Studies on renal mucopolysaccharides

This thesis is an account of studies made on the distribution and nature of acid mucopolysaccharides in the mammalian renal papilla. The papilla was found to be the only region of the kidney which has an appreciable extracellular space with acid mucopolysaccharide enmeshed in connective tissue fibres. Animals are unable to secrete urine which is either more concentrated or more dilute than plasma unless the tonicity of this region is greater than that of plasma. In the first part of this work (Chapter 2) the extent of extracellular acid mucopolysaccharide was correlated with the ability of the species to conserve water. It was found that the papilla could be sharply delineated by staining the acid mucopolysaccharide within it. This had the advantage over the less distinct cut surface appearances in that it was possible accurately to measure the length of the papilla in relation to that of the cortex and medulla in stained sections. An empirical relationship between the development of the papilla and the ability to secrete hypertonic urine was obtained for a number of species: Umax = 1.74 (Mandprime;/Candprime;) + 0.6, where Umax is the maximum concentration in Osmols/litre that can be achieved in the urine; Mandprime; is the length of the papilla, measured as the distance of the region containing extracellular acid mucopolysaccharide from the papillary tip towards the renal cortex; and Candprime; is the continuation of this line through the medulla and cortex. The correlation coefficient for the six species for which figures for maximum urinary concentration were known was 0.9. The significance of this expression is discussed in Chapter 2 in relation to a similar equation put forward by Black in 1965. The morphology of the connective tissue in the papilla of the guinea pig was examined by light and electron microscopy (Chapter 3) and by histochemistry (Chapter 4). The histological results were correlated with the electrophoretic and staining characteristics of acid mucopolysaccharides extracted from this region. There was very little collagen of the adult type to be found in the papilla. The main fibrillar component was reticulin which was well demonstrated by silver impregnation and the periodic acid - Schiff technique. Within the reticulin mesh another class of PAS positive fibril was found which, in addition, stained positively for anionic groups. These fibrils could not be impregnated with silver. In the electron microscope the second class of fibril was seen not to be banded and to be smaller than the reticulin fibrils. The histochemical results (Chapter 4) showed that the largest part of the amorphous extracellular acid mucopolysaccharide in the papilla was hyaluronic acid. In addition, two quantitatively small fractions were observed in the biochemical extracts which were associated with the extracellular acid mucopolysaccharide. Both these fractions stained metachromatically with thiazine dyes and were shown to incorporate radioactive sulphate. In sections metachromatic extracellular material was confined to the tip of the papilla: In this region of high in vivo osmotic pressures the interstitial cells also contained monochromatic granules. The exact nature of these two fractions was not elucidated, nor was an attempt made to identify the intracellular granules with its synthesis or destruction. The results from the morphometry of the kidneys of fourteen species (Table 2/1) showed that desert species have long papillae which contain a limited amount of connective tissue. The metachromatic staining of extracellular acid mucopolysaccharides extends further towards the cortex in those species which can secrete highly concentrated urine. On the other hand, the papillae of animals which habitually produce dilute urine were squat in outline and contained abundant connective tissue. The tip of the papilla in these species was not found to stain set metachromatically. The extent of metachromasia was thus found to be related to the development of high tonicity in vivo, and thus to be most prominent in that part of the papilla where the sodium concentration is greatest in life. It was observed histologically that acid mucopolysaccharide was associated with the lateral spaces between collecting tubule cells in the guinea pig. It had been suggested by Ginetzinsky in 1958 that the material found between these cells in the rat constituted the barrier between fluid in the collecting tubule lumen and the interstitium. He postulated that antidiuretic hormone released hyaluronidase from the apex of the collecting tubule cell and that the enzyme then digested the intercellular cement permitting tubular fluid to equilibrate osmotically with the interstitium. The material between collecting tubule cells the guinea pig was extracted and information about its nature was obtained from its behaviour in electrophoresis and from its staining characteristics. It was found to be a very soluble acid mucopolysaccharide which contained sulphate and PAS positive groups, both of which features distinguish it from hyaluronic acid. The histological technique had thus to be modified to preserve as much as possible of the material in the thin (3andnbsp;andmu;) wax sections required for the optimal demonstration of this region in the light microscope. The fixative developed to give the most satisfactory retention of acid mucopolysaccharide at the same time as preserving cytological structure reasonably well was as follows: Formol (40% CHO) 100 ml Cellosolve 225 ml Distilled water 45 ml Aminoacridine hydrochloride 1.6 gm. After fixation the tissue blocks were passed directly into pure cellosolve: Rinsing in aqueous solutions was avoided. Aminoacridine hydrochloride is soluble in cellosolve hence excess precipitant was removed during dehydration without running the risk that acid mucopolysaccharide might be leached out. The blocks were cleared in the usual way and then embedded in ester wax which was found to be superior to paraffin wax for a number of reasons discussed in Chapter 4. Radioactive sulphate was used to trace the synthesis of acid mucopolysaccharide by the collecting tubule cells. After injection of the label, radioactivity was first seen in sections to be associated with PAS positive, amylase resistant granules in the Golgi regions of the collecting tubule cells. After longer intervals radioactivity became associated with granules near the apical and lateral borders of the cells. The Golgi region as demonstrated morphologically (Chapter 3) was always seen to be between the nucleus and apical or lateral borders of the cell and never between the nucleus and the base. Similarly, material which had incorporated radioactive sulphate was never seen to pass across the base of a collecting tubule cell. The reasons for associating the development of an extensive Golgi region with the synthesis of acid mucopolysaccharide by these cells is discussed in connection with the results published by others working with embryonically similar gut cells. It proved possible by autoradiography to identify positively the material in the biochemical extract that had come from the collecting tubule cells. Autoradiography of tissue sections one hour after injection of the label showed that radioactivity was limited to the collecting tubule cells: Similarly only one band in the electrophoretic separation of acid mucopolysaccharides extracted from the papilla after this interval contained radioactivity. By noting how this band stained before and after the action of enzymes upon the material within it, it was concluded that the material associated with collecting tubule cells had the following features: It contained protein which could not be digested by papain. It contained a limited amount of 'neutral' polysaccharide which could be stained by the PAS routine. It could not be digested by amylase. It stained as strongly at pH 1 with Alcian Blue as it did at pH 3.5: This indicated that its anionic groups were sulphate and that carboxyl groups placed little part in the staining. It incorporated radioactive sulphate. The acid mucopolysaccharide component was partially degraded by commercial bovine testicular hyaluronidase. In Chapter 5 the changes in the morphology of acid mucopolysaccharides in the interstitium and between collecting tubule cells that arise when guinea pigs are dehydrated, injected with ADH or hyaluronidase, or given a water load are recorded. In animals given a water load the structural fibrils of the papillary interstitium become widely separated and, at the same time, the apparent space between tubules is increased. However, morphometry showed that the actual volume of the interstitium did not alter significantly whether the animal was water laden, deprived of water or injected with ADH. The significance of this observation is discussed in view of the contrary reports concerning changes in interstitial volumes: In the present study it was concluded that hydration of the acid mucopolysaccharide gel trapped within the fibrils accounted for their expanded appearance in diuresis. In antidiuresis papillary tubules were closely packed and a number of thin walled vessels, which were not normally observed by light microscopy appeared to be pulled open. This observation could be explained by contraction of interstitial cells under the influence of ADH, or by an increased force exerted on certain fibrils in the interstitium as a result of local changes in the physical propertied of acid mucopolysaccharides. No evidence could be found in this study to support active contraction of the interstitial cells. A model (Fig. 6/3) is presented to explain how a system of fibrils ad hyaluronic acid could enable water to be removed from the papilla in antidiuresis without a reduction in the tonicity of the papilla as a whole. The difference in volume of collecting tubule cells in hydration and dehydration was measured. In both cases the basal width of the cells averaged 9.4andnbsp;andmu; under the conditions of measurement. The average length of the cells in hydration was 13andnbsp;andmu; falling to 10andnbsp;andmu; on ADH infection or water deprivation. The change from columnar to cuboidal shape decreased the cell volume by 23%. Autoradiographic evidence was obtained which showed that the acid mucopolysaccharide associated with the apex of the collecting tubule cell was depolymerized in antidiuresis. The removal of a layer of acid mucopolysaccharide from this site could account for most of the volume change noted 1n sections not stained for acid mucopolysaccharides. It was possible that there was also some shrinkage of the cytoplasmic volume by osmosis in antidiuresis but the evidence for this was equivocal. There was nothing in the results from this study to support the hypothesis that hyaluronidase was released by apocrine secretion from the collecting tubule cells. 'Granules' of a size which could just be resolved optically (ie. of andfrac14; to andfrac12;andnbsp;andmu; in dia.) could be seen between collecting tubule cells (fig. 4/6). These 'granules' contained anionic groups which stained in the same way as the material at the apex of the cells. The resolution that is possible with light microscopical autoradiography was insufficient to prove that these particles incorporated sulphate in parallel with the apical material. The arrangement of the Golgi region in these cells (fig. 3/12) suggested that secretion took place into the lateral intercellular spaces as well as across the apex. However, granules were never seen between the cells in the electron microscope, and it is suggested that the granular appearance of this region in the light microscope arises from the plane of section of the cellular interdigitations. The amount of acid mucopolysaccharide between collecting tubule cells wan reduced when guinea pigs were deprived of water or given ADH. Removal of apical material with preservation of that between cells, or vice versa, was never observed. This result is discussed in Chapter 6, and a model is put forward to explain how such a system could regulate the flow of water across the collecting tubule wall. The evidence arising from studies by other workers which suggests a role for a hyaluronic acid/hyaluronidase system in the physiology of the kidney is reviewed in Chapter 6: This, and the criticism of the hypothesis of Ginetzinsky (1958) is discussed in the light of findings in the present study. It is concluded that renal acid mucopolysaccharides are involved in maintaining the constancy of the 'milieu interior' of the papilla, which determines that of the blood and ultimately of the whole body. It is thought that an understanding of the actual mechanisms by which they do this has probably been delayed by the modern emphasis on cellular activity with the result that the functional significance of connective tissue has tended to be overlooked.