Cyclosporine enhances the expression of TGF-beta in the juxtaglomerular cells of the rat kidney.

The mediators of cyclosporine (CsA) nephrotoxicity remain ill defined. In this study, we describe evidence of increased amounts of transforming growth factor-beta (TGF-beta) in the kidneys of adult male Wistar rats treated with CsA (5 to 25 mg/kg/day) for four weeks. Localization of TGF-beta was undertaken immunocytochemically at both light and electron microscope levels and Northern blot analysis was applied to detect changes in transcription of TGF-beta. In control rats, weak to moderate immunostaining for TGF-beta was observed, in the juxtaglomerular arterioles. CsA treatment resulted in a dose-dependent increase in the number of stained afferent and interlobular arterioles and in the intensity of staining. The number of stained afferent arterioles increased from a control value of 0.21 +/- 0.08/mm2 cortex to 0.84 +/- 0.15/mm2 cortex, P < 0.01, and to 1.12 +/- 0.10/mm2 cortex, P < 0.01, in rats treated with CsA 12.5 mg/kg/day and 25 mg/kg/day, respectively. The number of interlobular arterioles stained for TGF-beta increased from a control value of 0.07 +/- 0.05/mm2 to 0.31 +/- 0.02/mm2, P < 0.05, and 0.39 +/- 0.07/mm2, P < 0.01, in rats treated with CsA, 12.5 mg/kg/day and 25 mg/kg/day, respectively. At the electron microscope level, TGF-beta was localized exclusively within the granular cells of the juxtaglomerular arterioles. Northern blot analysis suggested that this enhanced staining is due to increased transcription of TGF-beta 1. We have therefore observed an association between TGF-beta and CsA-induced nephrotoxicity. While this does not establish a causal link, it leads us to postulate that TGF-beta, alone or in combination with other growth factors, may play a role in the pathogenesis of CsA induced nephrotoxicity.

Localization of PDGF-BB in the juxtaglomerular cells of cyclosporin-treated rats.

We have examined the ultrastructural localization of immunoreactive platelet-derived growth factor (PDGF) in Wistar rats injected with cyclosporin A (CyA, 12.5 mg/kg/day) for 4 weeks. CyA injections resulted in a significant increase in serum creatinine (47 +/- 3 vs. 35 +/- 2 mumol/l, p < 0.05) and a reduction in creatinine clearance (0.29 +/- 0.07 vs. 0.53 +/- 0.04 ml/min/100 g b.w., p < 0.01). CyA-treated rats displayed marked hypertrophy and hypergranularity of the juxtaglomerular cells. Morphometric studies showed a significant increase in the number (2.8 +/- 0.3 vs. 1.8 +/- 0.3, p < 0.05) and a 60% increase in the volume of secretion granules within these cells. PDGF was detected exclusively within the secretion granules of the juxtaglomerular cells of the afferent arterioles of CyA-treated animals. We conclude that CyA therapy or its induced haemodynamic alterations result in increased accumulation of PDGF-BB within the secretion granules of the juxtaglomerular cells.

Biphasic expression of IGF-I following subtotal renal ablation.

Insulin-like growth factor-I (IGF-I) has been implicated in the pathogenesis of experimental renal growth. This study was designed to investigate the quantitative and qualitative changes in renal IGF-I which occur during the course of progressive renal scarring in rats submitted to extensive renal ablation. Analyses were carried out at 7, 15, 21, 30, 90 and 150 days after subtotal nephrectomy. Compensatory renal growth occurred over the first 30 days, amounting to a 161% increase in the protein content per remnant kidney (272 +/- 22 vs. 104 +/- 3 mg at the outset, p < 0.001, means +/- SD, n = 6) but DNA per remnant kidney continued to rise, reaching a 285% increase by day 90 (5.12 +/- 0.06 vs. 1.33 +/- 0.06 mg at the outset, p < 0.001), probably reflecting the continued recruitment of inflammatory cells. Changes in extractable renal IGF-I (eIGF-I) were measured by radioimmunoassay. Seven days after subtotal nephrectomy eIGF-I had increased by 70% compared with controls (2.37 +/- 0.22 ng/mg protein vs. 1.39 +/- 0.2 ng/mg at the outset, p < 0.001) but had returned to normal by 30 days. This coincided with a transient increase in the immunostainable IGF-I (iIGF-I) within the cortical collecting ducts which, again, had subsided by 30 days. However, at 90 and 150 days, as severe scarring was detected, a different pattern of immunostaining (iIGF-I) was noted. Strong staining was evident in the injured cells of the distal tubules and within the fibrosed interstitium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of growth hormone on intestinal morphology of genetically dwarf rats.

Three groups of Lewis rat were studied: dwarf rats, genetically deficient in growth hormone; rehabilitated dwarf rats treated with exogenous growth hormone (GH); and normal wild-type rats. The small intestine of each animal was removed and simple random transverse sections were taken from the proximal and distal regions. The profile areas of villi, crypt and muscle were estimated by point count analysis and combined with intestinal length measurements to obtain absolute volumes. Villus and primary mucosal surface areas were estimated from intersection counts and linear measurements were made of epithelial cell height. Distally, villous volume and surface area were reduced by 42% and 39%, respectively, in the dwarfs compared with controls. These features were significantly smaller (P < 0.01) in dwarfs distally than proximally. Crypt volume and epithelial cell height were decreased equally in both proximal and distal regions of the intestine of dwarf rats. Following GH administration both features increased, crypt volume overshooting control values. These results indicate that GH deficiency has a subtle effect on intestinal morphology and that the intestine is more sensitive distally than proximally. Reconstitution with GH is capable of reversing many of these changes.

Increased platelet-derived growth factor in the kidneys of cyclosporin-treated rats.

We have examined the histological changes and the distribution of immunoreactive platelet-derived growth factor (PDGF) in the kidneys of Sprague-Dawley rats injected with cyclosporin A (CsA, 25 mg/kg/day). Control rats were injected with the olive oil vehicle alone. Groups of rats were killed after 1, 2, 3, 4 weeks of injection and 8 weeks after a 4 week period of injection. Additional controls included groups of rats injected with different doses of CsA and a group of rats subjected to chronic renal ischemia by partial of the aorta. CsA-treated rats gained weight more slowly than olive oil-treated controls (45%, P < 0.05). In rats injected with CsA, a significant increase in serum creatinine concentration (64 +/- 2 mumol/liter vs. 39 +/- 1 mumol/liter, P < 0.01) and a reduction in creatinine clearance rates (0.23 +/- 0.07 ml/min/100 g body wt vs. 0.43 +/- 0.07 ml/min/100 g body wt, P < 0.01) occurred after 3 weeks. After 1 week of CsA treatment, segments of the walls of some afferent and intralobular arterioles were thickened and stained strongly by the periodic acid Schiff (PAS) procedure. Immunostainable PDGF-BB and PDGF-AB were detected within short segments of the afferent and intralobular arterioles of the kidneys of CsA-treated rats and in the kidneys subjected to renal ischemia but not in tissue from other control animals. No PAS staining or immunostaining was evident in CsA treated kidneys eight weeks after the discontinuation of treatment. We conclude that CsA-induced ischemia results in increased accumulation of PDGF in the walls of renal arterioles.

Experimental diabetic renal growth: role of growth hormone and insulin-like growth factor-I.

We have compared the kidneys of two inbred strains of rats (Lewis and Lewis-Dwarf) 7 days after the induction of diabetes mellitus with streptozotocin, in order to examine the influence of a selective growth hormone (GH) deficiency on diabetic renal growth and insulin-like growth factor-I (IGF-I) content of the kidneys. Insulin-like growth factor-I (IGF-I) content of the kidneys. Insulin-like growth factor-I was measured by radioimmunoassay and its distribution within the kidney by immunohistochemical staining. We detected a significant increase in both the wet weight (32.9 +/- 5.3%, P = 0.0085) and dry weight (16.3 +/- 6.3%, P = 0.046) of the kidneys of diabetic Lewis rats but dwarf rats, selectively deficient in GH, did not show a significant increase in either parameter. Extractable IGF-I increased within the kidneys of diabetic rats of both strains but to a lesser extent in the dwarf rats (+105 +/- 28% and +65 +/- 21% respectively, P < 0.01). In diabetic Lewis rats a positive correlation was noted between the severity of glycaemia and kidney IGF-I content (r = 0.604, P < 0.05) but no such correlation was noted in dwarf rats. Inulin-like growth factor-I immunostaining increased in diabetic rats of both strains, mainly within cells of the thick ascending limb of the loop of Henle including damaged and vacuolated cells. However, morphometric analysis of the staining showed that it was significantly less widespread in the diabetic dwarf rats (P = 0.026).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I and experimental diabetic kidney disease.

Insulin-like growth factor-I (IGF-I) has been implicated in the pathogenesis of experimental diabetic renal growth. In this study, we have examined the serial changes of renal IGF-I, by radioimmunoassay (RIA) and immunohistochemistry, in rats made diabetic by a single intravenous injection of streptozotocin (STZ; 55 mg/kg). The kidney IGF-I content, as determined by RIA, increased within 48 h of the induction of diabetes mellitus, peaked on day 4 and returned to normal levels by day 30. Renal IGF-I correlated positively with the severity of hyperglycaemia on day 7 (r = 0.685, p < 0.001). Immunostaining showed IGF-I to be located within the cortical collecting ducts of normal rats. In diabetic rats, IGF-I also appeared within the cells of the thick ascending limbs of the loops of Henle, in particular those undergoing cytoplasmic vacuolation. The changes in immunoreactive IGF-I assessed stereologically were consistent with the amounts of extractable IGF-I. They were not observed in normoglycaemic, STZ-injected, rats treated with high-dose insulin. This study suggests that IGF-I is involved in diabetic tubular injury and growth.

Effect of diatrizoate on the function of the isolated perfused rat kidney.

The mechanism of the nephrotoxicity of water-soluble contrast media (WSCM) remains ill defined. We have studied the effect of diatrizoate on the isolated perfused rat kidney (IPRK). Emphasis was on the effect of low- and high-dose diatrizoate on glomerular filtration rate (GFR), renal perfusate flow (RPF), fractional excretion of albumin (FE Alb) and fractional reabsorption of sodium (FR Na). The addition of diatrizoate to the IPRK led to a dose-dependent biphasic change in RPF and GFR characterized by an initial transient increase followed by a marked and sustained decrease. Diatrizoate induced a diuresis and a parallel increase in urinary sodium excretion (fall of FR Na). Fe Alb was also increased in kidneys exposed to diatrizoate. Electron microscopy of a control kidney showed preservation of cellular architecture, which contrasted with the observed cytoplasmic vacuolation of proximal tubular cells after perfusion with diatrizoate. This study confirms a direct effect of WSCM on the function of the IPRK. In this experimental model, diatrizoate reproduces the effects observed in vivo on GFR and renal perfusion.

Electron microscopic immunocytochemical localization of proline-rich proteins in normal mouse parotid salivary glands.

Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with alpha-amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.

Peritoneal ultrafiltration after chronic exposure to dialysis fluid.

Eleven rats were given twice-daily intraperitoneal injections of 20 mL of dialysis fluid containing 4.5% glucose for 6 weeks. The peritoneal ultrafiltration capacity of this group was compared with that of a control group of 10 rats that had received no injections by measuring the volume and glucose concentration of the dialysate remaining in the peritoneal cavity 2 hours after injection. Animals that had received injections of dialysis fluid showed significant loss of peritoneal ultrafiltration: volume of dialysate remaining in the control group was 31 (13-35) mL, and in the experimental group was 25 (11-45) mL, with p less than 0.02 (Mann-Whitney). This was associated with enhanced glucose absorption: glucose absorbed by the control group was 382 (312-706) mg, and 595 (435-738) mg in the experimental group (p less than 0.002, Mann-Whitney).

Role of growth hormone in the development of experimental renal scarring.

Recent experimental data has implicated growth hormone in the development of glomerular sclerosis. In this study, we have examined the development and progression of glomerular and tubulointerstitial scarring in Wistar and Dwarf rats, selectively growth hormone-deficient, following subtotal nephrectomy. Wistar rats showed progressive proteinuria, hypertension and renal failure as well as severe renal scarring 120 days after subtotal nephrectomy. In contrast, growth hormone-deficient Dwarf rats had minimal proteinuria, mild renal functional impairment and moderate renal histological scarring. The difference in these functional and structural parameters between the two strains is highly significant, although both experimental groups had comparable food consumption and systemic blood pressure. The significantly smaller glomeruli and limited kidney hypertrophy over 120 days observed in Dwarf rats may account for some of the protection against glomerular sclerosis and tubulointerstitial scarring observed in that strain.

Beta-adrenergic regulation of beta-actin mRNA abundance in mouse parotid glands by a post-transcriptional mechanism.

In the first 24 h after a single injection of the beta-adrenergic agonist isoprenaline to mice, the level of beta-actin mRNA in the parotid glands increased significantly above that observed in untreated mice. The increase was transient, reaching 11 times the normal level 18 h after treatment and declining thereafter. Repeated daily doses of isoprenaline did not result in any further increase in beta-actin mRNA. Nuclear transcription experiments showed that there was no increase in the transcription rate of the beta-actin gene 8 h after an injection of isoprenaline, although beta-actin mRNA levels were increasing at this time. Immunoblotting revealed an increase in beta-actin protein in parotid gland samples after isoprenaline treatment, although the increase was not to the same extent as the mRNA, perhaps indicating that degradation of beta-actin had also increased. Using immunocytochemistry it was found that beta-actin was located mainly in the apical cortex of the normal acinar cell. There was a significant decrease in cortical beta-actin 24 h after isoprenaline treatment, suggesting that the beta-actin was under the process of redistribution. From these data we propose that isoprenaline caused an increase in beta-actin synthesis by a posttranscriptional mechanism and a redistribution of beta-actin in preparation for the well-known subsequent change in morphology and function of the parotid glands.

Mesothelial hyperplasia in response to peritoneal dialysis fluid: a morphometric study in the rat.

Five rats were given twice daily intraperitoneal injections of hypertonic dialysis fluid for 6 weeks. The structure of the hepatic peritoneum of this group was compared with that of a control group by applying morphometric techniques to transmission electron micrographs. The experimental group showed marked mesothelial hyperplasia with doubling of the number of cells and a significant increase in the length of intercellular junction per unit area of peritoneum. Since cell volumes in the two groups were similar, the increase in cell density in the experimental animals was the result of the cells assuming a more cuboidal shape. Experimental animals also showed an increase in the number of microvilli, pinocytotic vesicles and rough endoplasmic reticulum per unit area of peritoneum. Chronic exposure to dialysis fluid has profound effects on the number, shape and composition of peritoneal mesothelial cells in the rat.

Morphometric studies on the development and sexual dimorphism of the submandibular gland of the mouse.

A light microscopic morphometric analysis of the development of the mouse submandibular gland has been carried out from birth up to the age of 6 weeks. At birth the bulk of the gland consists of approximately equal volume proportions of acinar, terminal tubule and non-secretory cells. The granular convoluted tubule is absent at birth. The neonatal female gland resembles that of the male in many respects. With the regression of the terminal tubule at 2 weeks of age the duct system of the gland is seen to differentiate into excretory, striated and intercalated ducts. The volume proportions of the gland constituents of the female are similar to those of the male at 2 weeks. At this age, the acini occupy 55%, the striated duct 20% and the intercalated duct 15% of the total gland volume. Sexual dimorphism is clearly evident in the gland at 4 weeks of age when the duct system is seen to differentiate to form its granular convoluted tubule component. The granular tubule occupied 19% of the gland volume in the male but only 8% in the female at 4 weeks. The proportions of acini are only 41% in the total gland volume of the male mouse but 62% in the female at 4 weeks. In the male gland the proportions of granular convoluted tubule increase from 13% to 21% between 4 and 6 weeks and the secretory granule content of these cells from 6% to 24%. At 6 weeks of age the volume proportion of granular convoluted tubule in the male is 45% and that in the female is only 12%. At this age the acini occupy a proportion of 30% in the male gland as opposed to 57% in the female gland. At 6 weeks the volume of granular convoluted tubule cells is 40% lower in the female (1842 microns 3) than in the male gland (2995 microns 3).

The ultrastructure of human abdominal mesothelium.

Fresh specimens of human peritoneum collected from heart-beating cadaver organ donors have been examined by transmission electron microscopy. Samples were taken from the anterior abdominal wall and from the surfaces of the liver, stomach and diaphragm. The mesothelium consisted of a single layer of flattened cells generally 2.5 microns to 3 microns thick. These were joined by tight junctions and desmosomes to form a continuous sheet. The cells rested on a prominent basement membrane deep to which was a layer of fibrous connective tissue. This layer was more compact under the mesothelium from the abdominal wall and liver than elsewhere. Long microvilli projected from apical surface of the cells. In many cases these covered the entire surface but sometimes they were more profuse at the edges of the cells near the intercellular junctions. The cells possessed a well-developed cytoskeleton of intermediate filaments which coursed through the cytoplasm in thick bundles. The cells also had a well-developed rough endoplasmic reticulum and Golgi apparatus. Numerous smooth-surfaced and coated vesicles could be seen adjacent to the plasmalemma at all surfaces, providing evidence of considerable pinocytotic activity. There was little regional variation in the structure of the mesothelium. We found no evidence of pores passing through the layer although, on the liver, cisternae were present between the cells and these were often occupied by lymphocytes.

Changes in the size and number of secretion granules in the rat exocrine pancreas induced by feeding or stimulation in vitro. A morphometric study.

The size, number and volume per cell of secretion granules in rat exocrine pancreas have been measured using stereological techniques. The changes which occur as a result of feeding starved animals (90 min) or stimulating lobular fragments in vitro with carbachol are documented. In fasted animals mean acinar cell volume was estimated as 1670 micron 3 and the cells contained an average of around 450 secretion granules with a corrected mean diameter of 0.70 micron. They occupied around 7% of cell volume. After feeding mean cell volume was about 1300 micron 3 and the cells contained an average of about 190 granules per cell with a mean diameter of 0.58 micron. They occupied 3% of cell volume. A shift in the size frequency distribution of granule diameters occurred as a result of feeding. In vitro experiments in which lobules were induced to secrete with carbachol (10 microM, 3 h, 37 degrees C) had a similar effect. Mean cell volume was reduced from around 1760 micron 3 to 1360 micron 3, mean granule number from around 420 per cell to 180 per cell and the volume density of granules was reduced from about 8% to 3% of cell volume. There was no significant change in mean granule diameter or shift in the size-frequency distribution of granule diameters. Incubation of tissues with cycloheximide (1 mM, 3 h, 37 degrees C) did not prevent secretion by carbachol but it prevented replacement of granules. As a consequence, depletion by carbachol was greater in the presence of cycloheximide, the granules being reduced to around 110 per cell and to only 2.5% of cell volume. We conclude that feeding causes a preferential loss of larger granules and that during secretion replacement of granules occurs. Some of these granules are smaller than those evident in the glands of starved animals.

Effect of glucose on insulin biosynthesis and beta cell ultrastructure in cultured fetal rat pancreas.

Pancreatic rudiments from 14-day fetal rats were cultured whole for 8 days in medium containing 5.5 or 16.5 mmol glucose/l (1G or 3G medium). Rudiments grown in 3G medium (3G cells) contained more DNA and insulin than those grown in 1G medium (1G cells) but there was no alteration in the insulin/DNA ratio or the fractional area of the rudiment occupied by insulin-containing cells. Morphometric analysis of ultrastructure revealed that the beta cells grown in 3G medium were smaller and had smaller nuclei than those grown in 1G medium. The size of exocrine cell nuclei in 1G or 3G medium was similar. Insulin granules occupied a greater proportion of the cytoplasmic volume in rudiments grown in 3G medium although the mean absolute volume of insulin granules per cell grown in 1G and 3G media was similar. Hence the residual cytoplasmic volume (cell--nucleus and granules) of 3G cells was less than that of 1G cells. Insulin granules from 3G cells had smaller granule sacs and cores than those from 1G cells. It is concluded that glucose stimulates the growth of rat fetal pancreas in vitro and has important effects on beta cell ultrastructure.

Exocrine glands and protein secretion: a stereological viewpoint.

The exocrine pancreas and the parotid salivary gland have been widely used as models for studying the synthesis, intracellular transport and discharge of exportable proteins. This article briefly reviews quantitative morphological (stereological) studies which have been made of these glands and assesses their contribution to our understanding of the secretory process. A general stereological profile of these glands is presented and the way in which their morphology changes during development is outlined. Detailed consideration is given to the granule population of the cells, particularly the way in which granules are formed and discharged as a result of secretory stimuli. The membrane content of secretory cells and membrane dynamics during the secretory cycle are also examined. Throughout, the emphasis is placed on the interpretation of stereological data rather than on the methods themselves.

Membrane dynamics in the parotid acinar cell during regranulation: a stereological study following isoprenaline-induced secretion.

Recently weaned male rabbits were injected either with 150 micrograms/kg isoprenaline in saline containing 0.01 M ascorbic acid or simply with the drug vehicle. Groups of drug-injected animals were killed at various time after injection. Parotid gland tissue samples from all animals were fixed, embedded and thin sectioned, and micrographs were prepared at standard magnification. Estimations of membrane areas of each membrane type in parotid acinar cells were made. It was found that in animals killed 2 hours after induced secretion apical area was larger than in controls. In animals killed at successively later times the apical area was progressively less. No elevation of any internal smooth membrane areas was ascertained at any sampling time, though the areas of rough endoplasmic reticulum in 2-12 hour samples were larger. It is suggested that excess apical membrane, though probably removed by interiorization, is afterwards disassembled in side the cell to create fresh macromolecular building units (protein molecules), perhaps after passing through the Golgi apparatus. This cryptic pool of building units can provide about 900 micrometers2 of secretion granule membrane per cell, the supply apparently being exhausted in the first eight hours after degranulation, whilst granule numbers are being increased. Thereafter, apparently, limited granule fusion occurs, so that ultimately the cellular complement of secretion granule membrane comes to enclose a greater volume of secretory product, though the average granule number per cell is small.