The Ultrastructure of Nephrocalcinosis Induced in ...

Vet. Pathol. 18: 62-70 (1981)

The Ultrastructure of Nephrocalcinosis Induced in Chicks by Cestrum diurnum Leaves K. SARKAR, R. NARBAITZ, R. POKRUPA and H. K. UHTHOFF

Departments of Pathology, Anatomy and Surgery, School of Medicine, University of Ottawa, Ottawa, Ontario, Canada

Abstract. Powdered Cesfrurndiurnum leaves, mixed with two diets differing in calcium and phosphorus contents, produced nephrocalcinosis in young chicks regardless of serum calcium elevation. The calcific deposits, found in both proximal and distal portions of cortical tubules, began either in the cytoplasm or in lysosomal bodies as a unilaminar spheroid structure containing apatite crystals. The ultrastructural characteristicsof intraluminal concretions suggested that they were formed intracellularly but later were extruded into the lumen. The extent of calcific deposits increased with duration and with hypercalcemia. Although Cestrum contains an analog of 1,25-dihydroxycholecalciferol,neither mitochondria nor basal lamina contained calcific deposits described in nephroca1cinos:s secondary to hypervitaminosis D.

The appearance of nephrocalcinosis following large doses of vitamin D was first reported in 1928 [14]. In recent years, great advances have been made in the understanding of vitamin D metabolism, and its biologically active metabolite has been isolated. We now know that vitamin D is converted in the liver into 25hydroxycholecalciferol, which is the main circulating form of the vitamin in blood [3]. This metabolite is hydroxylated further in the kidney to 1,25-dihydroxycholeca1ciferol, but this conversion is regulated closely according to the need of the organism. Thus, when excess vitamin D is given to normal animals, it is converted to 25hydroxycholecalciferol without subsequent production of 1,25-dihydroxycholecalciferol. Consequently, the serum of animals with experimental hypervitaminosis D shows a high concentration of 25-hydroxycholecalciferol,while the concentration of 1,25-dihydroxycholecalciferolremains relatively low [4]. It has been established that 1,25-dihydroxycholecalciferol is the most active metabolite of vitamin D [3, 41, but induction of nephrocalcinosis by pure 1,25dihydroxycholecalciferol has not been reported. Renal calcification, however, is seen frequently among animals feeding on plants such as Solunum mulucoxylon [2] and Trisetumflavescens[6], which contain an analog of 1,25-dihydroxycholecalciferol.On the other hand, neither cattle nor horses feeding on Cesrrzim diurnum, another plant 62

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containing an analog of 1,25-dihydroxycholecalciferol[ 181, develop nephrocalcinosis. Hypercalcemia occurs in these animals, however, in association with excessive deposition of calcium in bone as well as in soft tissues such as tendons, ligaments and major vessels [ l 1, 121. During our previous studies on Cestrum-induced lesions in chicks [ 1, 131, we found that Cestrum mixed with a rachitogenic test diet produced hypercalcemia. Reexamining the material obtained from those experiments, we noted that calcific deposits occurred more readily in kidneys than in any other soft tissues. The present study of kidneys from chicks fed Cestrum with diets differing in calcium content was undertaken to clarify the ultrastructural localization and sequential progression of the calcific deposits.

Materials and Methods Newly hatched chicks were obtained, and the experiments were started when they were 4 days old. The Cestrum diurnum leaves were reduced to powder and mixed in an omnimixer with either normal Purina chick diet (6308 Bio Startena, Ralston Purina Canada Inc., Longueuil, Quebec; henceforth diet A) or a rachitogenic test diet (#170640, Teklad Test diets, Madison, Wis.; henceforth diet B). The Cestrum content in both diets was 3 g/100 g diet. Diet A contained 0.9% calcium and 0.7% phosphorus; diet B contained 1.45% calcium and 1.36% phosphorus [9]. The chicks were divided into three groups: one group ate diet A (Group 1); the second group had Cestrum mixed with diet A (Group 11); and the third group was given Cestrum in diet B (Group 111). Each group consisted of nine chicks, and the experimental protocol was repeated three times. In these experiments, no group of chicks was given diet B without Cestrum. In our previous experiments [I, 131, chicks fed diet B alone failed to develop hypercalcemia, and a reexamination of their kidneys showed no nephrocalcinosis. Three chicks from each group were killed after one, two and three weeks of feeding. They were anesthetized by an intraperitoneal injection of chloral hydrate-barbiturate mixture. Blood for calcium determination was obtained by cardiac puncture from two chicks of each group at death. Estimation of serum calcium thus was done on 18 of 27 chicks in each experiment. The kidneys, exposed by an abdominal incision, were flooded with half-strength Karnovsky’s futative [lo]. Random sections were taken from different regions of the left kidney, and immersed in the same futative for two to four hours at 4OC. The pieces were post-futed with 1% osmium tetroxide, dehydrated in graded ethyl alcohols and finally in propylene oxide, then embedded in epon. Sections for electron microscopy were stained with uranyl acetate and lead citrate. The serum calcium was determined by an automatic calcium analyzer (19401, Coming Scientific Instruments, Medfield, Mass.). Tissue for light microscopy was futed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE) and von Kossa’s stain.

Results It was apparent during the three weeks of the experimental period that chicks of Group I11 failed to thrive when compared with those in the other two groups. Serum calcium of chicks belonging to Groups I and I1 was from 9.2 mg to 10.5 mg/100 ml throughout the experiment. In contrast, the chicks of Group I11 were hypercalcemic at the end of the first week. Their serum calcium varied from 13.5 mg to 17.2 mg/100 ml, but showed no sequential rise from the first to the third week.


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Light microscopy showed basophilic, von Kossa-positive spheroid bodies representing calcific deposits, generally limited to the renal cortical tubules of both groups of Cestrum-fed chicks. These bodies often were laminated, and were present in the epithelial cells as well as in the lumen. In many of the tubular segments containing calcific spherules, the epithelial cells were multinucleated with nuclei in a row at the periphery, imparting a Langhans-type giant cell appearance (fig. 1). Multinucleated giant cells sometimes were seen in tubules with no readily discernible calcific deposit. In Groups I1 and 111, the extent of nephrocalcinosis increased with time, although it was much more pronounced in Group I11 than in Group 11. There was, however, hardly any change in the site of calcific deposits. With increased time, some tubular epithelial cells were eroded by large, multilobulated concretions. In only one section of tissue, from a chick of Group I11 after three weeks, did we find a band of calcific deposit in paravascular interstitial tissue (fig. 2). There were no significant changes in the glomeruli. Mononuclear cell infiltrate was found infrequently near larger vessels or randomly in the cortical interstitial tissue. The infiltrate did not seem to be directly related to deposit-containing tubular segments. Ultrastructurally, calcium deposits could be found both in the cytoplasm of epithelial cells and in the lumen of the tubules, as seen with the light microscope. The intracytoplasmic concretions occurred in proximal as well as distal portions of the cortical tubules, while those in the lumen were predominantly in the distal portions. The calcium deposits were spheroid bodies with an electron-dense layer surrounding a lucent core (fig. 3). These bodies could occur either singly or in clusters, apparently free in the cytoplasm or within lysosomal structures representing autophagosomes (fig. 4). The concretions often were multilobular and laminated (fig. 5 ) . Needle-shaped crystals resembling apatite usually were a component of these concretions, especially in the region of the outer lamina (fig. 6). In some of the larger concretions, however, the crystalline structures were not easily discernible. Many of the concretions were partially enclosed by a membrane (fig. 5). The cytoplasmic organelles in the vicinity of concretions showed no consistent change. Some mitochondria were swollen with vacuolated matrix (fig. 6). Matriceal granules in mitochondria, however, did not seem to be larger or more numerous. Some of the proximal tubular epithelial cells containing deposits showed disruption and focal effacement of brush border. The calcific deposits in the lumen occurred mostly as laminated concretions, but sometimes as aggregates of apatite crystals contained in a membrane-bound sac-like structure (fig. 7). The intraluminal deposits often were accompanied by cellular debris. In some epithelial cells of tubules containing both intracytoplasmic and intraluminal deposits, concretions protruded toward the lumen, giving an appearance of imminent extrusion (fig. 8). The nuclei in multinucleated cells were of irregular shape and size (fig. 9). Small vesicular structures were abundant in the cytoplasm of these cells. The basal lamina of tubules containing concretions was generally of uniform density and thickness,


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Fig. 1: Tubular architecture altered by calcific deposits. Aggregation of nuclei near deposit. HE. Bar = 50 pm. Fig. 2 Intensely basophilic area in interstitial tissue suggests calcific deposit (arrow). Bar = 50 pm. Fig. 3: Unilaminar spheroid body in cytoplasm of tubular epithelial cell contains apatite crystals. Uranyl acetate and lead citrate. Bar = 2 pm. Fig. 4 Multiple spheroid calcific deposits either in membrane-bound lysosomal body or free in cytoplasm. Uranyl acetate and lead citrate. Bar = 1 pm. 65


Fig. 5 Multilaminar, lobulated calcitic deposit enclosed by membrane (arrows). Uranyl acetate and lead citrate. Bar = I pm. Fig.6 Apatite crystals in outer lamina of deposit. Adjacent mitochondrion (m) swollen and vacuolated. Uranyl acetate and lead citrate. Bar = 0.5 pm. Fig. 7: Apatite crystals in membrane-limited sac-like structure in lumen. Uranyl acetate and lead citrate. Bar = 0.5 pm. Fig. 8 Large intracytoplasmic concretion protrudes towards lumen containing similar concretions. Uranyl acetate and lead citrate. Bar = 3 pm. 66


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Fig. 9: Multinucleated epithelial cell; numerous vesicular structures in cytoplasm. Electrondense body (arrow) suggests early concretion. Uranyl acetate and lead citrate. Bar = 1 pm.

and devoid of discernible deposits. The glomeruli and the cortical interstitium showed no ultrastructural abnormalities. Discussion

This study demonstrates that Cestrum diurnum leaves, which contain an analog of I ,25-dihydroxycholecalciferol,could be a potent nephrocalcinogenic agent in chicks, even in the absence of hypercalcemia. The most important action of 1,25-dihydrox-


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ycholecalciferol is to enhance intestinal absorption of calcium and phosphate [ 171. The degree of hypercalcemia resulting from large doses of 1,25-dihydroxycholeca1ciferol hence would depend upon increased availability of calcium and phosphorus from diet. This was evident in our study when Cestrum failed to raise serum calcium levels with normal Purina chick diet containing adequate amounts of calcium and phosphorus. When Cestrum was fed in the same concentration with a rachitogenic test diet with a higher content of calcium and phosphorus, however, the serum calcium of chicks was raised significantly. Although the calcification worsened with hypercalcemia, the fact that it could occur without alteration in serum calcium precludes Cestrum-induced nephrocalcinosis being of “metastatic” type. It is generally accepted that calcification secondary to hypervitaminosis D is of the “metastatic” type, because an elevation of serum calcium usually precedes calcium deposition in soft tissues [15]. This is also true in laboratory rodents, in which the morphological characteristics of vitamin D-induced nephrocalcinosis have been studied extensively 17, 8, 161. Some investigators have contended, however, that hypervitaminosis D could be directly toxic to renal tissues, which then would undergo calcification as a secondary process [19]. Whatever the mechanism is in either Cestrum- or vitamin D-induced nephrocalcinosis, there are significant differences between the two in the morphological site of calcific deposits. Vitamin D-induced nephrocalcinosis occurs predominantly in proximal tubules with deposits in cytoplasmic vacuoles, basal laminae and mitochondria (8, 161. In contrast, both the mitochondria and the basal laminae were free of calcific deposits following Cestrum induction. Both the proximal and the distal portions of cortical tubules contained calcific deposits. It is known that the major part of calcium is transported in the proximal tubule, but some transport occurs in the distal convoluted tubule and in the cortical collecting duct [51. In our sequential studies, the deposits started as unilaminar spheroid bodies either within lysosomal structures or free in the cytoplasm. The gradual expansion of the deposits resulted in multilamination and lobulation. The presence of apatite crystals in the outermost laminae of the concretions probably indicated a continued expansion, whereas their absence could signify an arrested state. Apart from occupying space in the cytoplasm, the deposits did not seem to affect the neighboring organelles adversely. We cannot explain the multinucleation of several tubular epithelial cells; the phenomenon has not been described in relation to any other type of experimental nephrocalcinosis. In the lumen, apatite crystals sometimes were enclosed within a membrane-bound vacuole. It can be argued that the presence of a limiting membrane denoted their intracellular origin. Even some luminal concretions suggested that they could have been extruded from their intracytoplasmic location, where similar bodies protruded toward the luminal pole. Cestrurn-induced calcinosis was first described in cattle and horses that graze on this plant in the southern part of the United States (11, 121. The animals were


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hypercalcemic, and excessive calcium deposition occurred in bone as well as in soft tissues such as tendon, ligaments and the major vessels. Nephrocalcinosis, however, rarely was seen in either horses or cattle [ 1 1, 121. The induction of calcinosis in the kidneys rather than in ligaments and tendons in chicks, then, could be attributed to a species difference. Also, the relatively high concentration of Cestrum in the chicks in ratio to body weight could be a factor. We conclude that the Cestrum diurnum leaves, when fed to young chicks, induce nephrocalcinosis, which increases in seventy with duration and with hypercalcemia. The calcific deposits occur in both proximal and distal portions of cortical tubules. In the intracytoplasmic location, the spheroid deposits could form either within lysosomal structures or freely in the cytoplasm. It appeared that the intraluminal concretions were extruded into the lumen following an intracytoplasmic origin.

Acknowledgements This study was supported by grants from the Arthritis Society and the Medical Research Council. Mr. Francis Wei and Ms. Kathleen Wilson provided technical assistance. Ms. Suzanne Noel typed the manuscript.

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from rachitic chicks before and after vitamin D or Cestrum diurnum leaves treatment. Rev Can Biol36: 105-1 12, 1977 PFANNENSTIEL, W.: Weitere Beobachtungen uber Wirkungen bestrahlten Ergosterins im Tierversuch. Munch Med Wochenschr 75 1 113-1 114, 1928 ROBBINS,S.L.; ANGELL,M.: Basic Pathology, p. 616.W.B. Saunders Co., Philadelphia, 1976 SCARPELLI, D.G.: Experimental nephrocalcinosis. A biochemical and morphologic study. Lab Invest 14 123-141, 1965 WASSERMAN, R.H.; CORRADINO, R.A.: Vitamin D, calcium and protein synthesis. Vitam Horm 31: 43-103, 1973 WASSERMAN, R.H.; CORRADINO, R.A.; KROOK,L.P.: Cestrum diurnum: a domestic plant with 1,25-dihydroxycholecalciferol-likeactivity. Biochem Biophys Res Commun 62 8591, 1975 YENDT,E.R.: Vitamin D: Part 11. In: Pharmacology of the Endocrine System and Related Drugs: Parathyroid Hormone, Thyrocalcitonin and Related Drugs, vol. I, pp. 143-144. Pergamon Press, Oxford, 1970

Request reprints from K. Sarkar, M.D., Department of Pathology, School of Medicine, University of Ottawa, Ottawa, Ontario KIN 9A9 (Canada).
