T H E EFFECTS O F T H Y R O I D H O R M O N E O N VITAMIN D - I N D U C E D NEPHROCALCINOSIS

R. J. NEWMAN Depar fment of Anatomy, University of Leeds

PLATES X AND XI

THE characteristic radiological changes in cretinism are : delay in the appearance and growth of ossification centres with deformity and fragmentation of epiphyses, hypoplasia of one or more vertebral bodies near the dorsolumbar junction, and lines of increased density in the metaphyses and upper and lower surfaces of vertebral bodies (Astley, 1958; Bateson and Chander, 1965).

A radiological feature that is not well known is the occasional presence of calcium in the urinary tract. This may be in the form of nephrocalcinosis or nephrolithiasis or both. The table enumerates published cases that showed these radiological findings and others diagnosed either by biopsy or at necropsy.

Reports of at least twenty-three cases of hypothyroidism associated with deposition of calcium in the kidney indicates a possible relationship between the two conditions. Lack of thyroid hormone may be conducive to calcification or, as has been suggested, hypothyroidism may increase sensitivity to vitamin D.

The aim of this study was to test the latter suggestion and to investigate also whether excess of thyroid hormone might affect sensitivity to vitamin D.

MATERIALS AND METHODS

Animals Eighteen litters of young adult male jirds (Meriones unguiculatus) were used in this investi-

gation. This animal has previously been used to investigate hypervitaminosis D in the euthyroid state (Hollins, unpublished).

Experimental technique Each litter consisted of about six animals and was divided into three groups. Group A

served as controls and received “modified diet 41B” (Oxoid Ltd). Group B received the same diet to which had been added Neo-Mercazole (carbimazole) at a concentration of 5 mg per g of diet. This drug has previously been shown to have marked antithyroid activity in rats (Lawson, Rimington and Searle, 195 1). Group C received “modified diet 41B” containing Tertroxin (triiodothyronine, T3) at a concentration of 2.5 pg per g of diet. The efficacy of these drugs in changing the animals’ thyroid status in the desired way was shown by I3II- uptake studies, thyroxine-binding globulin saturation tests (Clark and Brown, 1970a, 6, and c ; Radiochemical Centre, Amersham, 1969) and thyroxine assays (Chapman and Livesey, un- published technique).

On the 35th and 37th day of treatment animals were given a subcutaneous injection of 750 IU vitamin D2 (calciferol) in arachis oil per g of body weight (0.0075 ml of a 100,000 IU

Receiued 24 Aug. 1972; accepted 8 Feb. 1973. J. PATH.-VOL. 111 (1973) 13

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EFFECTS OF THYROID HORMONE ON NEPHROCALCINOSIS 15

per ml preparation per g of body weight). One animal from each group was killed by ex- sanguination under light ether anaesthesia on day 35. Half the remaining litter was killed on day 40 and the rest on day 42.

To investigate possible nephrocalcinogenic effects of the vitamin D solvent six animals from each group received injections of arachis oil (0.0075 ml per g body weight).

Histological studies The left kidney was bisected and fixed in cold 80 per cent. ethanol; 7-pm paraffin-wax

sections were stained with haematoxylin and eosin for general histology. Sites of calcification were demonstrated by a variety of techniques that included metal substitution (v. Kossa, 1901), alizarin red S (sodium and alizarin sulphonate) (McGee-Russell, 1958) and nuclear fast red (Kernechtrot) (McGee-Russell, 1955). Periodic acid-Schiff (PAS) (Pearse, 1959) and alcian blue (Steedman, 1950) techniques were used to stain polysaccharide complexes.

Quantitative studies The right kidney was dehydrated at 9OoC, treated with petroleum ether to remove fat and

a constant fat-free, dry weight obtained before the tissue was reduced to ash in a muffle oven at 600OC. The calcium content of the inorganic residue was estimated as a routine by the spectrophotometric method of Trinder (1960), and confirmed by the fluorimetric procedure of Kepner and Hercules (1 963).

RESULTS Histological studies

Day 35. The histological appearance of the kidneys was normal and was the same in all three groups.

Day 40. Pathological changes were present in the kidneys of all animals. The most striking observation in the kidneys of the carbimazole-treated group

was the presence of calcium in the form of intraluminal and interstitial casts in the inner cortex, medulla and papilla. Most of the casts were fully calcified, but those that were only partially calcified contained a PAS-positive and sometimes an alcian blue-positive amorphous substance (fig. 1). Clusters of degenerating cells rich in PAS-positive granules were occasionally found lying free in the lumen of tubules. These often appeared to be acting as a nidus for calcification. Many of the cells of the collecting ducts contained PAS-positive particles (fig. 2). The parietal layer of Bowman’s capsule of some Malpighian corpuscles showed a thickening that stained with PAS, and the glomeruli often appeared congested. Infiltrations of round cells surrounded many such corpuscles and many of the medullary casts.

The kidneys of the T,-treated animals showed very little cellular damage or inflammatory response. Calcification was restricted to the cortex, where it was found in the basement-membranes of the convoluted tubules and the parietal layer of Bowman’s capsule.

Kidneys from control animals contained fewer calcium deposits than those of the other two groups. They appeared in the form of intraluminal and inter- stitial casts, mainly at the juxtamedullary junction. Round-cell infiltrations were occasionally seen.

Day 42. The number of calcium deposits had increased in all kidneys. In the carbimazole-treated animals the calcium was located almost exclusively in

16 R. J. NEWMAN

the medulla and papilla. In the T3-treated animals the cortex was the site of calcification, whereas in the control kidneys the calcium was distributed with no regular pattern (fig. 3).

In the kidneys of those animals treated with carbimazole the calcium appeared as casts in the lumen of the loops of Henle and collecting ducts and as interstitial deposits. Round-cell infiltration in the vicinity of such casts was a frequent finding. Non-calcified casts that stained with alcian blue and PAS were often noted. Many of the cells of the convoluted tubules had sloughed off and were seen lying in the lumen lined by a denuded basement-membrane. Such cells were rich in PAS-positive material. Granules that also stained with PAS were often seen within cells of the collecting ducts. Some glomeruli showed disorganisation and congestion and a PAS-positive and alcian blue-positive thickening of the parietal layer of Bowman’s capsule. The basement-membranes in the medulla and papilla stained heavily with alcian blue.

In the T3-treated animals calcium was deposited in the basement-membranes of the convoluted tubules and often in the parietal layer of Bowman’s capsule (fig. 4). In those kidneys where calcification was most intense it was not possible to state with certainty that the calcium was restricted to the basement-membrane, and in several specimens it appeared to have been deposited basally within some of the cells. Alcian blue-positive material, which was previously almost absent from the basement-membranes in the cortex, was now present. No infiltrations of round cells were noted.

In the control kidneys the distribution of calcium was not constant, but was scattered throughout the sections. In the cortex it was in basement-membranes and in the medulla in the form of intraluminal and interstitial casts. Most Malpighian corpuscles showed no pathological changes and infiltrations of round cells were only occasionally noted.

Control animals given arachis oil without vitamin D. The kidneys of these animals presented an entirely normal appearance on days 35,40 and 42.

Other sites of calcification. Subintimal calcification of the arcuate arteries was common in the carbimazole- and T3-treated animals but rare in the controls. Two cases of calcification of the pelvis of the ureter were noted in animals treated with T3. Calcification of the renal capsule was not seen.

Quantitative studies After the injections of vitamin D the calcium content of the kidneys of all

three groups of animals increased. This increase was most pronounced in animals treated with carbimazole or triiodothyronine. Both at days 40 and 42 the differences in calcium content between the experimental and control animals were shown to be statistically significant by Student’s t-test (P < 0.05). Injec- tions of arachis oil without vitamin D were found to have no nephrocalcinogenic effect. These results are represented diagrammatically in fig. 5.

DISCUSSION The quantitative estimates of renal calcium were supported by the histo-

logical findings and demonstrated, at least in the jird, a hypersensitivity to

NEWMAN

EFFECTS OF THYROID HORMONE ON NEPHROCALCINOSIS

PLATE X

FIG. 1 .-Corticomedullary junction of carbimazole-treated animal on day 40 showing PAS-positive (arrow) and calcified casts. v. Kossa (silver) and PAS. x 160.

FIG. 2.-Papilla of carbimazole-treated animal on day 40 showing PAS-positive particles within cells of collecting ducts. v. K6ssa (silver) and PAS. x 210.

NEU MAN

EFFECTS OF THYROID HORMONE ON NEPHROCALCINOSIS

PLATE XI

FIG. 3.-The pattern of nephrocalcinosis on day 42 in (A) a carbimazole-treated animal; (B) a control animal; and (C) a T3-treated animal. v. Kossa (silver). x c. 6.

FIG. 4.-Cortex of T3-treated animal on day 42 showing calcification of basement-membranes and parietal layer of Bowman’s capsule. v. Kossa (cobalt) and neutral red. x 200.

EFFECTS OF THYROID HORMONE ON NEPHROCALCINOSIS 17

the calcinogenic effects of vitamin D in hypo- and hyper-thyroidism. An unexpected, but interesting finding was that the distribution of calcium in the kidney varied in different thyroid states (summarised in fig. 6). This raises the question of how these differences arise.

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Nephrocalcinosis in hypo- and hyper-thyroid animals It has been known for almost two decades that isolated intact mitochondria

can accumulate calcium from the surrounding medium against a concentration gradient (Slater and Cleland, 1953). Such a process requires energy and DeLuca and Engstrom (1961) and Vasington and Murphy (1962) were the first to show its dependence upon oxidative phosphorylation.

J. PATH.-VOL. 111 (1973) B

18 R. J. NEWMAN

Little thought has been given to the cellular significance of this active ion accumulation. Lehninger (1 964) however suggested that mitochondria serve as

H ypoih yroid

Control

FIG. 6.-Distribution of calcium deposits in kidneys of control and experimental animals after ad- ministration of vitamin D.

temporary “ion-buffering systems” capable of local and transient sequestration of ions in order to control disparities in the ionic composition of the cytoplasm. It is possible that in the hypothyroid state the capacity of the mitochondria to bind calcium is reduced, so leading to high cytoplasmic calcium concentrations. Epstein (1968) suggested that such concentrations are cytotoxic and lead to cell death. These concepts suggest a hypothesis that is consistent with the histological findings in the present study,

Administration of large doses of vitamin D causes an elevation of serum calcium (Eisenstein, Battifora and Ellis, 1965; Epstein), and consequently an increased calcium load is presented to the cells of the proximal and distal con- voluted tubules. If in hypothyroidism the mitochondria1 function of these cells is impaired then their cytoplasmic calcium concentration may rise to levels that

EFFECTS OF THYROID HORMONE ON NEPHROCALCINOSIS 19

cause cell death. The dead cells would then slough off from the basement- membrane and pass into the lumen of the tubules, forming a nidus for calcifi- cation. If the cells sloughed off from the proximal convoluted tubules, casts would collect in the loop of Henle. If, however, the cells were of distal con- voluted tubule origin the casts would form in the collecting ducts. In both cases casts would appear in the medulla.

This hypothesis would explain not only the experimental results but also the fact that the nephrocalcinosis reported to occur in hypothyroid patients is mainly the medullary-cast type.

In the hyperthyroid state accumulation of calcium within the cell and con- sequent cell death does not occur since the mitochondria1 metabolism is, if any- thing, increased and the excess calcium is transported across the cell and deposited in the basement-membrane. This site of calcium deposition is the same as that previously observed in euthyroid animals after the administration of calcium gluconate (Grimes, 1957; Fourman, 1959).

The significance of the PAS-positive particles within the cells of the collecting ducts in all the groups of animals, but especially in the hypothyroid group, is not clear. This observation has not been previously reported, but Engfeldt et aL (1958) and Grimes both found PAS-positive inclusions in the cells of the proximal convoluted tubule after the administration of parathyroid hormone. Grimes suggested that the inclusions were mucoprotein that had been released from bone, but this is not consistent with the fact that in this experiment most inclusions were found in the group with, presumably, a diminished osteoclastic activity.

In all groups of animals PAS-positive thickening was found in the parietal layer of some of the Bowman’s capsules. This was especially prominent in the carbimazole-treated animals and is in keeping with the findings in the post mortem and biopsy specimens taken from hypothyroid patients with nephro- calcinosis (see table).

SUMMARY Jirds (Meriones unguiculatus) were rendered hypo- or hyper-thyroid by the

addition of carbimazole or triiodothyronine to their diet, and in each of these states were found to be hypersensitive to vitamin D.

After two subcutaneous injections of the vitamin histological studies of the kidney showed that in the carbimazole-treated animals the calcium was dis- tributed as casts in the medulla and papilla. This pattern of nephrocalcinosis is the same as that previously reported in hypothyroid patients. In triiodo- thyronine-treated animals the calcium was deposited only in basement-mem- branes in the cortex. In the control kidneys calcium was distributed both as casts in the medulla and in basement-membranes of the cortex. PAS-positive particles within the cells of collecting ducts and infiltrations of round cells surrounding calcium deposits were most common in kidneys of carbimazole-treated animals.

It is suggested that mitochondria serve as temporary ion-buffering systems and this ability is reduced in the hypothyroid state. This leads to high cyto- plasmic calcium concentrations which cause cell death. In hyperthyroidism the highly active mitochondria prevent cellular damage.

20 R. J. NEWMAN

I am indebted to Dr Julia Fourman for her constant advice and encouragement throughout this research. I wish to thank Professor R. L. Holmes for allowing me the facilities of his Department and Dr W. J. Moore for assistance with the statistics.

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