v —‘——4 < - - _ r—— -‘L- 4 A COMPARISON OF RENAL TISSUE SODIUM AND CALCIUM OONCINTRATION CHANGES INDUCID BY PARATHYROID EXTRACT, GALCIUM EXCESS OR CALCIUM DIPRWA'flCfiN IN THE RABBIT Thesis for flu boat» of Ph. D. MICNIOAN STAYS UNNERSITY Charles Henry Walls II 1963 ——— THESIS This is to certify that the thesis entitled A COMPARISON OF RENAL TISSUE SODIUM AND CALCIUM CONCENTRATION CHANGES INDUCED BY PARATHYROID EXTRACT, CALCIUM EXCESS OR CALCIUM DEPRIVATION IN THE RABBIT presented by Charles Henry Wells II has been accepted towards fulfillment of the requirements for Ph.D. degree in Phxsiology and Pharmacology ‘ Major professor / Date February 27, I963 0.169 I LIBRARY Michigan State University ABSTRACT A COMPARISON OF RENAL TISSUE SODIUM AND CALCIUM CONCENTRATION CHANGES INDUCED BY PARATHYROID EXTRACT, CALCIUM EXCESS OR CALCIUM DEPRIVATION IN THE RABBIT By Charles Henry Wells II Similar renal lesions have been developed in experimental ani- mals by administering parathyroid extract (PTE) (Cantarow gt‘gl.. 1938), calcium salts (Mulligan, 1946) or by maintaining the animals on a calcium free diet (Buckner and Nellor, 1960, Dienhart, 1960). The few studies of renal functional changes which accompany the de- velopment of these lesions reveal further similarities. Glomerular filtration rate depressions have been reported in animals treated with PTE (Epstein gt al., 1959) or calcium gluconate (Chen and Neuman, 1955) while reductions in concentrating ability occur in PTE-treated (Epstein.gtl§l., 1959) and calcium gluconate treated animals (Freedman gt gl., 1958). Although no common precipitating factor is apparent, the above observations suggest that these factors converge upon a common path~ ological process. Further evidence for this hypothesis may be de- rived from the study of vitamin D intoxication, which likewise al- ters calcium metabolism, and is associated with renal tubular cell calcification and concentrating ability changes like those result- ing from PTE, calcium excess or calcium deprivation. The reduction in concentrating ability accompanying the vitamin D lesion has been shown to depend upon renal interstitial ion concentration reduc- tions. In order to obtain more evidence for use in assessing this theory, lesions were induced in immature rabbits by means of re- stricted calcium intake, parenteral administration of calcium salts, or by daily injections of parathyroid extract. Renal samples from these and from control animals were histologically examined to verify the presence of the characteristic lesions, and samples of renal cortex, medulla and papilla were analyzed for sodium and cal- cium concentrations. Daily urine volume and renal calcium excre- tion measurements were made for each animal before and during in- duction of the lesion. Microscopic examination of the renal samples revealed no tenable basis for differentiating the lesions of one treatment from those resulting from either of the others. The analyses of sodium and calcium concentrations in samples of renal cortex, medulla and papilla from animals subjected to the various treatments revealed similar cortical to papillary concentra- tion gradients for both ions. These gradients existed in all ani- mals studied; however the magnitude of the gradient was signifi- cantly (P=.05) less in animals subjected to PTE, calcium salt ex. case or calcium deprivation than in control animals. Cortical so- dium and calcium concentrations were significantly (P=.05) above control values in one or more groups subjected to each of the above treatments. A high degree of correlation was found to exist be- tween tissue sodium and calcium concentrations in the various renal samples. Urine volume changes induced by the various treatments were minimal, with a trend toward volume reduction. Urinary cal- cium excretion and urine calcium concentration were significantly decreased (P=.O5) in one or more groups subjected to each of the above treatments. These findings, by demonstrating further similarities between the disease states induced by PTE, calcium salts or a calcium free diet, support the view that these seemingly diverse treatments con- verge upon a common disease process. Indirect qualitative measures of interstitial fluid calcium concentration changes induced by these various treatments consis- tantly show marked reductions in papillary calcium concentrations, despite the formation of calcific lesions in this region. One may thus reason that the observed calcification must be dependent upon increased urine calcium concentration if it is to be explained by the popular metastatic theory, i.e., the precipitation of calcium in otherwise normal tissue because of massive increases in the cal- cium concentration of the fluids bathing the tissues. Although lesions were seen in the collecting duct epithelium of the papillae, the effluent urine either remained unchanged or was reduced. These observations imply that the tubular epithelial calcification asso- ciated with all of these various precipitating causes is not depen- dent upon a calcium induced metastatic process. A COMPARISON OF RENAL TISSUE SODIUM AND CALCIUM CONCENTRATION CHANGES INDUCED BY PARATHIROID EXTRACT, CALCIUM EXCESS OR CALCIUM DEPRIVATION IN THE RABBIT By Charles Henry Wells II A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology 1963‘ GEQWLS K). h._ I ’1 ’ ." e- 1 I! f ii ACKNOWLEDGMENTS The author wishes to express his indebtedness to Drs. J. E. Nellor, P. O. Fromm and E. R. Miller whose contributions of time and materials made this research possible and to Dr. W. D. Collings not only for his contributions in the preparation of this text but also for his counsel throughout the graduate program. INTRODUCTION . . . . . REVIEW OF LITERATURE . . ~ METHODS AND MATERIALS RESULTS . . . . . . . DISCUSSION . . . . . . summr AND CONCLUSIONS WCES o' o o o o o o 9 iii TABLE OF CONTENTS Page 15 23 67 7o iv LIST OF TABLES Calcium Concentrations in Renal Tissue . . . . Sodium Concentrations in Renal Tissue . . . . Alterations of Serum Calcium Concentrations . Urine Volume Changes . . . . . . . Urinary Calcium Excretion . . . . O O O O O O O O O O O 0 Correlations between Tissue, Plasma and Urine Calcium Concentrations . . . . Urine Calcium Concentration Changes Composite Presentation of Results Page 30 32 37 53 LIST OF FIGURES Page Experimentally Induced Calcium Concentration Changes 31 Experimentally Induced Sodium Concentration Changes . 33 Relation of Renal Tissue Sodium and Calcium Concentration . . . . . . . . . . . . . . . . . . 35 Plasma Calcium Adsorption on Glass . . . . . . . . . 38 INTRODUCTION Experimental animals develop similar renal lesions if given excesses of vitamin D (Goormaghtigh and Handousky, 1938), calcium salts (Mulligan, 1946) or parathyroid extract (PTE) (Cantarow 33 31., 1938) or if maintained on a calcium restricted diet (Buckner and Nellor, 1960, Dienhart, 1960). The advanced form of the le- sion is characterized by scattered calcification and necrosis of tubules, mostly in the renal cortex or corticomedullary zones. Less advanced lesions are characterized by the presence of calcified cytoplasmic granules and of a thickened, calcified tubular cell basement membrane. Complete functional studies of these various cases have not been published. However, a reduction in urinary concentrating ability is established for animals treated with PTE, vitamin D or calcium salts (Epstein gt gl., 1958, Carone gt_gl., 1960, Freedman, gt_§l., 1958) while reductions in renal plasma flow and in the glo- merular filtration rate have been reported for PTE induced changes (Epstein gt $1., 1959, Carone gt 51., 1960). Humans with parathy- roid adenomas or vitamin D intoxication also have decreased renal concentrating ability (Gill and Bartter, 1961, Dorhout, 1957). Renal plasma flow and glomerular filtration rate reductions have been reported in persons with parathyroid adenomas (Cohen gt gl., 1957, Edvall, 1958). The microscopic lesions in man were also much the same as those observed in experimental animals (Thorn gg‘gl., 1954, Boyd, 1953). Although it is apparent that calcium metabolism alterations l are common to all of these situations, a more specific common fac- tor is yet to be found. Examples of elevations and depressions of either serum calcium or parathyroid hormone levels can be found associated with.one or more of the precipitating factors. Never- theless.the remarkable similarity of the renal lesions and the changes in renal function which.occur with.any of these.apparently unrelated causes suggests the probability of similar, if not iden. tical, terminal steps in the pathological process. Further verification of the similarity of these processes and a.more complete comprehension of the mechanisms responsible for these changes is of importance not only to the understanding of the disease state but to its prevention and treatment as well. aw _F_ LITERATURE A number of conditions which alter calcium metabolism give rise to similar if not identical renal lesions and renal functional changes. These renal lesions are characterized by tubular basement membrane thickening and calcification, calcification and necrosis of tubular cells and the presence of interstitial calcification and occasional calcified tubular casts. Grossly, the lesions appear in all portions of the kidney, but are most heavily concentrated in the corticomedullary zone. They are remarkably scattered, with normal tubules lying beside others that are completely destroyed (Chown ‘gt‘gl.. 1939. Hueper. 1927, Hanes, 1939, Grimes, 1957). Lesions fitting the above description are commonly found in patients suffering from parathyroid adenomas (Thorn,g§‘gl., 1954) or hypervitaminosis D (Boyd, 1953) and have been.reported in pa. tients with peptic ulcers who have been on long-term high alkali, high calcium diets (Becker gtflgl., 1952). Other examples of lesions of the same apparent nature have been produced in experimental animals by administration of parathyb roid extract (PTE) (Cantarow 223;” 1938, Epstein §_t_ 3;” 1959. Hueper, 1927), excess vitamin D (Epstein g§,gl,. 1958, Goormaghtigh and Handousky, 1938, wallach and Carter, 1959), calcium salts (Mulligan, l9h6, Schneider gtflgl.. 1960) and by maintenance of the animals on low calcium diets (Buckner and Nellor, 1960, Dienhart, 1960). Most of the studies in which these lesions have been experi- 3 mentally produced have been acute, and it has been pointed out that differences between the acute experimental lesion and the chronic human variety do exist (Anderson, 1939). Chronic experimental le- sions, however, are histologically similar to chronic lesions in man. It is probable that the two merely represent different phases of development rather than actual differenced in the disease pro- cess (Epstein. 1960). The striking similarity of the lesions and associated renal functional changes, and the fact that all of the precipitating fac- tors involve calcium metabolism alterations, would lead one to sue- pect a common etiology. Despite their apparent involvement. neither the elevation of serum parathormcne nor the elevation of serum calcium levels could be a possible common etiological factor. Serum.parathyroid hormone levels are elevated in cases of PTE administration, and of a cal- cium restricted diet; renal lesions are seen in both situations (Nicolaysen‘gg‘gl., 1953, Buckner.andeellor, 1960). However there is no reason to suspect any parathyroid hormone elevation in the case of vitamin D or calcium.gluconate administration. On the con- trary, one might expect a depression of serum.parathyroid.hormone levels in these cases (Crawford gt,gl,, 1957. Patt and Luckhardt, 1942). Serum calcium.levels are elevated after vitamin D (Goodman and Gillman, 1955), calcium gluconate~(McJunkin‘gt‘gl.. 1932), or parathormone administration (Albright'gtugl.. 1929, Canary and Kyle. 1959. Todd 93 51,” 1962), but low to normal with the calcium free diet (Baumann and Sprinson, 1939). The effects of vitamin D and parathyroid hormone on calcium metabolism have been extensively studied, and there is agreement on the general effects of each. An inverse causal relationship exists between serum calcium levels and the rate of parathyroid hormone release (Patt and Luckhardt, 1942). Parathyroid hormone elevates serum calcium by its action on bone and kidney. It enhances osteo- clastic activity. and depolymerization of bone matrix, with a con- current dissolution of bone salts and thus a release of calcium to the blood stream (Engle, 1952, Johnston gt,gl.. 1961). Phosphaturia and calciuria are the usual result of parathyroid hormone excess. Phosphaturia does not result from an active tubu- lar secretion of phosphate but from a parathyroid hormone induced reduction in phosphate reabsorption by the tubules (Pitts gtfl§l.. 1958, Samiy gt‘gl.. 1960). Decreased serum phosphate increases serum calcium by encouraging bone salt dissolution. The complex physico-chemical properties of plasma preclude the possibility of a simple solubility product constant relationship between calcium and phosphate. Nevertheless an inverse relationship between serum calcium and phosphate exists. The calciuria which often accompanies parathyroid hormone ex. cess is not due to a reduction in the calcium reabsorptive ability of the renal tubular epithelium. On the contrary, recent studies indicate that parathyroid hormone enhances tubular calcium reabsorp- tion (Widrow and Levinsky, 1962, Kleeman gt 51.. 1958). The absence of a demonstrable tubular maximum for calcium (Chen and Neuman, 1955, Poulos, 1957, Epstein, 1960) places the dependence of the cal- cium.reabsorption rate upon the rate of calcium filtration. This has been experimentally verified by Canary and Kyle (1959). Para- thyroid extract administration results in calciuria because of an increased filtered calcium load. In this case the urinary calcium loss increases although the calcium reabscrpticn rate also increases (Canary and Kyle, 1959). The tubular site of maximum calcium reab- sorption has been shown to lie between the terminal portion of the proximal tubule and the proximal segment of the distal convoluted tubule (Widrow and Levinsky, 1962. Grollman 23.51,, 1962, Lassiter gtflgl., 1962, Nesson and Lauler, 1959). Parathyroid hormone is thought to have little effect on gastrointestinal calcium.absorp- tion. This calcium absorbing process is vitamin D dependent and accounts for serum calcium elevation.noted with vitamin D adminis- tration (Kimberg 23 g" 1961, Epstein gt 31.. 1958). unfortunately the physiological functions of vitamin D and parathyroid hormone give few clues to possible mechanism involved in development of lesions resulting from the administration of exp cessive amounts of either. waever a number of alternative hypo- ' theses have been proposed to explain the precipitation of calcium to fern these lesions. Many authors contend that the calcification process resulting from one or more of the treatments discussed above is metastatic, i.e., the calcium is precipitated in otherwise normal tissue. Others favor the concept that calcium is precipitated in.damaged tissue. This process is referred to as dystrophic calci- fication. The metastatic theory has been presented to explain the mechanism of calcification of lesions induced by PTE (Shelling gt 31., 1938, Schneider gt 31., 1960), vitamin D (Ham, 1932, Goormagh- tigh and Handousky, 1938, Mulligan, 1946), and calcium gluconate (Mulligan, 1946, Schneider 23.31., 1960). Gill and Bartter (1961) studied functional changes induced by excessive calcium intake, vi- tamin D excess and parathyroid hormone excess. They concluded that V calcium excess was the factor primarily responsible for renal func- tion changes which occur in these cases. McJunkin gt 31. (1932), however, after a study of calcium glu- conate and PTE-induced lesions, were convinced that cell destruction precedes calcification. "In no animal have we seen calcification unassociated with a destructive lesion.... They are toxic lesions which are usually associated with hypercalcemia. The obvious Tn— ferencc is that the calcium salts produce the lesions." The mecha- nism by which salts inflict damage is still obscure. They noted that injection of calcium salts into kidney tissue failed to pro- duce characteristic lesions and they suggested the possibility of elevated blood calcium resulting in elevated tissue lymph calcium. This was hypothesized to be the damaging agent. Further support of the theory that these lesions were of a dy- strophic nature came from numerous studies of lesion development by various agents. Becker gt‘gl. (1952) reported that lesions which developed in renal tissue of their patients who had been on long-term high alkali, high calcium diets were dystrophic. Cantarow 33.31. (1938) stated that calcification of PTE excess is dystrophic and not dependent upon serum calcium levels per-g9. Furthermore Epstein.gt_§l. (1959) cited cases where renal tubular cell destruc- tion was seen with calcification following vitamin D treatment. Other investigators, realizing that the dystrophic and metastap tic theories were concerned with only one aspect of tissue calcifi- cation, sought to clarify other aspects of the calcification pro- cess. Heuper (1927) noted the predominance of lesions in the kid- ney, stomach and lung and hypothesized that this was due to pHLal- terations. All of these organs release acid and.are consequently left somewhat.basic.t This reduces calcium solubility. This could be a-contributing factor whether the calcification process is dye strophic or metastatic. Another interesting facet was suggested by Engle.(l952). Treatment of an animal with PTE results in dis- solution of bone matrix as well as calcium.sa1ts, and thus results in an increase in serum mucopolysaccharide concentration. It has been observed that PTE depolymerizes renal tubular basement mem— brane substance, as well as bone matrix (Baker and Francisco, 1959, Grimes, 1957). Intratubular casts associated with PTE treatment have been shown.to be composed largely of mucopolysaccharide and are often calcified (Baker and Francisco, 1959). No one has 61b plained how this mucopolysaccharide, which,presumably lost its cal- ciumpbinding power in bone and thus allowed dissolution of bone salts, regains this property'when concentrated in renal tubules. Grimes (1957) suggested that mucopolysaccharide is absorbed into tubular cells. where 11-. is further depolymerized to calcium-binding forms by intracellular enzymes. Calcification of casts occurs be- cause of necrosis of tubular epithelium, with rupture and release of these enzymes to attack intratubular mucopolysaccharides. Grimes studied PTE and calcium gluconate-treated animals and found the tu- bular epithelial cytoplasmic calcification to be calcification of draplets which can be stained by the Periodic acid Schiff technique (P.A.S.). The P.A.S. positive staining indicates that the substance is a depolymerized polysaccharide (Hotchkiss, 1948), and in this case presumably the residue of bone matrix breakdown. The intratu- bular casts associated with calcium gluconate or PTE excess are also P.A.S. positive (Engle, 1952, Grimes, 1957). Interestingly, tubular basement membranes also show path areas of P.A.S. positive thickening following either treatment. It is felt that this is the basis for calcification of basement membranes seen in the above two cases. Baker and Francisco (1959), in reference to PTE treated ani- mals, stated “...it would be reasonable to conclude that the para,- thcrmone first produced the observed precalcific renal ground sub- stance alterations and thereby specifically prepared these geogra- phical tubule sites for the calcification that subsequently developed." The study of depolymerization of mucopolysaccharides by PTE produced evidence that both bone matrix (Johnston 31; al_.. 1961) and other hexeeamhe structures are depolymerized (Hausmann, 1960). The concept that depolymerized products may be associated with the renal calcification process is further reinforced by the observation of. 86ch 31; 51; (1954) that thetotal quantity of biocolloids in the urine of patients with calculous disease is 3 to 13 times normal. Although depolymerization of renal tubular ground substance has 10 been demonstrated following administration.of calcium gluconate or parathyroid extract, there is reason to suspect that differences axe ist in these depolymerization processes. Animals which had incorpo- rated radioactive sulfate into body tissues were treated with.para- thyroid extract and with calcium gluconate. The former group-re- leased.more sulfate than the latter. This was interpreted by the investigator as greater depolymerization of chondroitin sulfate by PTE than by calcium gluconate (Reaven e_t_ 51;, 1960). The relation, if any, of these findings to development of renal lesions following PTE or calcium gluconate administration is yet to be determined. .Another possibility in the development of these lesions was suggested by Schneider 9:: a_l_. (1960). They asserted that PTE had a direct effect on renal tubular epithelium, in addition to its at? fect.on tubular basement membranes and serum.calcium levels, which was responsible for development of the lesions. Furthermore, they challenged the single etiology hypothesis, and support their contenn tion with observations made on PTE and calcium gluconate-treated ant imals. They felt that the calcium gluconate lesion was almost ex.- elusively limited to the tubule basement membrance, with cell necro- sis being a secondary effect. In the PTE-induced lesions tubular epithelial necrosis wasreported to accompany, but not precede, cal- cification of cells while the basement membrane thickening was thought to be secondary to epithelial cell necrosis. They further stated that there is more cortical inyolvement with calcium gluco- nate-induced lesions than in.those ensuing from PTE treatment. Bows ever, the corticomedullary zone was the predominate lesion site for 11 either PTE or calcium gluconate induced lesions. The observations of Schneider‘gtugl. have not gone uncontested. Baker and Francisco (1959) noted basement membrane changes following administration of PTE, even in the absence of calcification of the membrane or tubular cells. Hanes (1939) and Baker 93 9;. (1954) reported that the process involved changes in the basement membrane first, followed by tubular cell necrosis for PTE induced lesions. Further similarity of the renal changes induced by excesses of PTE, calcium gluconate, vitamin D or by a calcium restricted diet is illustrated by the renal functional changes which accompany the development of these lesions. It is well established that a.de- pression of the glomerular filtration rate occurs concurrently with development of discernable pathological changes from excessive vi- tamin D (Whllach and Carter, 1959), from PTE (Carone 33.51,, 1960, Epstein gt‘gl., 1959) or from calcium gluconate (Chen and Neuman, 1955, Poulos, 1957, Whllach and Carter, 1959). Similar observations have been made with persons suffering from.parathyroid adenomas (Cohen gt 9;" 1957, Edvall, 1958). The tubular maximum (Tm) for PAH has been reported to be de- pressed in persons with parathyroid adenomas (Cohen 2’; g," 1957), and a similar depression of tubular function was observed by Dorhout (1957) in patients with hypercalcemia. Edvall (1958) felt that this depression of theTn PAH was a reflection of organic destruction. Bis studies of patients with parathyroid adenomas before and after surgical removal of the tumors, which failed to show any reversal of Tm PAH values toward normal, support this view. . 12 The concentrating ability of the kidney decreased following administration of excessive PTE (Carone g§_al., 1960, Epstein gt’gl., 1959), vitamin D (Epstein 23.31., 1958, Goormaghtigh and Handousky, 1938, Gill and Bartter, 1961, Mulligan, 1946), or calcium gluconate (Freedman{gt‘§;., 1958). Persons with parathyroid adenomas (Cohen pgt‘gl., 1957, Edvall, 1958, Gill and Bartter, 1961), or hypercalce- mia due to destructive bone lesions (Edvall, 1958) are reported to have similar functional changes. The best established current theory explaining the concentra» ting ability of kidney employs an osmotic gradient as the driving force for water reabsorption. The contributions of numerous inves- tigators to the formulation and verification of this theory have been summarized and discussed in a review article by Hemer Smith (1959). The following are essential features of the theory. Osmolar concentration of interstitial fluid increases from the cortex to the papilla. This high papillary osmolarity is developed and maintained by active tubular reabsorption of salts from the tubular urine in conjunction with a high degree of water impermeability exhibited by the ascending limb of the loop of Henle. The osmotic concentration of tubular urine approximates that of the surrounding interstitial fluid until urine reaches the ascending part of the loop of Henle. This segment, while essentially imperme- able to water, nevertheless remains active in salt reabsorption. U- rine reaches the distal convoluted tubule in a hypotonic state. The ultimate concentration of urine is thought to occur in the collecting 13 ducts. This process depends upon the maintenance of a high osmo- tic concentration in the interstitial space, especially at the tip of the papilla, and upon the permeability of the collecting duct. This latter factor is controlled by the antidiuretic hormone (ADH). Cohen gtflgl. (1957) observed that exogenous ADH administration in patients with parathyroid adenomas failed to alter the urine con- centrating ability of the kidney. This would suggest that the functional change may be an inability to concentrate ions in the renal interstitial fluids and thus develop the concentration gra- dient necessary for the reabsorption of water. Manitus gt 3;. (1960) studied sodium and urea concentrations of the papilla and medulla of rats hypercalcemic from vitamin D in- toxication. They concluded that the rats' inability to concentrate urine in this case was due, at least in part, to inability to de- velop and maintain a high concentration of sodium in the intersti- tial fluid of the medulla and papilla. Evidence that urine osmolarity often approximates that of the interstitial fluid of the papilla was derived from studies by U11-' rich and Jarausch (1956) in which the osmotic concentrations of both papillary fluid and urine were measured under different situa- tions. From their studies they concluded: "Harnstoff ist am An- stieg des osmotischen Druckes im Nierengewebe Von Dursttieren.Men- genmassig am starksten beteiligt, dann folgt Natriumchlorid. Die Konzentrationen der untersuchten Stoffe im Gewebe der Papillenspitze zusammengerechnet, ergeben einen osmotischen Druck3 der nur wenig von dem des ausgeschiedenen Urins abweicht....” 14 Early observations of the effect of calcium salts on renal con- centrating ability were made by Porges and Pribram (1908). They in- jected sodium salts and sodium-calcium salt mixtures into the blood streams of dogs and noted that calcium in the salt mixture caused a greater diuresis than injection of similar quantities of pure sodium salts. This might suggest that the circulating calcium con- centration is a factor which exercises some control over the urine concentrating mechanism. It is unlikely that the change in the urine concentrating ability is dependent upon the development of destruc— tive renal lesions in view of the report by Dorhout (1957) that the concentrating ability of the kidneys in persons with parathyroid adenomas improves rapidly following removal of the adenoma, while Tm PAH changes do not. A careful consideration of the evidence offered in explanation of the renal changes delineated in the above experiments dictates caution in the adoption of a particular theory. METHODS AND MATERIALS The previous discussion of literature reveals that similar re- nal lesions can be develOped by conditions which lead to either an elevation of serum calcium or of serum patathormone levels. If the similarity of the pathological manifestations from these dissimilar causes is to be explained by their convergence upon a common final process, the changes in renal function accompanying the development of the lesion should be alike for all precipitating causes. As a test of this hypothesis, renal tissue sodium concentrations were measured in animals subjected to hypercalcemia, PTE excess or both. The importance of decreased renal tissue sodium concentrations in the reduction of renal concentrating ability is established for vitamin D intoxicated animals (Manitus gt 51., 1960). Furthermore, a decrease in renal concentrating ability has been reported in hyperparathyroid- ism (Edvall, 1958), although its possible dependence upon reductions in renal tissue sodium concentration has not been established. Elevated serum.parathcrmone levels, in the absence of hyper- calcemia should result from prolonged maintenance on a calcium free diet. Elevated serum calcium levels, associated with a depression of sermm parathormone concentrations, should result from injection of calcium salts, while elevation of both serum parathormone and calcium levels should follow PTE injections. Two or more groups of animals were subjected to each of these treatments and these groups were sacrificed after differing spans of treatment so that more information could be accumulated about the lesions in their early stages of development. 15 16 PTE was used to induce lesions in two groups, each of which consisted of six immature rabbits. The members of the first group, numbers 43 through 48, were each given 50 units of Lilly Parathyroid Extract1 per kilogram body weight per day. These animals were sacrificed twenty-four hours after the third injection with a sharp blow to the base of the skull. A 10 ml. blood sample was immediately taken by heart puncture and centrifuged. After centrifugation the plasma was decanted and frozen for future analysis. All syringes and cone trifuge tubes used in the above prcdedure were rinsed with.ammonium heparin prior to use to avoid blood clotting. The kidneys were removed and samples of cortex, medulla and papilla were collected in tared vessels, weighed and frozen. Other renal tissue samples were preserved in 10% buffered neutral formu- lin for future histological study. Members of the second PTE-treated group, numbers 49 through 54, received 50 units of Lilly Parathyroid Extract U.S.P. per kilogram per day for five days, followed by 100 units per kilogram.per day for two days and 200 units per kilogram on the final two days of treatment. These animals were sacrificed on the day following the final treatment, and samples of kidney and plasma preserved in the same manner as described for animals of the first group. Lesions were produced in three groups of six immature rabbits each by the intraperitcneal administration of calcium salts. These salts were administered in the form of Cal-Dextro Solution.2 The l. Paratlvroid Extract U.S.P., Eli L,lly Co., Indianapolis, Indiana. 2. Cal-dextro, a product of the Fort Dodge Laboratories, Ft. Dodge, Iowa, is a commercial preparation of a d-Saccharate and Gluconate salts in sterile aqueous solution containing 1.68 gm per cent calcium. 1? dosage for animals 25 through 30 was 1 ml per kilogram per day, for animals 37, 38, 39, 40, 59 and 60 was 2.5 ml per kilogram per day and for animals 41, 42, 55 through 58, and 67 through 72 was 5 ml per kilogram per day. All animals in the calciumptreated groups were injected with ‘the salt solution on three consecutive days with the exception of animals 67 through 72 which received six consecutive daily injec- tions. These animals were sacrificed twenty-four hours after the final calcium salt injection and samples of blood and tissue were collected in the manner described for sample collection from the .Parathyroid Extract treated animals. All animals discussed so far were allowed access to'wayne Rabbit Ration and tap water at all times. The final two groups, however, were maintained on a restricted calcium intake. They had constant access to a calcium-free diet and distilled water through- out the experimental period. This diet was prepared by Dr. E. R. Miller of the Michigan State University Department of Animal Hus- bandry to have the following composition: Protein (casein) 20% Fat (lard) Carbohydrate (cerelose) 62% Fiber (cellulose) 10% Mineral 3f 7 P 0.6% Zn 55 ppm x 0.3% Mn #0 ppm Na 0.3% Cu 15 ppm Cl o.u5$ Co 15 PPm Mg 300 ppm‘ I 0-5 PPm Fe 100 ppm *parts per million 18 Vitamins (concentration per kilogram feed) A 2000 1.0. Niacin 20 mg D 500 1.0. Inositol 200 mg E 8 mg Para-aminobenzoic Acid 20 mg K 2 mg Folic Acid 400 ug Thiamine 5 mg Biotin 80 ug Riboflavin _ 5 mg B12 80 ug Pantothenic Acid 20 mg Choline 2 gm Pyridcxine 5 mg Ascorbic Acid 128 mg Animals numbered 61 through 66 were maintained exclusively on this diet and distilled water for twenty-four days, while animals 73 through 78 were similarly maintained for fifteen days. 'At the end of these periods the animals were sacrificed and samples of plasma and tissue collected in the manner previously described. Although it has been shown that renal lesions from PTE or cal- cium gluconate excess, or resulting from a restricted calcium intake are not associated with similar changes in serum calcium concentra- tions, this should not be interpreted as an indication that consis- tant changes in the renal tissue calcium levels do not occur. In or- der to study this possibility, the weighed samples of cortex, medulla and papilla, which were collected and frozen at the time of sacrifice, were homogenized separately with a motor driven Potter-Elvehjem tis- sue grinder and diluted to concentrations which fell within the mea- surable range of a Coleman Model 24 flame photometer. A11 dilutions were made with .02$ Sterox in triple distilled water, and the cal- cium analyses made with the flame photometer calibrated with a stan- dard solution containing 0.2 milliequivalents of calcium and 6.0 mil- liequivalents of sodium per liter. Mathematical adjustments were made for sample size and dilution so that the calcium concentrations could be reported in milliequivalents per kilogram tissue. 19 A similar analysis of renal tissue sodium.was undertaken so that correlations between tissue sodium and calcium concentrations could be established. Aliquots of the tissue homogenates prepared for the calcium de- terminations were diluted to concentrations of approximately one gram tissue per 200 cc solution and quantitatively analyzed by flame photometry. A11 homogenate dilutions were made with .02fi Steroxl in triple distilled water, and the direct reading scale for sodium de- terminations calibrated with a standard solution containing five mil- liequivalents sodium and 0.2 gm Sterox per liter of triple distilled - water. ZMathematical corrections for dilution and tissue sample size were made as with.the calcium determinations so that the results could be expressed in.milligrams calcium per kilogram tissue sample. It was felt that a knowledge of plasma and urinary calcium con- centrations at the time of sacrifice would also be valuable. The plasma samples collected and frozen at the time of sacrifice were later thawed and the calcium concentrations were determined with a Coleman.Model 24 flame photometer. The excretion of urine containing much of its salt in precipi- tated form, as is so common in rabbits, renders analysis of dissolved urine calcium.meaningless. Furthermore, one cannot be sure that the precipitate-tc-fluid ratio in bladder urine, taken by needle puncture, is representative of collecting duct precipitate to fluid ratios. These difficulties were overcome by maintaining the animals in - metabolism cages, measuring the urine volume for each twentyafour hour period, acidifying the urine until no precipitates remained and l. Hartman-Leddon Co., Philadelphia, Penn. 20 quantitatively assaying the urine-acid mixture for calcium. A the- oretical urine calcium concentration, assuming no calcium precipi- tation, can be calculated thus: 600 Z M” Vex-M0 Va where Coy equals the mean theoretical calcium concentration in the twenty-four hour urine sample, 6004‘, is the calcium concentration in the twenty-four hour urine-301 mixture, Vu-HCI represents the volume of the urine-m1 mixture and [/0 represents the twenty-four hour urine volume. . The calcium excretion per twenty-four hours (C034) can also be derived from these data: 6024 = coy-Ha X Va -HC/ These deteminations were made daily on all animals (except 25 through 30, 61 through 66, and 72 through 78) during the experimen- tal periods, and during a four-day control period which preceded treatment. The twenty-four hour urine volume-calcium excretion analysis schedule varied slightly for animals on the special dietary regimen. Analyses were made daily for the four-day pro-treatment control period, and on every third day thereafter. ‘ I In the execution of the above determinations it was found that careful washing of all drainage surfaces of the metabolism cages with El and collection of this fluid with the urine-331 sample was necessary to assure the inclusion of all precipitated urinary salts in the daily sample. 21 As a final aspect of the study, histological preparations of renal tissue obtained from the various experimental animals were made and studied. These samples were fixed for twenty-four to forty-sigh: hours in 10% neutral formalin, dehydrated in alcohols, , cleaned in xylene and imbedded in paraffin. Eight micron sections were obtained, stained for calcium by the Von Kcssa method and counterstained with Kernechtrot. The fixing, dehydrating and staining procedure outlined on page 152 of the Armed Forces Insti- tute of Pathology publication “Manual of Histologic and Special Staining Technics", (1960) was followed without modification. Several tests for statistical significance were applied to (the data obtained in this study. Serum calcium concentration and tissue homogenate concentrations were compared with control values by the Student 7' test. T:_ 57% ‘ [Z16 - %&)7*(Z§; (Ex 24 (”I’VE/’7; +/72'.'3) In portions of the study where animals could serve as their own controls, such as measurement of the twenty-four hour urinary calcium excretion, or the twenty-four hour urine output, a variation of the Student 7' test was used,.which assesses significance of the differ- ences in the means of control and experimental values for each animal. 22 7'- d? ' 270’ a; —%zl % (”XX/71H) As employed here, JX represents the mean of the differences between the control and experimental mean values for all animals in the group, and n; represents the number of control minus experimental mean value differences included in the analysis. , The formula . ny __ (Xx)(z1L rx .— ] (th “LA? (5)" NM) was used in calculation of all correlation coefficients except for the correlation of sodium and calcium concentration changes. This calculation. employed the follwwing formula: Z(dx)(dy) — W d’d’: [(Za- 355-”)726/674- if)? As applied to this study, d2. and 0? represent, respectively, the differences between corresponding control and experimental group tissue homogenate calcium and sodium concentrations. RESULTS I. Microscopic Examination Plates 1 (PTE induced), 2 (calcium salt induced) and 3 (spe- cial diet induced) are low magnification photomicrographs showing advanced stages of cortical destruction, as produced in this study. Plates # and 5 are similar photomicrographs showing advanced tubu- lar destruction in the medulla and papilla respectively. Plates 6, 7 and 8 show Von Kossa positive inclusions in tubu- lar cell cytoplasm. Darkened, Von Kossa positive, tubular cell margins may be seen in plates 9, 10 and 11. Plate 12 is an en- larged view of three tubular cell nuclei, showing black, and thus presumably calcified, intranuclear inclusions. .23 24 Plate 1 Plate 2 Plate 3 Plate 6 25 Plate 5 I - 1 Q 1 ' V l {77' 2‘ \ ". ‘ '1 l e v % ~ 1A7_,_”_‘_ Plate 7 Plate 8 "‘v 't Plate 9 Plate 10 Plate 11 Plate 12 28 II. Sodium and Calcium Concentrations in Renal Tissue Table 1 presents the results of calcium analyses of tissue homogenates, and the comparison of the mean cortical, medullary and papillary calcium concentrations of the various experimental groups with those of the control groups. Figure l graphically presents the same data. It will be noted that the calcium con- centration of renal tissue in the control animals increased A progressively from the cortex to the papilla. All of the experi- mental groups showed similar calcium concentration gradients from cortex to papilla although in three cases (Groups n, 5 and 7) the cortical calcium concentrations exceeded those of the medulla. The papillary calcium concentrations of the experimental groups were universally below the corresponding control group values, with depressions significant at the .05 level occurring in groups 3, 4, 5, 6 and 7. All three experimental treatments, i.e., PTE, calcium excess, and calcium-free diet, are represented in these groups. The mean cortical calcium concentration of each of the ex- perimental groups was elevated above the corresponding control value, although these changes attained .05 significance only with one PTE (Group 8), one special diet (Group 5) and two calcium treated groups (Groups 1, 4). Figure 2 and table 2 present a summary of determinations of the mean sodium.concentrations of the papillary, medullary and cortical tissue homogenates. Table 2 also contains comparisons of the mean tissue sodium concentrations from controls and various 29 experimental groups. Mean tissue sodium concentrations, of experi- mental groups and their comparisons with correSponding control values, followed much the same pattern as the calcium concentra- tions in the same tissues. The mean cortical sodium concentrations of the experimental groups were elevated above control values in most cases, and ele- vations significant at the .05 level occurred in groups h (calcium treat), 6 (special diet) and 8 (PTE treated). Depressions of papillary sodium concentration means below correSponding control values were consistently seen with depres- sions significant at the .05 level occurring in all cases except groups 1 and 2. TABLE 1. Group No. of No. Animals Treatment Control 10 None 1 6 Cal-dextro 1cc/Kg/day for 3 days Cal-dextro_ 2.50c/Kg/day for 3 days Cal-dextro Soc/Kg/day for 3 days Cal-dextro Soc/Kg/day for 6 days Calcium re- stricted diet for 15 days Calcium re- stricted diet for 24 days PTE doses 50 units/day for 3 days ' PTE doses in- creasing from 50 to 200 'units/day over 7 days Mean Tissue Hbmogenate Cortex 3.34 3.85‘ 3.65 3-53 5.17* 5-73‘ 3.60 4.82 4.08* Medulla 4.22 5.18"I 5-15* 3.96 5.00* 5.05* 4.60 4.20 4.82* Calcium Conc. (Meq/Kg tissue) Papilla 9.41 8092 8.08 5-39* 7-77* 5.30* 5.20* 6.90* 8.62* *Significantly different from control values (P=.05). Cortical and papillary samples were tested by a one-tailed test and the medullary samples by a two-tailed test. 31 FIGUREl Experimentally Induced Calcium Concentration Gradient Changes 10 C . C 8 "’ ‘ CD . 6 A ‘ “e Meq Ca/Kg ‘ I ‘ ‘ ' ‘ I . I I I I 2 . I Cortex ‘Hedulla , CDPapilla Control 1 2 3 4 5 6 7 8 Group Number Groups 1-4: Calcium Treated Groups 5—6: PTE-Treated . Groups 7-8: Dietary Calcium Deprivation For specific group therapies, see table 1. 32 TABLE 2 Mean Tissue Hbmogenate Sodium Conc. (Meq/Kg tissue) Group No. of No. Animals Treatment Cortex Medulla Papilla Control 10 61.1 123.9 206.9 1 6 Cal-dextro 65.5 125.8 194.7 lcc/Kg/day for 3 days 2 4 Cal-dextro 56.6 132.5 191.8 2. 5co/Kg/day for 3 days 3 8 Cal-dextrc 62.0 108.8* 182.0’ Sec/Ks/day for 3 days 4 6 Cal-dextro 68.7"I 119.7 168.8* Sec/Ks/day for 6 days stricted diet for 15 days 6 5 Calcium re- 68.6"I 120.0 179.8‘ stricted diet for 24 days 7 ‘ 6 PTE doses 61.0 111.2"l 173.3' 50 units for 3 days 8 6 PTE doses in- 68.8* 114.5 179.2* creasing from 50 to 200 units over 7 days *Significantly different from corresponding control values at the .05 level. Cortical and papillary samples were tested by'a one-tailed test and the medullary samples by a two-tailed test. Meq Na/Kg 33 FIGURE 2 Experimentally Induced Sodium Concentration 150 100' 50 Gradient Changes CD GD ‘. . Cl 9°.'. AA‘ ‘ ‘ A A A‘ I' 'Ill'l. I'Cortex ‘Medulla .Papilla Cantrell 2 3 a 5 6 7 8 Group Number ' Groups 1-4: Calcium Treated Groups 5-6: PTE-Treated Groups 7-8: Dietary Calcium Deprivation For specific group therapies, see table 2. 34 Figure 3 is a graphic presentation of the relationship of sodium and calcium concentrations obtained from cortical, medul- lary and papillary samples of all animals studied. Relation 150 125 100 Meq Ca/Kg 75 50 25 35 FIGURE 3 of Renal Tissue Sodium and Calcium Concentration o o o o - e 9a o o o 0 0 & o o o o 0 .o o o o °Qa° °e - o o' '8 Y?’ (D ‘0 ‘0 ch9¢¥> {>Qb ‘0 o 000£;& O . «>0.on °°°’ <0 0 00 6 0° so 100 150 200 ‘7 zsr Meq Na/Kg 36 III, Experimental-Alterations of Serum Calcium Concentrations Changes in plasma calcium concentration, which were brought about by PTE, calcium salt excess or by a calcium restricted diet, are presented in table 3. The decreased plasma calcium in animals maintained on the calcium restricted diet (groups 5 and 6) and increased plasma calcium in calcium salt treated (Group 4) and PTE treated animals (Groups 7 and 8) were significantly different from the control group plasma calcium concentrations (P=.05). ‘ The calcium concentrations in plasma samples decline with time if the samples are stored in glass. Figure 4 is a composite of the concentration changes noted in three plasma samples. Cal- cium concentrations, expressed as percent of the original value, are plotted against time since the collection of the sample. Group No. Control 3 I Treatment None Cal-dextro Sec/Kg/day for 3 days Cal-dextro Sec/Ks/day for 6 days Calcium re- stricted diet for 15 days Calcium re- stricted diet for 24 days PTE doses 50 units for 3 days PTE doses inp creasing from 50-200 units over 7 days 37 TABLE 3 No. Animals 6 4 Mean Serum Ca Conc (meq/l) 6.10 6.15 6.43* 5.66* 5.80* 6.72* 6.43‘ *Significantly different from control at .05 level. 38 FIGURE 4 Calcium Adsorption From Plasma 100 98 f of Initial Ca Gone. 96 92 90 O 2 4 6 8 10 12 Adsorption Time (Days) 39 IV. Urine Volume Changes A summary of urine volume measurements reported in milliliters per 24 hours per kilogram body weight is presented in table 4. 40 TABLE 4 Urine Vol/24 hr/Kg body wt. Group Animal Control Mean Experimental Mean No. Treatment No. Minus Control Mean 1 Cal-dextro 25 35.85 - 7.43 lcc/Kg/day for 3 days 26 71.38 -l5.83 27 62.71 -l7.82 28 78.78 -10.03 29 27.66 - 7.62 30 28.16 6.23 2 Cal-dextro 41 50.20 - 8.87 2.5cc/Kg/day for 3 days 42 67.00 1.67 60 51.25 ~40.92 3 Cal-dextro 37 54.20 -12.87 ' See/Ks/day for 3 days 38 27.80 42.20 39 77 O 75 -39 e “‘2 40 37.80 -19.13 55 63.50 -58.50 56 37.50 -27.50 57 15.25 20.08 58 31 e 50 -19 0 l7 41 TABLE 4 (can't) Urine Vol/24 hr/Kg body wt. Group Animal Control Mean Experimental Mean No. Treatment No. Minus Control Mean 4 Cal-dextro 67 59.00 - 7.83 Soc/Ks/day for 6 days 68 9.00 - 2.33 69 114.00 -70.83 70 17 075 “11092 71 34.25 ~14.75 5 Calcium.re- 73 70.50 7.70 stricted diet for 15 days ' 74 45.75 4.08 76 113 .75 46.92 78 “'9 0 5O " 7 o 83 6 Calcium reg 61 69.25 - 0.70 stricted diet for 24 days 62 41.00 -21.37 64 21.00 14.56 6 5 ‘ 130.00 -9o.1u 27000 “’33 66 42 TABLE 4 (can't) Urine Vol/24 hr/Kg body wt. Group Animal Control Mean Experimental Mean No. Treatment No. Minus Control Mean 7 PTE doses 43 104.20 3.80 50 units for 3 days 44 91.80 -39.47 45 73.60 25.07 46 101.20 ~12.53 “7 “5.80 - 6e80 48 45.00 - 2.67 8* PTE doses in- 49 39.25 ~13.75 creasing from 50-200 units 50 129.25 -27.75 over 7 days 51 65.00 -37.37 52 53.50 -37.87 53 5&050 -neoo 5n 102.25 ~39.50 ‘"Significantly different from control at .05 level. 43 V. Urinagy Calcium Excretion Table 5 presents a summary of calcium excretion before and after the various treatments. Significant (P=.05) reductions in calcium excretion occurred in 6 of the 7 groups studied. _TABLE 5 . Ca Excretion (meq/Z4hr/Kg body wt) Group Animal Control Mean Experimental Mean No. Treatment No. Minus Control Mean 2* Cal-dextro 41 19.55 - 8.30 2.5cc/Kg/day , for 3 days 42 18.22 - 0.99 59 18.60 -l3.43 60 13.75 - 8.28 3* Cal-dextro 37 15.44 - 6.12 5cc/Ks/day for 3 days 38 14.24 - 9.43 39 20038 '15e66 40 17.02 -l3.50 55 12.98 - 9.08 56 15.35 -13.62 57 5.74 1-34 58 15.30 ~10.98 4* Cal-dextro 67 14.98 - 5.08 Soc/Ks/day for 6 days 68 11.98 - 8.94 70 12.43 -11.13 71 13-75 - 7-33 72 12.88 - 3.99 *Significantly lower than control values at .05 level. 45 TABLE 5 (can't) Ca Excretion (meq/24hr/Kg body wt) Group Animal Control Mean ‘ Experimental Mean No. Treatment No. Minus Control Mean 5* Calcium re- 73 19.23 -18.35 stricted diet - for 15 days 74 20.25 -19.13 75 13.60 ' -12.72 76 15.15 -12.80 77 15.65 -13.50 78 15.75 -14.28 6* Calcium re- 61 18.28 -17.28 stricted diet for 24 days 62 9.21 - 6.86 63 11e63 -10e60: 61+ 10095 - 9.14 65 16.63 -14.27 66 12.55 -10.63 7 PTE doses 43 12.23 - 4.36 50 units for 3 days 44 9.42 - 6.02 45 9.48 0.85 46 7.86 2.51 47 9.24 0.06 48 11.27 - 6.14 *Significantly lower than control values at .05 level. 46 TABLE 5 (con't) Ca Excretion (meq/24hr/Kg body wt) Group Animal Control Mean Experimental Mean No. Treatment No. Minus Control Mean 8* PTE doses in- 49 , 16.83 - 3.44 creasing from 50-200 units 50 11.68 - 1.75 over 7 days 51 15.98 _- 2.08 53 14.23 1.17 5“ 11040 - 1.81 *Significantly lower than control values at .05 level. 47 VI. Correlations between Tissue, Plasma and Urine Calcium Concentrations An attempt was made to demonstrate interrelations between calcium concentrations in urine and in renal tissue or between those of plasma and renal tissue. A summary of the results of this analysis is presented in table 6. Group No. 3 No.of Ani- mals u TABLE 6 Treatment Cal-dextro Sec/Ka/day for 3 days Cal-dextro Sec/Kg/day for 6 days Calcium re- stricted diet for 15 days Calcium re- stricted diet for 24 days PTE doses 50 units for 3 days PTE doses in- creasing from 50 to 200 units over 7 days *Significant at the .05 level. Correlation Coefficient for: Plasma Calcium- Papillary Calcium- Papillary Calcium Urine Calcium Concentration 0e926* -O.456 0.258 0.803 0.048 -0.695 Concentration 0.597 0.505 0.715 0.469 0.636 0.444 49 VII. Urine Calcium Concentration Changes A summary of urine calcium concentration studies, before and during treatment, is presented in table 7. 50 TABLE 7 Urine Ca Conc.(meq/l) Group Animal Control Mean Experimental Mean No. Treatment No. Minus Control Mean 2 Cal-dextro' 41 .438 - .158 2.5cc/Kg/day for 3 days 42 .341 - .075 59 .633 .068 60 .299 .936 3 Cal-dextro 37 .289 - .013 Soc/Ke/day ~for 3 days 38 .579 - .490 39 0301 - 011‘8 40 .462 - .207 55 .209 ‘ 1.337 56 .421 - .238 57 .343 .081 58 0507 - .108 4 Cal-dextro 67 .267 .020 Sec/Ks/day for 6 days 68 1.331 - .871 69 .150 - .033 70 .793 - .549 71 .40“ "' e001 72 .077 .192 5* Calcium re- 73 .283 - .271 stricted diet for 15 days 74 .447 - .415 75 0597 - 0502 *Significantly lower than control values at .05 level. Group No. Treatment 5*,(con't) 6* Calcium re- stricted diet for 24 days 7 PTE doses 50 units for 3 days 8* PTE doses in- creasing from 50-200 units over 7 days 51 TABLE 7 (con't) Animal No. 76 77 78 61 62 63 64 65 66 43 44 45 46 47 48 49 50 51 52 53 5a Urine Ca Cone. (meq/l) control Mean. .141 .766 .377 .270 -239 .246 .616 .130 .698 .116 .108 .132 .080 .215 .249 .433 .098 .255 .267 .283 .110 Experimental Mean Minus Control Mean .472 - .676 L .304 - .247 - .089 — .207 - .546 - .069 - .627 - .043 .316 - .028 .045 .008 - 0120 .577 - .001 .683 .495 .095 .048 *Significantly different than control values at .05 level. g; 52 VIII. Compgsite Presentation of Results Table 8 is included to give the reader a composite view of the results of this study. The values included represent those which were significantly different from corresponding control values at the .05 level. The symbol + indicates an increase of experimental over control values and - a decrease in experimental values; "0" indicates that the observed changes were not of sufficient magnitude to achieve .05 significance. It should not be interpreted as meaning that the control and experimental values were proven to be in the same population. Serum.Ca1cium Urinary Ca1- cium Excre— tion/24 hr Post Treat- ment Urine volume/24 hr Post Treat- ment Mean Urine Calcium Concentration Papillary Calcium Concentration Medullary Calcium Concentration Cortical Calcium Concentration Papillary Sodium Concentration Medullary Sodium Concentration Cortical Sodium .Concentration TABLE 8 Calcium Calcium Parathyroid Deprivation Administration Extract 1? days 24 days 3 doses 6 doses 3 doses 7 doses - - 0 + + + - - - - o - 0 0 0 0 0 - + 0 + + 0 + + 0 0 + 0 + 0 + 0 + O + 53 It“ DISCUSSION I. Microscopic Analysis A rough.microscopic examination of the renal lesions resulting from a calcium free diet (Plate 1), from PTE excess (Plate 2) or from calcium excess (Plate 3) revealed little that would be of value in distinguishing one from another. The lesions were located pri- marily in cortical tubule segments, although areas of tubular cal- cification and destruction frequently appeared in the medulla (Plate 4). The occurrence of advanced tubular destruction and calcifica- tion in the papilla was less common but did occur (Plate 5). Advanced destructive lesions, such as those in plates 1-5, have developed to such a degree that little could be determined about the nature of the original lesion. Various authors have reported calcification of cytoplasmic gran- ules (Grimes, 1957) and of tubular cell basement membranes (Hanes, 1939, Schneider 23 2%., 1960) following PTE or calcium salt treat-' ment. Basement membrane calcification was reported by Hanes (1939) and Schneider‘gt‘gl., (1960) to be the initial change in the devel- , opment of these lesions. Animals of this study, whether treated with calcium, PTE or the special diet, developed calcified cytoplasmic granules (Plates 6,7,8) and calcium deposits at the tubular cell margins that could be in- terpreted as calcification of basement membranes (Plates 9, 10, 11). These changes could be found in all parts of the kidney, and on oc- casion each appeared without the other. This observation not only ' 54 55 precludes the possibility of differentiating the lesions from one precipitating factor from those of another, but also casts doubt on the concept that basement membrane calcification is universally the initial change in the formation of these lesions. Tubular cell nuclear inclusions, presumed to be calcium because of positive Vbn Kossa staining, were also found in animals treated with PTE, calcium salts or a calcium restricted diet. These inclup sions were seen in cortical, medullary and papillary cells, and of- ten appeared without other demonstrable changes in the tissue. waa ever, they were more frequently found in cells with either basement membrane calcification or calcified cytoplasmic inclusions, or in ' cells with both. ‘< The degree of nuclear involvement varied from the presence of one or two small dense intranuclear inclusions (Plate 12) to the development nuclei which stained a dense uniform black by the Vbn Iossa method (Plates 6, 7, 8). The more completely calcified nuclei were found predominantly in the papillary region, although varying degrees of renal tubular cell nuclear involvements were found through- out the organ. 1 The occurrence of these nuclear inclusions, in the absence of other demonstrable alterations, and their frequent appearance in cells with pathological changes varying from mild cytoplasmic or basement membrane calcification to total cell destruction would make one suspect that this was the first sign of cell calcification. However, the occurrence of calcified cytoplasmic granules, and base- ment membrane calcification in the absence of nuclear calcification 56 casts doubt on this interpretation. The significance of the stain- able nuclear inclusions cannot be deduced from results of this study. Renal lesions, developed in this study by PTE, calcium salt excess or by a calcium free diet, were so similar both in general distribution and in the occurrence of basement membrane, cytoplasmic and nuclear calcific changes that no tenable basis for differentia- ting one from another could be discovered. Little doubt exists that these lesions were of the same nature as those reported by others (Beuper, 1927, Mulligan, 1946) to have resulted from PTE or calcium salt excess or from a restricted calcium intake despite the appearance of intranuclear inclusions which had not been previously reported. II UrineI Plasma and Tissue Sodium and Calcium Concentrations The mean papillary sodium concentrations of all experimental groups were depressed below control values (Tables 2, 8, Figure 2). The importance of these changes was suggested in a report by Manitus .gt‘gl. (1960) who studied the reduction of sodium concentration in the papilla of vitamin D intoxicated rats. They reported "that the impaired renal concentrating ability observed in hypercalcemic rats resulted, at least in part, from impaired reabsorption of sodium by the renal tubules and a diminished ability to concentrate and main- ° tain a high concentration of sodium in the interstitial fluid of the medulla and papilla.” Further support of the concept that the papil- lary sodium concentration is directly related to the urine osmolarity can be derived from the studies of Ullrich and Jarausch (1956). They 57 showed, with remarkable precision, a linear relationship between papillary tissue homogenate sodium concentrations and the osmolarity of the effluent urine. The sodium concentration changes found in figure 2 would lead one to speculate that reductions in papillary sodium concentrations are the cause of decreased renal concentrating ability reported to occur in animals treated with PTE (Epstein ‘gt‘gl., 1959) or calcium gluconate (Freedman ethal., 1958). Although the relation of changes in renal concentrating ability to altered papillary sodium concentrations is of interest, the obser- vation that similar changes in papillary sodium concentrations occur following PTE, calcium excess, restricted calcium intake (Table 2) or vitamin D (Manitus gt ;a_l_.., 1960) is probably of more significance. No evidence on the mechanism of reduction in papillary sodium is ad- vanced by this study (nor has any been found in the literature) yet it is clear that hypercalcemia is not the cause, nor even a common factor (Table 3). Furthermore, there appears to be a strong logical and experimental basis fer the assumption that serum parathyroid hor- mone elevations occur in animals maintained on a calcium free diet (Buckner and Nellor, 1960) and in animals given PTE excess, whereas the blood hormone level is reduced following vitamin D (Crawford ‘gt‘gl., 1957) or calcium salt therapy. Consequently, alterations in blood parathyroid hormone level pgg‘gg could not be the cause of these similar tissue concentration changes, nor of the formation of the histological lesions. A plot of tissue sodium versus calcium concentrations (Figure 3) suggests a positive, if somewhat variable, interrelation between the 1f ‘5‘ 88 1b” 58 two ion concentrations. A correlation coefficient of 0.641 was cal- culated from these data. Its significance, in excess of the .001 level, despite the seemingly low correlation coefficient, is explained by an‘p_of 169. This high over-all positive correlation exists de- spite the strong negative correlation contributed by the general de- pression of medullary sodium with concurrent elevation of medullary calcium in most of the experimental groups (Tables 1, 2). Mean me- dullary calcium concentrations in groups 1, 2, 4, 5 and 8 were signi- ficantly elevated above the corresponding control values at the .05 level, whereas the mean sodium concentrations of groups 3, 5 and 7 were significantly less (P=.05) than their corresponding control means. Plasma calcium concentration increases-occurred in PTE and cal- cium salt treated animals, while decreases were observed in the cal- ciumpfree-diet animals (Table 3). The plasma samples from which these analyses were made, were frozen in glass tubes and stored for one to five days prior to analysis. It has since been found that plasma samples stored in this manner undergo a progressive decrease in calcium concentration with time, presumably from calcium adsorp- tion on the glass. If this is correct, the rate and quantity of cal-. cium adsorption will depend on several highly variable factors, such as the glass surface area to fluid volume ratio and the initial cal- cium concentration of the plasma. Thus an accurate determination of (the calcium loss from the plasma is impossible, although the order of’magnitude of the change is easily found. It is unlikely that a plasma calcium.loss of,more than 7% or less than 3% occurred from ( 59 any of the samples whose analyses are compiled in table 3. It is probable that the serum calcium losses were approximately equal for all groups. However, this has not been proven, and consequently one should be aware of this limitationwhile interpreting these plasma calcium data. Daily urine volume output (Tables 4, 8) consistantly showed n1 small decreases following the initiation of any of the treatments 4 studied. ‘These changes, however, were of significant magnitude at the .05 level only in one of the PTE groups (Group 8). The daily’ ,5 ‘ ' lv urinary calcium excretion (Tables 5, 8) also decreased in all‘experb imental groups. These decreases proved to be significant at the .05 level for six of the seven groups studied. The biological signifi- cance of these changes cannot be established without further study, however, a tentative explanation will be presented later. Studies of the relationships between calcium concentrations in urine and renal tissue and between those in plasma and renal tissue yielded inconclusive results (Table 6). The correlation coefficients derived from the plasma calciumppapillary calcium concentration rela- tionships varied from.-0. 695 to 0.926. Despite the indicated signi- ficance of this last value, these data give little support to the con- . cept that a strong relationship exists between these two parameters. ’ Noneiof the correlation coefficients calculated for papillary 'calcium concentrationpurine'calcium concentration relationship were significant at the .05 level. However, the consistantly similar values would suggest that a significant correlation might be shown if sufficiently large samples were employed. 60 III. Relation of Tissue Sodium and Calcium with Renal Lesions Consideration should be given to the manner in which the data so far presented relate to the formation of the renal lesions. The metastatic theory for the formation of these renal lesions is based on the development of some condition which allows calcium to precipi- tate in otherwise normal tissue. For this type of lesion the cause is frequently implied to be increased calcium in interstitial fluid, or perhaps in tubular urine. A study of the changes in calcium con- centration of these fluids in states which give rise to these lesions would be of value. Since neither samples of tubular urine nor in- terstitial fluid were available, extrapolations must be made from indirect evidence. Mean daily urine calcium concentrations cannot be directly equated with intratubular urine calcium concentrations because of calcium and water reabsorption which occurs to a variable degree in transformation of the latter into the fonmer. Tubular u- rine is not, of course, an entity to which any single set of values can be applied. Its concentration and volume varies constantly as it passes down the nephron. Changes in tubular urine calcium con- centration may be the cause of the observed lesion through a process of metastatic calcification. If this is true, the concentration change must be reflected in the effluent urine since lesions deve- lop in the collecting ducts of the papilla as well as in cortical tubular cells. Data presented in table 7 raises some doubt about this hypothesis. No significant increases in urinary calcium con- centrations were seen, whereas significant reductions in urine 6l calcium occurred with both dietary groups. The interstitial fluid calcium concentration could, of course9 lead to a metastatic calcification of tubular cells. An increase in tissue homogenate calcium does not necessarily represent a similar increase in interstitial fluid calcium, although such an interpreta~ tion may be preferred. Alternative possibilities are increased in- ‘ tracellular calcium concentration and changes in the cell-to-inter- stitial fluid ratios. The volume contributions of blood and tubular urine to total renal mass are quite low (weaver gt‘gl., 1956) and thus neither could account for the marked ion concentration changes which occur. Similarly, explanation of these marked concentration changes on the basis of changed intracellular ion concentrations is unlikely. The intracellular ion concentration changes necessary to bring about such changes in total tissue calcium and sodium concen- trations are undoubtedly beyond the range tolerated by normal cells. blood pressure dependent changes in renal volume, presumably due to alterations in the interstitial fluid volume, are well established. Although such changes would alter the calcium concentration of tissue homogenates by changing the cellular fluid-to-interstitial fluid ratio, it could not account for the simultaneous increase in cortical calcium concentration and decrease in papillary calcium concentration as shown in tables 1 and 2. The most logical assumption to explain the data of tables 1 and 2 would be that tissue homogenate ion concentration changes reflect in- terstitial fluid ion concentration changes. If one is to accept this hypothesis, the relationship of the changes in interstitial fluid 62 calcium concentration, which can be inferred from table 1, to the development of the observed lesion casts further doubt on the meta- static calcification hypothesis. The increased cortical calcium con- centration is quite compatable with the metastatic theory; however, development of lesions in the papilla, where drastic reductions in tissue calcium occur, is not. These findings do not eliminate the possibility of these lesions being metastatic. They are, however, in opposition to the popular concept that tissue calcification in such cases could be due to elevations of calcium concentration in the fluids bathing the tubular cells. Iv. Relation of Sodium and Calcium in Renal Tissue Aside from the discussion of pathology and associated changes, the correlation of sodium.and calcium concentrations in renal tissue “(Figure 3), and the striking changes in calcium and sodium concen; tration from the cortex to the papilla (Tables 1,2) deserve discus- sion. The observation that tissue sodium concentrations vary in this manner has been substantiated by micropuncture studies of Las- siterqgtwgl. (lé6l) and by the tissue homogenate studies of Ullrich and Jarausch (1956). It is currently believed, as discussed pre» viously, that this concentration gradient.is maintained by the reab- sorption or sodium from.tubu1ar urine by the tubular cells. The calcium concentration gradient in renal tissue presented in table 1, can be explained on a similar basiss. Contributions to the inter- stitial fluid ion concentrations made by the reabsorption of sodium from the tubular urine should be closely paralleled by tubular urine 63 calcium reabsorption. As far as can be determined, the same areas of ' the nephron are responsible for sodium and calcium reabsorption (Wes- son and Lauler, 1959. Globus 25 gl., 1959, Vander, 1961, Grollman‘gt g;., 1962). Normal calcium reabsorption rates of approximately 99% of the filtered load have been reported (Chen and Neuman, 1955). These closely parallel the sodiumreabsorption fraction (Poulos, 1957). These observations would lead one to anticipate interstitial sodium to calcium ratios approximately equal to the serum sodium to calcium ratio. These factors could, of course, explain the highly sig- nificant correlation of tissue sodium and calcium which was reported earlier. Reductions in calcium.and sodium concentration gradients which accompany any of the calcium metabolism alterations induced in this study lack the firm base for their explanation that the correlation of tissue sodium and calcium concentrations enjoyed. Nevertheless a largely speculative explanation of the phenomenon can be given. The currently favored theory explaining urine formation assumes not only that a cortical to papillary osmotic gradient exists, but that urine is isotonic with the surrounding tissue fluids except for the contents of the ascending limb of the loop of Henle. Through, out this segment ions are removed from the urine by the tubular cells and the urine reaches the cortex in a.markedly hypot%pic state (Gottschalk and Mylle,.l958). At this point the urine reaches tu- bule segments which are freely permeable to water, and osmotic equilibrium is re-established. It is necessary to extend the ac- cepted theory and hypothesize the existence of cortical interstitial fluid somewhat hypotonic to plasma. This should result from reab- ‘\ , 64 sorption of large amounts of water from the hypotonic tubular urine presented to it. If this is the case, then the reduction in renal sodium and calcium concentration gradients by an increase in cortical_sodinm and calcium concentrations and a depression in papillary sodium and calcium ion concentrations, can be accomplished by any factor which reduces the glomerular filtration rate (GFR). A reduction in filtrate formation will reduce the amount of sodium and calcium reabsorption, and if the rate of capillary reabsorption of renal interstitial fluid is unaltered, the concentration of so- dium and calcium in the papilla must fall. At the same time, a re- duction in the amount of tubular urine delivered to the cortex will reduce the amount of water reabsorbed, and thus reduce dilution of cortical interstitial fluid. Cortical sodium and calcium ion con- centration equilibria will thus be reached at lower levels than normal. From this theory one would predict a high correlation between tissue sodium and calcium concentration changes. The differences in concentration produced between experimental groups and controls for calcium were correlated with those which occurred for sodium. h.correlation coefficient of 0.81 was calculated for the changes in papillary sodium and calcium concentration between the eight experimental groups and.their controls. This value is significant at the .05 level. The correlation coefficients for cortical and medullary sodium and calcium concentration changes were not signifi- cant however. The calcified lesion, because of its predominantly cortical 65 distribution, undoubtedly contributes significant amounts of cal- cium to the cortical homogenate, while contributing progressively less to the medullary and papillary homogenates. This concept is reinforced by the lack of a demonstrable correlation between sodium and calcium concentrations changes experimentally induced in the cortex and medulla, and also by the occasional elevation of corti- cal calcium.above that in the medulla (Table 1, Groups u, 5, 7). Although the tissue homogenate calcium concentration increases could be explained by this mechanism alone, such a theory could not account for the concurrent rise in cortical sodium concentration. From the results of this study it is impossible to assess the rela- tive contributions of the calcified cortical lesion, and of the reduction in formation of hypotonic cortical interstitial fluid to the apparent rise in cortical homogenate calcium concentrations. Nevertheless, since neither explanation is adequate alone, both must contribute to the observed cortical calcium elevation. no indications of the factors leading to the depressed GFR can be derived from the findings of this study, but the occurrence of the phenomenon has been reported in animals given calcium salts (Hallach and Carter, 1959, Poulas, 1957), and PTE (Epstein gt‘gl., 1959), as well as in patients suffering from parathyroid adenomas (Edvall, 1958, Dorhout, 1957, Cohen gt,él,. 1957). The marked reduction in urinary calcium excretion, as shown in table 5 and figures 14—20, can also be best explained by a reduc- tion in the GPR. Such a reduction in the absence of other changes will result in a larger intratubular dwell time for the urine, and 66 thus a greater extraction of calcium by the tubular cells. The de-' ‘crease in the filtered calcium load which accompanies a drOp in the GFR will contribute still further to the reduction in urinary calcium excretion. Although such an explanation is speculative, it fits the experimental findings more closely than do alternative explanations. A reduction in the filterable calcium for all of'the experimental groups could explain the results we see. However, Canary and Kyle (1959) have shown a sharp increase-in filterable calcium.results from the administration of PTE. An increase in the calcium reabg sorptive ability of the tubular cells could also explain the de. crease in calcium excretion, but could not simultaneously explain the reduction in interstitial calcium concentrations. SUMMARY AND CONCLUSIONS 1. No basis could 'be found for distinguishing lesions of PTE excess from those of calcium excess or from those resulting from a restricted calcium diet. In all cases the lesions were located primarily in the cortex,‘withnheavily calcified tubules lying be- side others that appear normal. Distinct calcified cytoplasmic granules and calcification of tubule cell margins, that could be interpreted as basement membrane calcification, were also univer- sally seen. Previously unreported calcified nuclear inclusions were also seen in all groups studied, although the significance of these changes cannot be determined from this study. 2. Analysis of the sodium and calcium concentrations in renal cortical, medullary and papillary homogenates revealed an elevation of both ions in the cortex and a depression of both in the papilla of animals maintained on any of the three experimental treatments, i.e., calcium salt, PTE or a calcium restricted diet. This high papillary sodium concentration is generally thought to be developed and.maintained by active tubular reabsorption of sodium from the tubular urine in conjunction with a high degree of water imper- meability exhibited by the ascending limb of the loop of Henle. The calcium gradient shown in this study can be explained by a similar mechanism, i.e., active calcium reabsorption from the tubu- lar contents. This theory is reinforced by significant correlation coefficients derived from sodium and calcium relations throughout the organ and from the sodium.and calcium changes that occur under 67 68 the various treatments . 3. Cortical sodium and calcium increases associated with papillary sodium and calcium decreases were observed in all of the experimental groups. On the basis of the results in this study it is hypothesized that the above phenomenon resulted from a reduction in glomerular filtration rate which occurred in an unspecified.manner from PTE, calcium salt or calcium free diet treatment. The maintenance of a concentrated papillary intersti- tial fluid and a hypothesized hypotonic cortical interstitial fluid depends upon an adequate flow of salts for reabsorption in the papilla and water for reabsorption in the cortex. Further- more, the papillary sodium concentration decreases can be equated with a reduction in renal concentrating ability, yet no diuresis resulted in this study. A reduction in GFR readily explains this phenomenon. h. Significant reductions in urinary calcium excretion oc- curred in six of the seven groups studied. These groups were comprised of PTE treated, dietary treated and calcium salt treated animals. This, too, is explained by'theinferred reduction in GFR. Under such conditions the filtered calcium load is not-only decreased, but the concommittant increase in intratubular urine dwell time allows for more complete ion reabsorption. 5. The renal calcification resulting from PTE or calcium salt treatment is frequently reported to be metastatic. It is implied that this is due to increases in calcium concentration in the fluids bathing the tubular cells. The calcium increase must, 69 then, be either in tubular urine or in interstitial fluid. Indirect measures of the interstitial fluid calcium concentration change associated with calcium restricted diet, calcium salt or PTE treats ment, indicate that calcium concentration increases do occur in the cortex.- However a sharp reduction in papillary calcium concentra- tion is a consistant finding following any of these treatments and lesion formation occurs here also. If the lesion induced in these cases is of metastatic origin, based on increased tubular urine calcium concentration, that concen- 3tration rise must be seen in the effluent urine since lesions occur in the papillary segments of the collecting duct. The calcium con- centration of the effluent urine was found to be either decreased or unchanged for animals on any of the three_treatments studied. These findingsstrongly imply that metastatic calcification, based upon elevations of interstitial fluid or tubular urine calcium con- centration increases, is not involved in the formation of these lesions. 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