion of control and acetazolamide treated rats fed zinc-deficient and zinc-supplemented diets for six weeks Bicarbonate Excretion (qu/minute) Diet Acetazolamide dose (mg/kg) Feeding pattern 0 5 20 Zinc-deficient .0005E.000Ta 3.94:.35b 4.16:.62b Ad libitum (6) (3) (3) Zinc-supplemented .0004t.0006 3.33:.37b 3.45:.22b Pair-fed (5) (3) (3) Zinc-supplemented .OOl4i.001 3.43:.27b 9.061‘1.06b’c Ad libitum (8) (3) (3) aValues represent mean E S.E.M. (n) bSignificantly different from control (0 mg/kg) cSignificantly different from zinc-deficient animals and pair-fed controls 64 (Table 8). At a high dose of acetazolamide no differences in GFR were found between pair-fed controls (.77 E .06 ml/ minute) and zinc-supplemented animals (.82 i .06 ml/minute), but GFR in zinc-deficient animals remained depressed (.32 E .03 ml/minute). No differences in GFR were found between similar dietary groups in zinc-deficient or zinc-supple- mented animals. GFR in pair-fed controls given a high dose of drug was significantly greater than the GFR in untreated animals or those given a low drug dose. FUROSEMIDE Blood pH No differences in blood pH were found between control animals and those treated with a low or high dose of furo- semide for any dietary treatment. Differences between the three dietary groups within each treatment were not appar- ent (Table 9). Blood pC02 Untreated animals had significantly higher blood pCOZ levels than animals receiving a low or high dose of furo- semide regardless of dietary treatment (Table 9). No differences were noted between similar dietary groups for any treatment. 65 Table 8. Glomerular filtration rate (GFR) in control and acetazolamide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks _ GFR (ml/minute) Diet Feeding pattern Acetazolamide dose (mg/kg) 0 5 20 Zinc-deficient .27 E .06a .29 E 04 .32 E .03 Ad libitum 5) (3) (3) Zinc-supplemented .22 E .05 .38 E .03 .77 E .oec’d Pair-fed 6) (4) (5) . b b b Zinc-supplemented .82 E .06 .89 E 10 .82 E .06 Ad libitum 6) (3) (5) aValues represent mean S.E.M. (n) bSignificantly different from zinc-deficient animals and pair-fed controls cSignificantly different dSignificantly different animals given a low drug from zinc-deficient animals from control dose (0 mg/kg) and 66 EPEENEE EONEELEIEECE EEC; «acumen—E EPEEEOFC_Em+m E Acv .z.u.m A game acmmmgawg mmsz>E ENE .m. EEE ENE ENE EEE EEEEEEE EE NE.NAEE.EN EN.NAEN.EN EEE..AEE.NE NE.N_E.N NE.AEE.N EE.ANE.N EEEEEEEPEEEE-EEEN AN. Amy AEE AME Amy AEE EEE-L.EE mm._d_E._N EE._A_E.EN EEE.NANE.NE mm.a.m.~ NE.ANE.N EE.AEE.N EEEEEEE_EEEW-EEEN AEE Amy NEE EEE AME EEE EEEEE.P EE EE.NAE_.NN EE.AEE.NN EEP.NAEE.EE E_.AEE.N EE.NNE.N EEE.AEE.N EEEeEEEEE-EE_N EN N E EN N E Aax\msv Omen OENEOmOLEE Amx\asv Omen mE—Emmocau :CONEEE EEEEEOE AEEEEE NEEE EEE.E EE EEE_E EEEE mxmoz x_m so» muwvu umacms Iw—EEEEIOE—N Co u:o.uwwwEIuE_~ uoC mung uoummgu mEmEmmoCEC EEE ~ECNEOO Co Noun coopn EEE :E coopm .a mpnmh 67 Plasma Sodium Concentration Plasma sodium concentration in the three dietary groups did not differ between control animals or those receiving furosemide. Differences among dietary groups for any drug treatment were not apparent (Table 10). Plasma Potassium Concentration No differences in plasma potassium concentration were found among dietary groups for any drug treatment or between drug-treated and control animals for any dietary treatment (Table 10). Plasma Chloride Concentration Plasma chloride concentration did not differ among drug-treated or control animals for any dietary treatment, or between dietary groups for any drug treatment (Table 10). Urinary Sodium Excretion Animals in each dietary group treated with furosemide excreted a significantly greater amount of sodium than control animals (Figure 12). At both a low and high drug dose, sodium excretion in zinc-deficient animals was signi- ficantly less than in pair-fed controls and zinc-supplemented animals. In contrast, sodium excretion in untreated zinc- deficient animals (1.49 E .01 qu/minute) was significantly greater than in pair-fed controls or zinc-supplemented animals (.46 E .08 qu/minute and .69 E .04 qu/minute 623 REV .z.m.m A game acmmmgamg NEEPE>E ENE ENE .EE AN. any “EV ENE Amy EEE .N.NA EE.NA EE.A E_.H EN.A EN.N EN._A EE..A EN.“ EEE,EE_ EE EE.EE_ EE.NEF E..EE_ .E.N EE.N E_.E NN.NEP EE.EE_ EN.EE_ EEEEEEE_EEEE-EEEN .EE ENE ENE ENE Am. EEE AN. ENE EEE mm.pN FN._N ea.“ m—.N m¢.N om.N op.pN mm.NN me. A EECICNEE EN.NE. N_.EE. NE.EE. NE.N EE.E NN.E NE.NE. EE.EE_ EE.NE_ EEEEEEEPEEEA-EE_N ENE ENE ANV .N. Am. .EE ENE ENE NEE EN.“ NN.NN E_._A EE.A NE.N NN.A .E.A N...“ EE.EN EEE.E__ EE EE.EE_ EN.NE_ EE.EE_ mE.N NE.E EE.E .E.EE_ mE.EE_ EEN.EE. EEEEE.EEE-EE_N EN N E EN N E EN N E Amxxmev «woe mu_somogsu EEELEPEE Aux\aev OEOE EENEEEECEE EENENENOE Amx\asv wmou OENEOEECEE EEEEEN ccouuaa mcpummu «Ewe mxmm: x—m EEC mum—c EONEEEOPEEENIOENN so EEONONCOEIuc—N EEC mums cognate mc_Eoch:~ EEE FECNEOO EN EE'EE—go EEE Eavmmmuoa .EENEON Co NEE—uncucmucou EEEEPE .o. Open» Figure 12. 69 Urinary sodium excretion in control and furosemide treated rats fed zinc—deficient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). Stars denote a statistical difference from all other dietary groups at that drug dose (p < .05). 70 .N 15 - ZlNC-DEFICIENT g "'-__-_'=__‘_=-. PAIR-FED CONTROL \u. E:] ZlNC-SUPPLEMENTED lull 3‘10 2 Q '5 __ o: 8 E u E X E III :- 5 6 I a E O E a, _ z: 4 , :— g * z: E :— E- : 2 E z 2 ° 11! 20 FUROSEMIDE DOSE (mg/k9) 71 respectively). Urinary Potassium Excretion Zinc-supplemented animals and pair-fed controls treated with furosemide excreted a significantly greater amount of potassium into the urine than control animals in these dietary groups (Figure 13). No differences were found between untreated zinc-deficient animals and those recei- ving a low drug dose, however, potassium excretion in both of these groups was significantly less than in zinc-deficient animals administered a high dose of furosemide. Zinc-defi- cient animals given a low or high dose of drug excreted a significantly smaller amount of potassium into the urine (.51 E .01 qu/minute and 1.06 i .05 qu/minute respecti- vely) when compared to pair-fed controls (1.28 i .08 UEq/ minute and 1.92 E .07 qu/minute) or zinc-supplemented animals (2.18 E .23 qu/minute and 3.22 i .07 qu/minute) at low or high doses, respectively (Figure 13). Urinary Chloride Excretion Chloride excretion did not differ among dietary groups in untreated animals (Figure 14). No differences in chloride excretion were seen between untreated zinc-defi- cient animals and those given a low drug dose. Pair-fed controls and zinc-supplemented animals excreted a signifi- cantly greater amount of chloride into the urine than zinc- deficient animals when given a low or high dose of Figure 13. 72 Urinary potassium excretion in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). Stars denote a statistical difference from all other dietary groups at that drug dose (p < .05). URINARY POTASSIUM EXCRETION (qu/min) 73 - ZINC-DEFICIENT E PAIR-FED CONTROL E ZINC-SUPPLEMENTED o 2 FUROSEMIDE DOSE (mg/kg) ** 2O Figure 14. 74 Urinary chloride excretion in control and furosemide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statisti- cal difference from control values for that dietary group (p < .05). Stars denote a statistical difference from all other dietary groups at that drug dose (p < .05). URINARY CHLORIDE EXCRETION (qu/min) 5'3 J:- O on '5 F3. I l I T N 75 - ZINC-DEFICIENT E PAIR-FED CONTROL E ZlNC-SUPPLEMENTED 2 FUROSEMIDE DOSE (mg/kg) lllIl|llllllllllllllHllllllllllllllllll“11111111111111” 20 76 furosemide. Chloride excretion was significantly greater in animals receiving a high drug dose when compared to untreated animals or those receiving a low dose of drug for any dietary treatment (Figure 14). Urinary Bicarbonate Excretion Urinary bicarbonate excretion was significantly greater in all dietary groups given a low or high dose of furose— mide when compared to untreated control animals. No dif- ferences were found among dietary groups in untreated animals (Table 11). In contrast, bicarbonate excretion in zinc-deficient animals was significantly greater than in pair~fed controls or zinc-supplemented animals when a low or high dose of furosemide was administered (Table 11). Glomerular Filtration Rate GFR was significantly increased with drug treatment in all dietary groups when compared to untreated animals (Table 12). When a low dose of furosemide was administered to zinc-supplemented animals, GFR was significantly higher (1.29 E .16 ml/minute) than in zinc-deficient animals (.75 E .09 ml/minute) or pair-fed controls (.78 E .13 ml/ minute). No differences were found among dietary groups given a high dose of furosemide (Table 12). 77 Table 11. Urinary bicarbonate excretion in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks Bicarbonate Excretion (qu/minute) Diet Feeding pattern Furosemide dose (mg/kg) 0 2 20 . . . a b.c b.c Zinc-def1c1ent .0005i.0001 .062i.002 .054i.007 Ad libitum (6) (3) (3) Zinc-supplemented .0004E.0006 .014E.003b .O3i.009b Pair-fed (5) (3) (3) Zinc-supplemented .OOI4E.OOI .008E.002b .03E.005b Ad libitum (8) (3) (3) aValues represent mean E S.E.M. (n) bSignificantly different from control (0 mg/kg) CSignificantly different from pair-fed controls and zinc- supplemented animals 78 Table 12. Glomerular filtration rate (GFR) in control and furosemide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks GFR (ml/minute) Diet Feeding pattern Furosemide dose (mg/kg) 0 2 20 . . . a b b ZTnC-defTCTent 27 E .06 75 i .09 1.15 i 10 Ad libitum (5) (3) (3) Zinc-supplemented 22 E 05 78 E 13b 1 12 1 12b Pair-fed (6) (3) (3) Zinc-supplemented 82 E 06 l 29 E .st’c l 31: 07b Ad libitum (6) (3) (5) aValues represent mean E S.E.M. (n) b Significantly different from control (0 mg/kg) CSignificantly different from zinc-deficient animals and pair-fed controls 79 HYDROCHLOROTHIAZIDE Blood pH No significant differences in blood pH were found between drug treated and control animals for any dietary treatment, or among dietary groups for any drug treatment (Table 13). BIOOd pCOZ Animals treated with a low dose of hydrochlorothia- zide had significantly higher blood pC02 values than control animals or those given a high drug dose, regard- less of dietary treatment. No differences were noted between dietary groups for any drug treatment (Table 13). Plasma Sodium Concentration Plasma sodium concentration did not differ between dietary groups in animals receiving hydrochlorothiazide when compared to untreated animals, nor were any differ- ences found among dietary groups for any drug treatment (Table 14). Plasma Potassium Concentration Plasma potassium concentration of untreated animals did not differ from animals receiving a low or high dose of hydrochlorothiazide for any dietary treatment. Dif- ferences among dietary groups for any drug treatment were not apparent (Table 14). 80 «we: asst say; E =O>Nm m—EENEE so Amxxme ov mPEENEE posucoo sogu acogouw_o Apucmu.mpcm.mn AEV .z.m.m A game acmmogawg NOEPE>E Nev AN. NE. NE. EEC EEE.EEP EE _.._ANE.EN ENE.NN_E.NN EE..NEE.NN NE.N_E.N EE.NNE.N EEEEEEE_EEEE-EE.N .E. Ac. AEV NE. AEE .EEE-L_EE NP.NAEN.EN EEE.NNNN._E EE.NANE.NN EE.ANE.N NE.AEE.N EEEEEEE_EEEA-EEEN AN. ENE EEE ENC EEE EEE_EE_ EE ow.~Noo.mm ao~.pN~—.om mp.~Nom.cm po.N~m.n Emo.Am¢.N acmNupumo-E:_N EN m E EN E Aux\mev omoe ouwnnpguogopgoosoaz Amx\aev «not OENNENEHECEPEOECEN: :Lmuuma mcvvmmu «op: Aazeev Ncua woopm Exam: x_m Lou mumwu cmucosOPEEEEIOENN LE NEENONCOEIOENN EEC New; Emuemgo EENNE—guocopguogvx; EEE pogucou CE EEE EEEPE EEE EE EEE_E .N_ E_EEA 81 AEV .z.m.m N cams ucwmmgamg mmzpc>m RE AN. NEE- AEV AN. .E. Nev Amy AEV om.pw —¢.NH mm.+ op.“ pp.“ cw.“ we.“ NF.“ vs.“ saw—app u< c~.mcp sm.~c— wp.ecp mm.m mm.m ¢—.e ww.mvp n~.~e— a~.¢ep voucmsmpaasmiucv~ EEE Amy ENE- EEE NEE .EE .EE AEV Amy a—.pw Nu.“ mm.+ mp.“ on.“ mn.N w—.~« mo.~w we.Nu vmwugvaa O¢.ec— sm.mop mm.mop mm.m cm.m m~.¢ mm.~ep mw.me— mo.ne— tmucmsmpnazmuucpN z E E E E E E E E EE.NA NE.NA E_._A EN.“ E_.A NN.N EE.N Nm._d EE.EA EEE’EE. EE NN.EE. E_.NE_ EE.EE_ mm.m EN.E EE.E N_._E_ EE.NE. EEN.EEP EEE.E.EEE-EE.N om m c cu m 6 ON m c .mx\msv once Amx\msv owe: Amx\msv omen EENNENENECEPEOECEA: OE_NE_EEELEFEEECE>: EENNENEHOCEPEOENEE: ECONNEE mc_EmmE EEELE_EE Ezvmmuuoa Ez—com wove mxwo: x_m so» NEONE coucmeo—EEEEIOENN CE NEENONCmoioc—N to» meat cmuuocu oc—NE_ENEEE—suogux; EEE pogucoo E. oE_CE_:o EEE Ezpmmauoa .EEPEEE Co mcopuaeuzmucou mama—E .E. EPEEH 82 Plasma Chloride Concentration No differences were found among dietary groups for any drug treatment, or between control animals and those given hydrochlorothiazide for any dietary treatment (Table 14). Urinary Sodium Excretion Urinary sodium excretion was significantly greater in zinc-deficient animals, regardless of drug treatment, when compared to pair-fed controls and zinc-supplemented animals (Figure 15). No differences in sodium excretion were found between untreated pair-fed controls and those given a low dose of hydrochlorothiazide, however both were significantly less than animals given a high drug dose. Similar com- parisons were seen in zinc-supplemented animals. Urinary Potassium Excretion Potassium excretion in all dietary groups was sig- nificantly greater after administration of hydrochloro- thiazide when compared to untreated animals (Figure 16). No differences were found among dietary groups for any drug treatment. Urinary Chloride Excretion Chloride excretion was significantly greater in all animals receiving a high dose of hydrochlorothiazide, regardless of dietary treatment, when compared to control Figure 15. 83 Urinary sodium excretion in control and hydrochlorothiazide treated rats fed zinc-deficient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). Stars denote a statistical difference from all other dietary groups at that drug dose (p < .05). URINARY SODIUM EXCRETION (qu/min) 84 - ZINC-DEFICIENT 6 E PAIR-FED CONTROL :1 ZINC-SUPPLEMENTED a: a: *- lllllllllHHIHIHHHHHIHH.. . 111111111111 ° 1111111 20 HYDROCHLOROTHIAZIDE DOSE (mg/kg) Figure 16. 85 Urinary potassium excretion in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). N (a) #- URINARY POTASSIUM EXCRETION (qu/min) 86 - ZlNC-DEFICIENT E PAIR-FED CONTROL [:1 ZINC-SUPPLEMENTED o 5 20 HYDROCHLOROTHIAZIDE DOSE (mg/kg) 87 animals (Figure 17). No differences in Chloride excretion were found among dietary groups in untreated animals. In contrast, zinc-deficient animals given a low or high drug dose excreted a significantly greater amount of chloride than pair-fed controls or zinc-supplemented animals at these doses (Figure 17). Urinary Bicarbonate Excretion Animals in any dietary group treated with a high dose of hydrochlorothiazide, excreted a significantly greater amount of bicarbonate into the urine than control animals or those given a low drug dose (Table 15). Bicarbonate excretion did not differ among dietary groups for any drug treatment. Glomerular Filtration Rate GFR was significantly increased in pair-fed controls given a low or high dose of hydrochlorothiazide (1.08 E .18 ml/minute and .85 E .09 ml/minute) when compared to untreated animals in this dietary group (.22 E .05 ml/ minute). GFR in zinc-deficient animals remained depressed with drug treatment (Table 16). No differences in GFR were found between control and drug treated animals fed the zinc-supplemented diet ad libitum. 88 Figure 17. Urinary chloride excretion in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks. Each bar represents the mean E S.E.M. for a minimum of three animals. Asterisks denote a statistical difference from control values for that dietary group (p < .05). Stars denote a statistical difference from all other dietary groups at that drug dose (p < .05). URINARY CHLORIDE EXCRETION (qu/min) 89 - ZlNC-DEFICIENT * =":': PAIR-FED CONTROL 1:! ZINC-SUPPLEMENTED * * *- H1lHll1111ll1111111111111111111111”!!!11111111l. ° lllllllllllllllllllllll- .. HHHHHHUIHHIHI 20 I .< D an O n I l- O :o O —. E > E U l'l'l O O VD l'l'l 3 Q \ r ‘9, 90 Table 15. Urinary bicarbonate excretion in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks Diet Bicarbonate Excretion (qu/minute) Feeding pattern Hydrochlorothiazide dose (mg/kg) 0 5 20 Zinc-deficient .OOOSE.OOOIa .019E.005 .67E.10b’c Ad libitum (6) (3) (3) Zinc-supplemented .OOO4E.0006 .OOZE.OOO6 .72E.06b’c Pair-fed (5) (3) (4) Zinc-supplemented .0014E.001 .003E.0003 .50E.11b’c Ad libitum (8) (3) (4) aValues represent mean E S.E.M. (n) bSignificantly different from control (0 mg/kg) CSignificantly different from animals given a low drug dose 91 Table 16. Glomerular filtration rate (GFR) in control and hydrochlorothiazide treated rats fed zinc- deficient or zinc-supplemented diets for six weeks GFR (ml/minute) Hydrochlorothiazide dose (mg/kg) Diet Feeding pattern o 5 20 Zinc-deficient 0.27:.06a O.48E.02 0.47:.04 Ad libitum (5) (3) (3) Zinc-supplemented 0.22E.05 1.08E.18b 0.851.09b Pair-fed (6) (3) (4) Zinc-supplemented 0.82E.06 0.91E.07 1.15:.14 Ad libitum (6) (3) (4) aValues represent mean t S.E.M. (n) bSignificantly different from control (0 mg/kg) DISCUSSION In this study, zinc deficiency in the young rat was characterized by a cessation of growth followed by an extended period during which body weight varied very little. Concurrent with this growth failure, daily food intake decreased and this depression in food intake was sufficient to inhibit virtually all growth of pair-fed control animals given the same diet with supplementary zinc. These data are in agreement with those of other investigators repor- ting a persistant stunting of body growth in zinc—deficient animals and pair-fed controls (Macapinlac g3 g1., 1966; Chesters and Will, 1973). Chesters and Quarterman (1970) have shown that force feeding of the deficient animal to raise food intake to that of a normal animal offered a zinc-supplemented diet ad libitum, rapidly induced signs of severe illness followed by death. This observation demonstrates that the poor rate of growth of zinc-deficient rats is not due to a low food consumption per se, but is more likely caused by failure of the growth process at a specific step which requires zinc. The primary metabolic disturbances underlying this effect of growth retardation point to events critical to cell division and nucleic acid metabolism. Studies on 92 93 DNA metabolism in the alga, Euqlena qracilis, have left little doubt as to the involvement of zinc in cell repli- cation. All phases of the growth cycle, from RNA and DNA synthesis, to mitosis in this organism can be blocked by zinc deficiency (Vallee, 1976). Thus it appears that zinc is essential to multiple processes in the growth cycle, critical to the development, division and differentiation of cells. It is however, conjectural at present as to which phase of this process a deficiency of zinc will prove to be growth limiting. Similar to the results reported by Fosmire and Sand- stead (1977), kidney weights were significantly reduced in zinc-deficient animals and pair-fed controls, when compared to animals fed the zinc-supplemented diet (Table 2). Since body weights of these two groups were also significantly reduced, it appears that the overall growth retardation affected the kidneys in a similar manner. Kidney weights as a percentage of body weight for the three groups of rats are shown in Table 3. The percent body weight of the kidney was lowest in zinc-supplemented animals fed ad libitum and greatest in zinc-deficient animals. It was observed however, that the absolute weights of the organs were highest in the ad libitum fed controls and lowest in zinc-deficient animals. Similar results were reported by Prasad and Oberleas (1971). 94 Caloric restriction affected the growth of the kidney in pair-fed controls, in that when expressed as percentage of body weight, the ratio was slightly reduced at six weeks. Kidney weight to body weight ratio remained un- changed, between three and six weeks in zinc deficient rats, indicating that zinc deficiency severely retarded kidney maturation. A normal increase in body weight over time can account for the reduction in the ratio of KW/BW in animals fed the zinc-supplemented diet ad libitum. Subnormal plasma zinc concentrations were observed in all zinc-deficient animals in this study (Figure 5). In addition, plasma zinc concentration responded quickly to the dietary zinc inadequacy suggesting that a gradual deterioration of the overall zinc status of the animal was occurring. Plasma zinc concentration of animals fed zinc- supplemented diets remained in the normal range throughout the three and six week experimental periods. After six weeks, plasma zinc concentration of pair-fed controls had dropped significantly below values reported for animals fed the diet supplemented with zinc ad libitum. At three weeks no differences were found between the two groups (Figure 5). Contrary to these findings, Brown gg g1. (1978) reported the plasma zinc concentration of pair-fed animals remained in the same range as those fed ad libitum. Thus the data reported in this study suggest that nutritional insult, such as severe malnutrition and hence less than 95 optimal zinc intake, may produce subnormal plasma zinc concentrations. Although the decline in plasma zinc was invariably evident in pair-fed animals, values never fell to those seen in zinc-deficient animals indicating that the zinc content of plasma is responsive to changes in dietary zinc intake. The significance of lower plasma zinc concentrations in day zero controls when compared to animals fed the zinc-supplemented diet ad libitum for three and six weeks is questionable. Reported levels are not indicative of a deficiency state and may therefore be due to the zinc content of the diet used to feed the animals prior to obtaining them for experimental purposes. Kidney zinc concentration of zinc-deficient rats after six weeks was significantly less than in animals fed diets supplemented with zinc (Figure 6). Similarly, Kang t l. (1977) found a reduction in kidney zinc concentration m after four weeks of feeding a zinc-deficient diet. These data indicate that homeostatic mechanisms within the body were capable of maintaining kidney zinc concentration within a normal range only until the nutritional insult became severe. Although plasma zinc levels fell early after institu- ting the zinc deficient diet, whole blood zinc did not fall and at no time was there any difference between the defi- cient and supplemented groups. These results are consistent with those reported by Walker and Kelleher (1978). From 96 the data reported here, there appears to be no correlation between plasma and whole blood zinc levels, implying that the latter is of doubtful value as an index of zinc status. The increase in blood carbonic anhydrase activity reported here is in sharp contrast to many previous studies on this enzyme in zinc-deficient rats. Roth and Kirch- gessner (1974) found reduced activity of carbonic anhydrase in blood after four days of zinc depletion. Huber and Gershoff (1973) reported lower carbonic anhydrase activity in packed red cells after four weeks, whereas Day and McCollum (1940) found the enzyme activity of red cells unchanged. In the present study, carbonic anhydrase acti- vity of zinc-deficient rats and pair-fed controls was significantly greater than in zinc-supplemented animals after three weeks (Figure 7). These results imply that differences noted between dietary groups after three weeks must, for the most part, be related to differences in food intake. To account for differences solely on the basis of zinc deficiency, a significant difference in enzyme acti- vity would have to occur between zinc-deficient animals and pair-fed controls. This comparison was not evident from the results obtained, hence the noted increase in enzyme activity was not due to zinc deficiency per se. In contrast, after six weeks carbonic anhydrase activity of zinc-deficient animals was significantly greater than either group fed the zinc-supplemented diet (Figure 7). 97 These findings imply that caloric restriction is still the primary factor responsible for the differences seen in carbonic anhydrase activity between groups fed the zinc- supplemented diet. However, an additional variable is present, over and above malnutrition, which may have affected enzyme activity in the zinc-deficient animal. Enzyme activity in blood and tissue homogenates is often expressed as units per milligram of protein. Al- though the amount of protein in tissues examined in this study was not quantified, a change in this component may reflect the observed Changes in enzyme activity. No differences in kidney carbonic anhydrase activity were seen after three weeks of feeding for any dietary group (Figure 9), but a significant reduction in enzyme activity was noted in rats fed the zinc-supplemented diet for six weeks (Figure 8). Since enzyme activity did not differ between zinc-deficient animals and pair-fed con- trols, it appears that the greater enzyme activity found in these groups compared to zinc-supplemented animals fed ad libitum is related to the difference in caloric intake rather than zinc deficiency per se. The major pharmacological action of acetazolamide is the inhibition of the enzyme carbonic anhydrase, which in turn decreases hydrogen ion secretion and thus reabsorp- tion of bicarbonate in the proximal tubule (Maren, 1967). As a result, the increased urinary excretion of bicarbonate 98 leads to a decrease in the extracellular fluid bicarbonate concentration, and metabolic acidosis occurs. In the present study a significant fall in blood pH was noted in all animals given acetazolamide (Table 5). In addition to its effect on pH, the administration of acetazolamide caused a significant rise in blood pCO2 (Table 5). As a carbonic anhydrase inhibitor, the phar- macological action of this drug is not limited to renal mechanisms but will also effect the carbonic anhydrase activity of circulating erythrocytes. Acetazolamide may therefore create a disequilibrium in the carbon dioxide transport system of blood, giving rise to increased C02 tension. Plasma concentrations of sodium and potassium in any dietary group were unchanged during acetazolamide diure- sis, however, Chloride concentration was significantly increased in all animals receiving the drug. These fin- dings are similar to those of Maren g£_gl. (1954). If water composition of the body were unaltered during the diuresis, sodium concentration would be expected to de- cline. However, the diuretic increases urinary volume and sodium concentration in the plasma tends to be maintained. Since plasma potassium concentration was also unaltered, it seems that initial renal losses must be drawn from the extravascular pool with rapid reequilibration to sustain the plasma concentration. 99 The significance of hyperchloremia noted in the rat during carbonic anhydrase inhibition is questionable, since renal bicarbonate loss is inevitably matched by cations. The resultant change in hydrogen ion concentration of the blood in metabolic acidosis, leaving an anion deficit, may be a physiological stimulus for chloride retention. During diuresis caused by acetazolamide, the urine contains an excess of sodium, potassium and bicarbonate (Maren, 1967). In the results presented here, urinary bicarbonate and sodium excretion were significantly greater following acetazolamide administration when compared to untreated animals. This event is most likely attributed to the inhibition of bicarbonate reabsorption by the renal tubule. The carbonic anhydrase of proximal tubule cells is responsible for furnishing an intracellular supply of hydrogen ions, which then act as a mediator in bicarbonate reabsorption (Rector g3 g1., 1965). When the intracellular action of carbonic anhydrase is inhibited, bicarbonate reabsorption is diminished and hydrogen ion is unavailable for luminal exchange with sodium. As a result, the urinary excretion of bicarbonate and sodium is enhanced. Urinary potassium excretion increased in zinc-supple- mented animals and pair-fed controls after a low dose of acetazolamide was given, and in all dietary groups given a high drug dose (Figure 10). This increase was expected since sodium delivery to the distal nephron is also 100 increased as a result of diminished proximal reabsorption. The filtered sodium is then reabsorbed at this site in exchange for potassium secretion. Hence, an increase in potassium excretion is noted following drug administration. Chloride excretion did not differ among animals re- ceiving a low dose of acetazolamide when compared to con- trol rats. These results are in agreement with those of Weinstein (1968). Since acetazolamide reduces hydrogen ion availability in the proximal tubule, the increase in chloride reabsorption could be a compensatory mechanism to maintain electroneutrality. A significant increase in Chloride excretion was found in zinc-supplemented rats given a high drug dose. Since the urinary excretion of sodium,potassium and bicarbonate was also greatest in this dietary group at either a low or high dose of acetazola- mide, it seems most likely that the enhanced Chloride excretion is only an attempt to prevent an abnormal anion gap from occurring. In the present investigation, the glomerular filtra- tion rate of acetazolamide treated animals did not differ from control values in zinc-deficient or zinc-supplemented groups. The GFR of pair-fed controls given a high dose of acetazolamide however, was significantly greater than values reported for other doses in this dietary group. Since glomerular filtration rate varies with body size, differences seen between zinc-deficient animals and 101 untreated pair-fed controls, or those given a low dose of drug, when compared to zinc-supplemented animals are most likely due to this aspect. The physiological significance 'of an increase in GFR in pair—fed controls given a high dose of acetazolamide is questionable. Body weight of these animals was comparable to zinc-deficient animals, suggesting that the noted increase may be a hemodynamic effect. The diuretic action of furosemide is to inhibit sodium and chloride reabsorption in the ascending limb of the loop of Henle (Burg and Stoner, 1976). Unlike acetazolamide which inhibits carbonic anhydrase and thus produces drama- tic effects on acid-base balance, the diuretic effect of furosemide is to a great extent independent of such alter- ations. In the present study, no significant differences were noted in blood pH of untreated animals when compared to those given furosemide, indicating that the drug had no profound effects on acid-base status (Table 9). While a statistical difference was found in blood pCOZ between animals treated with furosemide and those not receiving the drug, the physiological significance of this is ques- tionable. Blood pC02 was slightly depressed in drug- treated animals suggesting that a metabolic alkalosis may have occurred if the drug had been given by sustained infusion. This being the case, a reduction in blood pCOZ would be expected due to the loss of fluid containing 102 sodium and chloride in approximately equal amounts and virtually devoid of bicarbonate. However, this is only speculative since furosemide was given as a bolus injec- tion in this study. Plasma sodium, Chloride and potassium concentrations did not vary among dietary groups during furosemide diure- sis (Table 10). This finding was not unexpected since fluid and electrolyte losses were replaced during diuretic therapy to offset changes in the composition of extracel- lular fluid. Animals in each dietary group treated with furosemide excreted a significantly greater amount of sodium, potas- sium and chloride into the urine than control animals. These findings are consistent with other investigators (Deetjen, 1966; Bowman g3 g1,, 1973). The saluretic res- ponse seen with furosemide in these animals is due to inhibition of active chloride transport in the ascending limb of the loop of Henle. Inhibition-at this site in the nephron causes an increase in the delivery of salt to the distal tubule, to a point that may exceed the normal trans- port capacity of that segment. The increase in potassium excretion noted in all drug-treated animals is a result of its distal secretion which occurs in exchange for sodium ions at this site (Peters and Roch-Ramel, 1969). A significant increase in bicarbonate excretion was seen in all animals in response to drug treatment. 103 Although the loop of Henle has been identified as the prin- ciple site of drug action, furosemide has been shown to exhibit proximal effects as a weak carbonic anhydrase inhi- bitor (Beyer and Baer, 1961). However, bicarbonate excre- tion in furosemide treated rats was much less than that seen after administration of acetazolamide, providing fur- ther evidence that this action is only secondary to its major pharmacological effect. In the present investigation, glomerular filtration rate was shown to increase in response to treatment with furosemide. This finding might be explained on a hemo- dynamic basis, since furosemide, by its vasodilator action has been shown to increase renal blood flow (Hook g5 g1., 1966). A similar transient increase was found in non- diuretic dogs given furosemide, but not when the drug was administered in water diuresis (Suki g5 g1., 1965). These results imply that the increase in GFR may have been a wash-out effect. In the present study, rats were volume expanded at 4% of body weight for 20 minutes prior to drug administration, possibly alleviating the wash-out phenome- non. This observation lends support to the theory that changes in GFR may be a consequence of a change in renal hemodynamics. The dose-response curves of animals given a low or high dose of furosemide show patterns of electrolyte excre- tion between dietary groups which are similar to those of 104 acetazolamide. This is apparent in that zinc—deficient animals and pair-fed controls fail to elicit the magnitude of response seen in animals fed the zinc-supplemented diet ad libitum. These differences are most likely due to the compromising effects of severe malnutrition on renal function. In zinc-deficient animals given furosemide, urinary sodium, potassium and chloride excretion was sig- nificantly less than in pair-fed controls or zinc-supple- mented animals. Moreover, urinary bicarbonate excretion was significantly greater in zinc-deficient animals at both drug doses than in the other dietary groups. It appears frOm these findings that zinc deficiency, in it- self, has had a detrimental effect on renal function over and above that seen during starvation. The diuretic activity of the thiazide diuretics, including hydrochlorothiazide, is a result of their inhibi- tory action of sodium and chloride reabsorption in the distal segment of the nephron (Mudge, 1975). The renal activity of this diuretic is also associated in part with inhibition of carbonic anhydrase, however this effect is secondary and hydrochlorothiazide is generally not used for this purpose. In the present study hydrochlorothiazide produced no change in blood pH of animals given either a low or high dose, indicating that the drug had little effect on acid- base status in the animal. These results are similar to 105 those reported by Duchin and Hutcheon (1977). Blood pCO2 was significantly higher in all animals given a low dose of hydrochlorothiazide when compared to untreated rats, or those given a high drug dose. The significance of these results are questionable since a concomitant change in blood pH was not detected, and other investigators (Duchin and Hutcheon, 1977) have reported no changes in arterial pCO Furthermore, hydrochlorothiazide did not produce 2. changes in plasma sodium, Chloride or potassium concen- trations since fluid and electrolyte losses in the urine were replaced during the course of these experiments. These findings suggest that the noted increase in blood pCO2 is not a result of drug administration per se, and may be due to procedural error. In contrast to results seen with acetazolamide or furosemide, urinary sodium and chloride excretion in zinc- deficient rats was significantly greater than in pair-fed controls or zinc-supplemented animals after a low or high dose of hydrochlorothiazide was given. The principle saluretic effect of hydrochlorothiazide is due to its action in the distal nephron, hence in this respect the increase in urinary sodium excretion after drug adminis- tration was expected. Since urinary sodium excretion in untreated zinc-deficient rats was significantly greater than in untreated pair-fed or zinc-supplemented animals, it appears as though administration of hydrochlorothiazide 106 had a dose-dependent effect. Greater sodium excretion in zinc-deficient animals implys that the reabsorptive capa- city of the kidney is less than optimal. Thus it appears that when hydrochlorothiazide is given, the effect is amplified and sodium excretion is greatly increased. Since sodium excretion was significantly enhanced in drug-treated animals, excretion of its attendant anion, chloride, was also greatly increased. This ChloruretiC effect seen during hydrochlorothiazide diuresis is due to inhibited reabsorption in the distal tubule (Mudge, 1975). In the present study, a significant increase in potas- sium excretion was noted following administration of hydrochlorothiazide. Due to an increase in sodium con- centration and delivery to the distal tubule, renal mecha- nisms governing potassium secretion are stimulated. In contrast to patterns of sodium and Chloride excretion in the zinc-deficient animal, kaluresis, although signifi- cantly enhanced by drug treatment, did not differ among dietary groups. With small doses of hydrochlorothiazide, there is little Change in bicarbonate excretion. However, bicar- bonate excretion increases at higher doses presumably due to inhibition of renal carbonic anhydrase (Maren, 1967). In the present study bicarbonate excretion only differed from control animals when a high dose of hydrochlorothiazide was administered, implying that the lowest dosage of 107 hydrochlorothiazide used was not sufficient to elicit a response. Furthermore, the data imply that zinc deficiency had no profound effect on bicarbonate excretion since responses did not vary between dietary groups. Many investigators have shown that GFR.either remains unchanged or decreases with the naturetic effect of hydrochlorothiazide (Beyer and Baer, 1961; Earley and Orloff, 1962; Peters, 1965). In the present study, GFR did not change in zinc-deficient or zinc—supplemented animals after hydrochlorothiazide was given. However, a significant increase was noted in pair-fed controls given either a low or high drug dose. The physiological signifi- cance of this increase is doubtful. Many factors can exert an influence on the measurement of glomerular fil- tration rate: water intake and blood pressure are two examples. Water intake of pair-fed controls was not visually distinguishable from that of zinc-deficient animals, hence an increase in blood pressure may have been a contributing factor to the noted increase in GFR. The present experiments show a differential effect of acetazolamide, furosemide and hydrochlorothiazide on sodium excretion in the rat. These studies demonstrate that urinary sodium excretion in the zinc-deficient rat after administration of acetazolamide and furosemide was less than that of zinc—supplemented animals whereas sodium excretion was significantly enhanced in zinc-deficient 108 rats treated with hydrochlorothiazide. By expressing the data as qu of sodium excreted per minute, gross differences in body weight such as those seen between zinc-deficient and zinc-supplemented animals, may be a factor responsible for differences in sodium excretion. However, sodium excretion in untreated zinc- deficient animals was significantly greater than in either group fed the zinc-supplemented diet. This finding sug— gests that a defect in renal function, and not differences in body weight, may be a contributing factor to the in- crease in sodium excretion of zinc-deficient animals. To further eliminate confounding variables which may contribute to the noted differences in urinary sodium, fractional sodium excretion was calculated for control and drug-treated animals (Table 17). Fractional sodium excre- tion is a measure of the sodium which is filtered at the glomerulus and ultimately lost in the urine. Therefore, by expressing the data in this manner glomerular filtra- tion rate is taken into consideration. The clearance pro- cedure used in this experiment was modified in that all animals were volume expanded, and no equilibration period was allowed between volume expansion and drug injection. The latter suggests that the animals may not have been in a steady state, in which the inulin being infused equals the rate at which it is excreted. If this assumption is true, the Clearance of inulin may not be an accurate 109 Table 17. Fractional sodium excretion in control and drug-treated rats fed zinc-deficient or zinc-supplemented diets for six weeks Fractional Sodium Excretion (%) Drug/ Diet/Feeding pattern Dose Zinc- Zinc- Zinc- deficient supplemented supplemented Ad libitum Pair-fed Ad libitum Control 4.75E.543’b 1.21E.l4 .55E.02 Acetazolamide c c c 5 mg/kg 14.58El.l4 12.55E.79 6.05E.50 20 mg/kg I3.oTE.59C 5.04:.29C 8.93:1.15C Furosemide C c 2 mg/kg 3.54:.08 3.71E.81 3.05E.23 20 mg/kg 4.64:.38 5.25:.16C 8.56E T7c Hydrochlorothiazide 5 mg/kg 5.03E.49c 1.45:.08 .88E.l6 20 kg/mg 8.58E1.12c 2.05:.33 1.24:.14 a Values represent mean E S.E.M. of three animals bSignificantly different from zinc-supplemented animals CSignificantly different from control for that dietary group 110 estimate of glomerular filtration rate. The calculated fractional sodium excretion of un- treated zinc-deficient animals was significantly greater than in pair-fed controls or zinc-supplemented animals fed ad libitum (Table 17). These results are similar to those seen when absolute sodium excretion was used as a basis for assessment of renal function. Thus it appears that zinc-deficiency has had a compromising effect on the reab- sorptive capacity for sodium in the kidney. A low dose of acetazolamide increased fractional sodium excretion in all dietary groups (Table 17). The ability of the kidney to reabsorb filtered sodium in un- treated zinc-deficient animals was impaired, and when acetazolamide was given sodium excretion increased accor- dingly. Pair-fed controls and zinc-supplemented animals responded similarly to drug administration by an increase in fractional sodium excretion, however the magnitude of reSponse was not as great as that seen in zinc-deficient animals. When a high dose of acetazolamide was given, fractional sodium excretion in the zinc-deficient animal did not change significantly from that seen after a low drug dose. These results suggest that a maximal naturetic effect was observed at the low drug dose and administra- tion of a higher dose produced little change in response. In contrast, zinc-supplemented animals responded to the high drug dose with a greater percent of the filtered 111 sodium excreted, demonstrating that the drug does produce a dose-response effect in the normal animal. Fractional sodium excretion in pair-fed controls given a high dose of acetazolamide was significantly lower than that seen after a low drug dose. It was observed however, that the GFR in this group significantly increased at a high dose of drug. The reasons for this increase are unclear. A similar increase in GFR was not seen in zinc-deficient or zinc—supplemented animals at this dose suggesting the dif- ference may be due to procedural error. An increase in fractional sodium excretion was noted in pair-fed controls and zinc-supplemented animals treated with furosemide (Table 17). In addition, as the dose of furosemide was increased, fractional sodium excretion in- creased, demonstrating that the drug had a dose-dependent effect. In contrast, fractional sodium excretion in zinc- deficient animals given a high dose of furosemide did not differ from that of untreated animals and was slightly depressed after a low drug dose. These results imply that the zinc-deficient animal was unable to respond to the diuretic. A significant increase in GFR was noted in zinc-deficient animals following treatment with furose- mide. This suggests that the depression in fractional sodium excretion was the result of an increase in sodium reabsorption in the renal tubule. These results are in contrast to those reported in untreated zinc-deficient 112 animals, in which a significantly greater part of the filtered load of sodium was excreted. Since fractional sodium excretion was enhanced by treatment with acetazo- lamide, similar results were expected with furosemide. The discrepancies noted in this study may be due to pro- cedural techniques employed to estimate GFR. Fractional sodium excretion during hydrochlorothia- zide diuresis was minimal in all zinc-supplemented animals (Table 17). Since hydrochlorothiazide inhibits sodium chloride reabsorption in the distal tubule, its naturetic effect is much less in comparison to acetazolamide and furosemide. Only 5-10% of the filtered sodium remains in the filtrate when it reaches the distal tubule; hence, fractional sodium excretion is expected to be less than that seen with the other drugs used in this study. In this respect, zinc-supplemented animals in both dietary groups may have responded normally, since only a slight increase in sodium excretion was noted following drug administration. In contrast to results seen with furose- mide, administration of hydrochlorothiazide to zinc-defi— cient animals produced a significant increase in fractional sodium excretion. Furthermore, the drug had a dose-depen- dent effect. As dosage was increased, fractional sodium excretion increased concomitantly. Since sodium excretion was significantly greater in untreated zinc-deficient animals, it appears that naturesis was enhanced by drug 113 administration. The results of this study clearly demonstrate that dietary zinc-deficiency may produce profound physiological and morphological changes in the rat as others have also shown. Subsequently, these changes may have a deleterious effect on renal function. When studying the effects of zinc deficiency on growth and development, careful atten- tion must be given to the effect of secondary inanition. Since anorexia is an early symptom of ensuing zinc defi- ciency, pair-feeding experiments are of value in distin- guishing between the primary effects of zinc deficiency and secondary effects due to reduced food intake. Thus, great care must be taken in interpreting results which show differences between zinc-deficient animals, pair-fed controls and animals fed a zinc-supplemented diet ad libi- tum. Although the results may show a statistically sig- nificant difference between dietary groups, the biological significance of this difference must be considered to rule out any existing doubt that the observed effects are the result of caloric restriction and not zinc deficiency per se. Alterations in carbonic anhydrase activity of blood and kidney in the zinc-deficient animal suggest that these Changes may be compensatory mechanisms to maintain a homeostatic state during severe physiological stress. Diuretic therapy designed to assess renal function 114 demonstrated that the zinc-deficient animal was unable to respond normally. These changes suggest that compensatory mechanisms may be inadequate, making diuretic therapy in a zinc-deficient state particularly hazardous. 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