Effect of Onpi-to (TJ-8117) on TGF-beta 1 in rats with 5/6 nephrectomized chronic renal failure

We and others have already reported that Onpi-to (TJ-8117), a Kampo medicine composed mainly of Rhei Rhizoma, has a beneficial effect on an adenine-induced renal failure model and 5/6 nephrectomized renal failure. However, little is known about the detailed mechanism of this medicine when used for renal failure. The present study was designed to clarify whether or not TJ-8117 affects TGF-beta 1 production or activation in glomeruli of 5/6 nephrectomized rats. TJ-8117 (400 mg/kg/ day) and captopril (50 mg/kg/day) were administered as drinking water from the day immediately after the operation and continued throughout the experiment. All rats were sacrificed at 4 weeks and renal cortical tissue was removed to quantify the protein and activity of TGF-beta 1 and activities of metalloproteinase. TIMP expression and extracellular matrix (collagen type I, IV) in the glomeruli were analysed histologically. TJ-8117 inhibited proteinuria, and the accumulation of collagen type I and IV in glomeruli of nephrectomized rats. In addition, TJ-8117 inhibited the TGF-beta 1 positive area in the glomeruli and the elevation of mature TGF-beta 1 level in the renal cortex of nephrectomized rats. In the TJ-8117 treated group, activities of metalloproteinase 1, 2 or 9 in the renal cortex were elevated compared with the control group. Captopril failed to affect the TGF-beta 1 level. We also found that the constitutive herbs in TJ-8117, Rhei Rhizoma and Ginseng radix, inhibited the process from inactive TGF-beta 1 to mature TGF-beta 1.     

Analysis of hepatocyte distribution and survival in vascular beds with cells marked by 99mTC or endogenous dipeptidyl peptidase IV activity

The present study was designed to clarify the effects of TJ-8117 on apoptosis in the glomeruli of 5/6 nephrectomized rats. TJ-8117 (400 mg/kg/day), captopril (50 mg/kg/day) and nicardipine (50 mg/kg/day) were administered as drinking water from the 56th day after renal ablation, and continued throughout the experiment. All rats were sacrificed at 13 weeks and renal tissues were removed to quantify histopathological and apoptotic parameters in glomeruli. TJ-8117 inhibited proteinuria, matrix index and decrease in the number of total cells in glomeruli of nephrectomized rats. In addition, the increase in apoptotic body and DNA fragmentation was significantly inhibited in the TJ-8117-treated group. Captopril and nicardipine failed to inhibit both parameters. In 400 mg/kg of the TJ-8117 treated group, the number of Bcl -2 positive cells in the glomeruli was elevated compared with the 5/6 nephrectomized control group. In addition, the number of Bax positive cells in the TJ-8117 group was significantly suppressed in a dose-dependent manner. These results suggest that TJ-8117 may inhibit apoptosis in the late stage of this model by suppressing matrix accumulation and progression of glomerulosclerosis. The apoptosis-preventing effect may be mediated by decreased expression of Bax in the glomerular cells.     

Sequential alterations in clinical biochemical indicators of renal function in 5/6 nephrectomized rats--basic study for renal toxicity using 5/6 nephrectomized rats

It has been established that progression of renal lesions in 5/6 nephrectomized rats, in which the total right kidney was removed and two poles of the left kidney were excised surgically, and are used as an animal model of renal failure in man, can be morphologically divided into three stages. In the present study, for establishing a renal toxicity study using this animal model on the physiological side, changes of biochemical parameters were sequentially investigated in 20 male 5/6 nephrectomized Wistar rats until 26 weeks after nephrectomy. Creatinine clearance (CLcre) and water as well as electrolyte reabsorption (FRwater and FR Na, K, Cl) were reduced at weeks 2-4, then increased slightly from weeks 6 to 10, and reduced again thereafter. On the contrary, urinary protein was elevated throughout the experimental period, while albumin fraction was increased after week 2 and low-molecular tubular protein increased after week 6 by electrophoresis. Urinary LDH also demonstrated high levels throughout the observation period, but ALP only increased after week 18. The present study thus confirmed that renal function after 5/6 nephrectomy is indeed changed in three stages with clinical biochemical parameters, especially CLcre and FR Na, K, Cl being good indicators to distinguish the three stages of glomerular and tubular dysfunction, respectively. In addition, urinary protein-fractions by electrophoresis in this animal model were examined for the first time, proving useful approach to glomerular and tubular dysfunction.     

Zinc therapy in acrodermatitis enteropathica

Albino Holtzman, albino Wistar and hooded HS rats were injected fortnightly for 14 weeks with human glomerular basement membrane (GBM) emulsified in Freund's complete adjuvant. Half of the rats were pretreated with Freund's complete adjuvant and some were unilaterally nephrectomized. Anti-GBM antibody glomerulonephritis, characterized by proteinuria (greater than 100 mg/16 h) and a diffuse linear deposition of host immunoglobulin along the glomerular basement membrane, was first detected in Holtzman rats 4 weeks after treatment with GBM had begun, and had developed in 69% of these rats by 15 weeks. In contrast, none of the similarly treated Wistar or HS rats became proteinuric at any time, although a few showed weak glomerular fluorescence at the end of the experiment. Thus Holtzman rats are susceptible, and HS and Wistar rats are resistant to experimental anti-GBM antibody glomerulonephritis. Pretreatment with Freund's complete adjuvant apparently shortened the induction period of the experimental disease in the Holtzman rats whereas unilateral nephrectomy appeared to decrease their susceptibility to it.     

Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats

The 5/6 nephrectomy model is used to study pathogenetic mechanisms underlying chronic renal failure. We previously demonstrated that increased mesangial cell proliferation and glomerular PDGF B-chain expression precede glomerulosclerosis in this model. In the present study we have assessed the concomitant changes in the cortical tubulointerstitium. A wave of tubular and interstitial cell proliferation (as determined by immunostaining for PCNA) occurred at week 1 after 5/6 nephrectomy. This wave preceded the peak glomerular cell proliferation by one week. Tubulointerstitial cell proliferation decreased thereafter and reached control values by week 10. In situ hybridization and immunostaining for PDGF B-chain and beta-receptor in sham-operated controls showed labeling of distal tubules and collecting ducts, while no signal was present in the interstitium. PDGF B-chain mRNA and protein expression was markedly increased in tubules at weeks 2 and 4 after 5/6 nephrectomy and in the interstitium (particularly in areas of inflammatory infiltrates) at weeks 2 to 10. Similar changes occurred with PDGF receptor beta-subunit immunostaining. Interstitial expression of desmin and alpha-smooth muscle actin (markers of myofibroblasts) progressively increased after week 1. Interstitial influx of monocytes/macrophages with focal accentuation started at week 2. Counts of lymphocytes, neutrophils and platelets showed only minor changes. In parallel to the monocyte/macrophage influx, progressive interstitial accumulation of collagens I and IV, laminin, and fibronectin occurred. All of these changes were correlated with the increase in serum creatinine, proteinuria and an index of tubulointerstitial damage. We conclude that tubulointerstitial changes after 5/6 nephrectomy show similarities with those observed in the glomeruli. Tubular and interstitial overexpression of PDGF B-chain and its receptor may play a role in mediating fibroblast migration and/or proliferation in areas of tubulointerstitial injury.     

Sairei-to inhibits the production of endothelin-1 by nephritic glomeruli(2): alisols, possible candidates as active compounds

We have previously reported that Sairei-to (TJ-114), a Japanese herbal medicine, prevented the production of endothelin-1 in anti-GBM nephritic rats, and that Alismatis Rhizoma (Takusha in Japanese), one of the twelve herbs composing TJ-114, might be responsible for the action. In order to further clarify the antinephritic components of TJ-114, we investigated the effects of Takusha extracts on various parameters, including endothelin-1 production of glomeruli in vitro and in vivo using anti-GBM nephritic rats. MeOH-100% MeOH and MeOH-50% MeOH fractions (31.3 microgram/ml or higher) strongly inhibited an increase in endothelin-1 concentration in culture medium when they were added to a culture of glomerular cells derived from nephritic rats. In addition, oral administration of the MeOH-100% MeOH fraction (30 mg/kg) ameliorated the proteinuria, increase in systolic blood pressure and changes in histopathological parameters in nephritic rats. Oral administration of the MeOH-100% MeOH fraction inhibited increase in endothelin-1 expression in the glomeruli of nephritic rats and in endothelin-1 production by a culture of glomerular cells derived from the nephritic rats. Alisols A and B, the main constituents of the MeOH-100% MeOH fraction, inhibited in vitro endothelin-1 production by glomerular cells derived from the nephritic rats. Oral administration of alisol B (30 mg/kg) prevented the endothelin-1 expression by glomeruli and the increase in endothelin-1 production by cultured nephritic glomerular cells. Oral administration of alisol B also ameliorated the proteinuria, the increase in systolic blood pressure and the changes in histopathological parameters in the nephritic rats. These results indicate that the antinephritic action of TJ-114, resulting from the inhibition of endothelin-1 production, may be attributed to the alisols in Takusha.     

The influence of nephrectomy, ureteral ligation, and of estradiol on plasma renin substrate in unilaterally nephrectomized rats

The effects of three experimental conditions on the concentration of plasma renin substrate were studied with special reference to plasma renin concentration in unilaterally nephrectomized rats. After simultaneous bilateral nephrectomy the maximum increase in plasma renin substrate was 17 times higher than normal within 24 h, while in rats which were unilaterally nephrectomized 10 days previously, followed by the removal of the remaining kidney (two-step bilateral nephrectomy), the maximum increase in plasma renin substrate was markedly suppressed (6-fold of normal). The maximum increases in plasma renin substrate by estradiol treatment in normal and unilaterally nephrectomized rats were about the same, associated with similarly decreased plasma renin concentrations. The similar increase in plasma renin substrate was found after ureteral ligation in unilaterally nephrectomized rats and bilateral ligation of the ureters in normal rats. This was the case where the plasma renin concentrations changed differently after ureteral ligation. After two-step bilateral nephrectomy plus estradiol treatment the maximum increase in plasma renin substrate was found to be higher than that found after two-step bilateral nephrectomy, but was lower than that after simultaneous bilateral nephrectomy. It is suggested that under the pathological conditions that stimulate renin substrate production, the plasma rein substrate concentration is less affected by circulating renin.     

Effects of zinc deficiency on endogenous antioxidant enzymes and lipid peroxidation in glomerular cells of normal and five-sixths nephrectomized rats

We evaluated the effects of zinc deficiency on the activities of endogenous antioxidant enzymes and lipid peroxidation in rat glomerular cells (GCs). Male Sprague-Dawley rats (n = 48) were fed a zinc-deficient diet and deionized distilled water for 1 week to induce zinc deficiency. Half of the rats (zinc-deficient group) continued on this diet for 4 weeks, and the other half (zinc-replete group) were maintained on the same diet but with zinc-supplemented water (150 mg/Lzinc sulfate solution). Half of each group underwent five-sixths nephrectomy, while the other half underwent a sham operation. Another 12 normal rats (controls) were fed standard rat chow (containing 23.4% protein and 70 ppm zinc) and drank deionized distilled water. The zinc-deficient rats, including sham and five-sixths nephrectomized rats, showed severe growth retardation and poor appetite. Their mean plasma zinc concentrations were half that of normal control rats, but their plasma copper concentration was significantly higher than that of the control rats. Zinc supplementation corrected the abnormality of plasma zinc and copper concentrations and the loss of body weight in zinc-deficient rats. Zinc-deficient rats exhibited lower renal creatinine clearance and higher GC-malondialdehyde (GC-MDA) than zinc-replete rats. The remnant kidney of all five-sixths nephrectomized rats, including zinc-deficient and zinc-replete rats, showed a compensatory elevation in renal creatinine clearance and increased GC-MDA concentrations. Zinc concentrations in the renal cortex were decreased in zinc-deficient rats and the activities of GC-superoxide dismutase and GC-glutathione peroxidase were increased, while zinc-replete rats exhibited normal activities of GC-superoxide dismutase and GC-glutathione peroxidase. We suggest that zinc deficiency enhances the formation of reactive oxygen species but does not affect the activities of endogenous antioxidant enzymes in glomerular cells.     

Mechanisms by which Hange-shashin-to reduces prostaglandin E2 levels

To determine the mechanisms by which Hange-shashin-to (TJ-14) reduces prostaglandin E2 (PGE2) levels, the effects on blood corticosterone levels were examined in vivo and the effects on cyclooxygenase (COX) activity in vitro assessed. TJ-14, orally administered to rats at dose levels between 125 and 1000 mg/kg, caused a dose-dependent increase in blood corticosterone levels. We also showed that Glycyrrhizae Radix and Ginseng Radix, constituents of TJ-14, are involved in the increase in blood corticosterone. The activity of COX-1 was not inhibited by TJ-14 even at a dose of 1000 microg/ml, while COX-2 was inhibited at dose levels between 10 and 1000 microg/ml. The constituents Scutellariae Radix, Glycyrrhizae Radix and Coptidis Rhizoma were believed to be involved in COX-2 inhibition. These results suggest that the effect of TJ-14 in decreasing PGE2 is partially mediated by corticosterone and inhibition of COX-2.     

The effects of hange-shashin-to on gastric function in comparison with sho-saiko-to

The effects of "Hange-shashin-to (TJ-14)" on gastric function were examined in comparison with "Sho-saiko-to (TJ-9)". Oral treatment with TJ-14 (125-500 mg/kg) caused dose-dependent suppression of ethanol-induced gastric injury, while it did not suppress gastric lesions induced by water-immersion stress. TJ-9 (125-500 mg/kg, p.o.) suppressed both water-immersion stress-induced gastric lesions and ethanol-induced gastric injury in a dose-dependent manner. Intraduodenal administration of TJ-14 even at 500 mg/kg did not affect gastric juice secretion, while TJ-9 at 125 to 500 mg/kg dose-dependently suppressed gastric juice secretion. TJ-14 (125-500 mg/kg, p.o.) accelerated gastric emptying in normal rats and improved the delayed gastric emptying induced by BaCl2 in a dose-dependent manner, whereas such effect was not noted with TJ-9. Oral treatment with TJ-14 at 500 mg/kg significantly suppressed apomorphine-induced vomiting, but it did not affect copper sulfate-induced vomiting. These results suggest that TJ-14 exhibits an anti-ulcer action (probably based on its ability to protect the gastric mucosa), improvement of gastric emptying and an anti-emetic action. TJ-9 also showed anti-ulcer effects, probably based on its ability to suppress gastric secretion and to protect the gastric mucosa. Thus, the present study demonstrated the effectiveness of TJ-14 and TJ-9 against gastric disease, and provided basic data which explain the differences in clinical application between these two kampo medicines.     

Effect of 1,25 (OH)2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats

In the past, there has been considerable concern that treatment with active vitamin D might accelerate progression independent of hypercalcemia and hypercalcuria. Nevertheless, 1,25(OH)2D3 has known antiproliferative properties and has also been shown to inhibit renal growth. Since glomerular growth is a permissive factor for the development of glomerulosclerosis, we reasoned that 1,25(OH)2D3 might even attenuate progression. To test this working hypothesis we performed two experiments of 8 and 16 weeks duration, respectively, to compare subtotally nephrectomized (SNX) rats treated with ethanol and SNX treated with 1,25(OH)2D3. Control animals were sham operated and pair-fed with SNX animals. 1,25(OH)2D3 (3 ng/100 g body wt/day) was administered by osmotic minipump. 1,25(OH)2D3 had no significant effect on systolic blood pressure and only a transient effect on weight gain. SNX reduced the number of glomeruli (left kidney) from an average of 3.3 x 10(4) to 1.2 x 10(4) per kidney. Mean glomerular volume was 3.87 +/- 0.71 x 10(6) microns 3 in sham operated animals and significantly (P < 0.05) higher (10.1 +/- 1.75 x 10(6) microns 3) in untreated animals 16 weeks after SNX. Glomerular volume was significantly (P < 0.05) less in 1,25(OH)2D3 treated SNX [10.1 +/- 1.75 in ethanol vs. 7.04 +/- 1.78 in 1,25(OH)2D3 treated SNX]. In parallel, there was significantly (P < 0.01) less glomerulosclerosis [glomerulosclerosis index 1.16 +/- 0.14 in the ethanol treated SNX vs. 0.80 +/- 0.16 in SNX treated with 1,25(OH)2D3] in the eight week experiment. Albuminuria was significantly (P < 0.01) lower in 1,25(OH)2D3 treated than in ethanol treated SNX (mean 0.785 mg/24 hr, range 0.43 to 1.80, vs. 3.75 mg/24 hr, 1.29 to 14.2). The morphological data were directionally analogous in a second 16 week experiment. Only slight changes of the vascular sclerosis index and tubulointerstitial index were seen in SNX and were not affected by 1,25(OH)2D3 further. To prove that the effect of 1,25(OH)2D3 was independent of PTH, parathyreoidectomized SNX rats without or with 1,25(OH)2D3 treatment were examined seven days post-SNX. PCNA staining showed suppression of cell proliferation. Furthermore, in situ hybridization for transforming growth factor-B (TGF-beta) showed less vascular and tubular expression in 1,25(OH)2D3 treated rats. We conclude that 1,25(OH)2D3 has antiproliferative actions during the compensatory growth of nephrons in response to subtotal nephrectomy. These effects are independent of PTH. The data document that 1,25(OH)2D3 reduces renal cell proliferation and glomerular growth as well as glomerulosclerosis and albuminuria as indicators of progressive glomerular damage.     

An experimental model of the effect of uremia on cyclosporin A availability

BACKGROUND: The aim of the study was to determine whether or not uraemia has an effect on cyclosporine A intestinal resorption. METHODS AND RESULTS: Model experiments were conducted in rats to monitor the effect of acute uraemia (bilateral nephrectomy) on the kinetics of cyclosporine A. Using intragastric tube, Cyclosporine A was administered to one group of rats in the form of Consupren (Galena, Czech Republic) and to another group in the form of Sandimmune (Sandoz, Switzerland), at a dose of 10 mg/kg/24 h either case. Blood levels of cyclosporine A were determined using RIA and specific and non-specific antibodies (cyclosporine and its metabolites). Cyclosporine A kinetics in nephrectomized rats was compared with that in control rats and in rats undergoing sham nephrectomy. The blood levels of cyclosporine A were significantly lower, and the area under the curve (AUC) of blood cyclosporine A in nephrectomized rats significantly smaller than in control rats. No significant differences in the evaluated parameters after Consupren or Sandimmune were observed. CONCLUSIONS: Our findings support the hypothesis that uraemia decreases cyclosporine A availability. The results suggest that the changes in cyclosporine A kinetics in nephrectomized rats following Consupren and Sandimmune administration are of the same character.     

Inhibition of intercellular adhesion molecule-1 with antisense deoxynucleotides prolongs renal isograft survival in the rat

BACKGROUND: Delayed graft function from ischemia-reperfusion injury has a negative impact on long-term renal graft survival. We tested the utility of antisense oligodeoxynucleotide (ODN) against intercellular adhesion molecule-1 (ICAM-1) in the pretransplant treatment of renal isografts in improving long-term graft survival. METHODS: Three groups of 16 inbred Lewis rats each underwent unilateral nephrectomy and were then transplanted with a kidney from a Lewis donor rat, which had received antisense ODN, reverse sense ODN, or saline vehicle six hours prior to nephrectomy. The kidneys were subjected to one hour of warm ischemia and 30 minutes of cold ischemia, which when untreated results in delayed graft function. The remaining native kidney was removed 10 days later. Serum creatinine and urinary protein excretion were measured in surviving rats at weeks 2, 4, 6, 8, 12, 16, and 20 after native nephrectomy. RESULTS: A Kaplan-Meier analysis revealed that by week 6 one half of the animals receiving reverse sense ODN and saline vehicle treatment had died, while all but 2 rats in the antisense ODN-treatment group survived to 20 weeks. Serum creatinine concentrations and urine protein excretion of surviving reverse sense and saline vehicle-treated rats were significantly higher than antisense treated rats at every time point. Histology at week 20 revealed marked interstitial fibrosis, focal glomerular sclerosis, vascular intimal and medial thickening and tubular atrophy in reverse sense and saline vehicle-treated kidneys, while antisense ODN-treated kidneys showed only modest changes. Immunohistochemistry showed macrophage and lymphocyte infiltration, as well as substantial up-regulation of MHC class II, in reverse sense and saline vehicle-treated kidneys compared to antisense ODN-treated kidneys. CONCLUSIONS: These results suggest that by ameliorating acute nonimmunological renal isograft injury, the long-term chronic nonimmunologic processes are improved as well. Furthermore, the data suggest that an antisense ODN strategy directed against ICAM-1 may have utility in human kidney transplantation.     

Increased expression of intercellular adhesion molecule-1 (ICAM-1) in diabetic rat glomeruli: glomerular hyperfiltration is a potential mechanism of ICAM-1 upregulation

Mononuclear cells, including monocytes/macrophages and T-cells, are considered to be involved in the progression of diabetic nephropathy, although the mechanism of their recruitment into diabetic glomeruli is unclear. The intercellular adhesion molecule-1 (ICAM-1) promotes the infiltration of leukocytes into atherosclerotic lesions as well as inflammatory tissues. In the present study, we investigated the expression of ICAM-1 in the glomeruli of streptozotocin-induced diabetic rats. The expression of ICAM-1 was increased significantly during the early stage of diabetes. The number of mononuclear cells, primarily monocytes/macrophages and lymphocytes, was significantly increased in diabetic glomeruli. Mononuclear cell infiltration into diabetic glomeruli was prevented by anti-ICAM-1 monoclonal antibody. Insulin treatment decreased ICAM-1 expression and mononuclear cell infiltration. The ICAM-1 expression on cultured human umbilical vein endothelial cells was not induced under high glucose culture conditions. Glomerular hyperfiltration is a characteristic change in the early stage of diabetic nephropathy. Treatment with aldose reductase inhibitor, which prevented glomerular hyperfiltration without changes in blood glucose levels, decreased ICAM-1 expression and mononuclear cell infiltration. Moreover, we examined the ICAM-1 expression in the glomeruli of the 5/6 nephrectomized rat, which is a model for glomerular hyperfiltration without hyperglycemia. The ICAM-1 expression and infiltration of mononuclear cells was significantly increased in the glomeruli of 5/6 nephrectomized rats. We conclude that ICAM-1 is upregulated and promotes the recruitment of mononuclear cells in diabetic glomeruli. Moreover, glomerular hyperfiltration that occurs in the early stage of diabetic glomeruli may be one of the potential mechanisms of ICAM-1 upregulation in diabetic nephropathy.     

Candesartan cilexetil reduces chronic renal allograft injury in Fisher-->Lewis rats

OBJECTIVES: To determine the effects of the angiotensin II receptor antagonist, candesartan cilexetil, on glomerular and systemic blood pressures and the development of renal injury in Lewis rat recipients of a single Fisher kidney (F334--> LEW transplantation), an established rat model of chronic renal allograft failure. DESIGN: Recent studies have shown that chronic injury of renal allografts in F334-->LEW rats may be virtually abrogated by supplying the Lewis recipients with two Fisher kidneys or, alternatively, by retaining a native kidney. These findings imply a major contribution from processes associated with nephron loss to the pathogenesis of chronic renal allograft failure, a notion supported by the observation that transplanting two kidneys also normalizes glomerular capillary pressure (PGC) in F344-->LEW rats. Thus, a pharmacological reduction in PGC, by blocking the effects of angiotensin II, should also lessen renal injury in F344-->LEW rats. MATERIALS AND METHODS: Bilaterally nephrectomized F344--> LEW rats were treated with the angiotensin II receptor blocker candesartan cilexetil (TCV-116) at 40 mg/l or with vehicle, administered in drinking water. Proteinuria and systolic blood pressure were assessed monthly, and histological studies were carried out after 24 weeks. The glomerular filtration rate and glomerular pressures were determined after 10 weeks in additional rats by clearance and micropuncture studies. RESULTS: Treatment with candesartan cilexetil lowered systemic blood pressure, normalized PGC at 10 weeks and greatly reduced proteinuria and allograft glomerulosclerosis at 24 weeks. CONCLUSIONS: These data indicate that the development of renal injury in F344-->LEW renal allografts can be prevented by the pharmacological blockade of angiotensin II receptors using candesartan cilexetil. This suggests that angiotensin-dependent processes contribute significantly to chronic injury in this model of late renal allograft failure.     

Tissue-specific regulation of IRS-1 in unilaterally nephrectomized rats

Insulin stimulates the tyrosine kinase activity of its receptor, resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-1). IRS-1 is also a substrate for different peptides and growth factors, and a transgenic mouse "knockout" for this protein does not have normal growth. However, the role of IRS-1 in kidney hypertrophy and/or hyperplasia was not investigated. In the present study we investigated IRS-1 protein and tyrosine phosphorylation levels in the remnant kidney after unilateral nephrectomy (UNX) in 6-week-old male Wistar rats. After insulin stimulation the levels of insulin receptor and IRS-1 tyrosine phosphorylation were reduced to 79 +/- 5% (P < 0.005) and 58 +/- 6% (P < 0.0001), respectively, of the control (C) levels, in the remnant kidney. It is possible that a circulating factor and/or a local (paracrine) factor playing a role in kidney growth can influence the early steps of insulin action in parallel. To investigate the hypothesis of a circulating factor, we studied the early steps of insulin action in liver and muscle of unilateral nephrectomized rats. There was no change in pp185 tyrosine phosphorylation levels in liver (C 100 +/- 12% vs UNX 89 +/- 9%, NS) and muscle (C 100 +/- 22% vs UNX 91 +/- 17%, NS), and also there was no change in IRS-1 phosphorylation levels in both tissues. These data demonstrate that after unilateral nephrectomy there is a decrease in insulin-induced insulin receptor and IRS-1 tyrosine phosphorylation levels in kidney but not in liver and muscle. It will be of interest to investigate which factors, probably paracrine ones, regulate these early steps of insulin action in the contralateral kidney of unilaterally nephrectomized rats.     

Growth and nutrition of uremic piglets

Piglets aged 6 days were rendered uremic by subtotal nephrectomy and their growth and dietary intakes studied over the next 21 days. Eleven control piglets fed a voluntary intake of a sow's milk substitute (group A), 11 nephrectomized piglets fed a voluntary intake of the same feed (group B), 6 nephrectomized piglets tube fed the same milk (group C), and 11 nephrectomized piglets fed a voluntary intake of a low protein, isocaloric food (group D) were studied. After nephrectomy the piglets had an initial rapid rise in blood urea concentration which had fallen by day 7 and then leveled out around 13 mmol/liter in group B and 8 mmol/liter in group D. After operation control piglets (group A) ate more from day 4 and were larger from day 7 than the nephrectomized piglets (group B). Those piglets tube fed (group C) were of a similar size to the controls but all died between day 7 and day 11 with associated high blood urea concentrations. Piglets fed the low protein, isocaloric feed (group D) were smaller than both the controls and group B. They also ate less food than the controls and those nephrectomized piglets in group B which were on a voluntary intake of the normal feed.     

Severe experimental uraemia does not decrease the population of rat pituitary somatotrophs

BACKGROUND: Growth hormone (GH) secretion by the anterior pituitary has been shown to be depressed in severely uraemic rats. Changes in the population of pituitary somatotrophs might be partially responsible for this decrease. METHODS: To analyse the population of pituitary somatotrophs in severe uraemia, immunocytochemical detection and quantification of GH-producing cells were carried out on paraffin sections from young rats either 5/6 nephrectomized, sham-operated fed ad libitum or sham-operated pair-fed with the nephrectomized animals. Results: Nephrectomized rats were severely uraemic and growth retarded. The overall cell density (total pituitary cells/mm2) was higher in 5/6 nephrectomized animals in comparison with the two sham-operated groups. Thus, although the percentage of GH cells was slightly lower in nephrectomized than in control rats, no difference in either the density (cells/mm2) or the cross-sectional area of GH cells was found among groups. CONCLUSIONS: These results suggest that severe experimental uraemia interferes with the maturation process of the pituitary gland and support the contention that differences in either the number or the size of pituitary somatotrophs cannot explain the reduced GH secretion previously reported in severely uraemic rats.     

Do the kidneys play an important role in the pathogenesis of "essential" hypertension?

The Prague Hypertensive Rat (PHR) is a model of genetic hypertension derived from the Wistar strain, in which a normotensive parallel, the Prague Normotensive Rat (PNR), was also bred from the same parent pair. Thus, it is possible to transfer organs between both parallels without signs of rejection and without the use of immunosuppressive drugs. Unilateral nephrectomy and transplantation of one kidney within the PHR and PNR groups did not affect the systolic blood pressure (SBP). Transplantation of one kidney from PNR to a bilateral nephrectomized (BNX) PHR normalized the high SBP; and transplantation of one kidney from PHR to BNX PNR led to an elevation of SBP: hypertension "travels" with the kidney. When the development of high SBP in PHR was prevented for 2 months after weaning by antihypertensive drugs, transplantation of one kidney from these rats to BNX PNR always induced a sustained hypertension in the recipient. If a PHR was left with one original kidney in situ after transplantation of a "normotensive" kidney, the high blood pressure persisted until the original "hypertensive" kidney was removed. These results support the view that the kidney of PHR produces a "hypertensinogenic" substance, the secretion of which is genetically determined and is not influenced by the magnitude of the SBP.     

The impact of early normalization of haematocrit by erythropoietin on renal damage in the remnant kidney model

BACKGROUND: Correction of anaemia in moderate to advanced renal failure is still a matter of debate because of postulated detrimental effects of erythropoietin on the progression of renal damage. METHODS: The renal effects of early normalization of haematocrit (Htc) by erythropoietin (rHuEpo) were investigated from the time of 5/6 nephrectomy up to 8 weeks post-intervention in three groups of remnant kidney model rats: untreated controls (CON), rats receiving 100 UI/kg body-wt of rHuEpo i.p. twice a week (EPO), and rats receiving rHuEpo in which periodic phlebotomies maintained Htc similar to the value of the control group (PHL). The latter group was included to evaluate the direct effects of rHuEpo on renal damage, i.e. independent from Htc correction. RESULTS: Two weeks after renal ablation (basal), Htc decreased in CON and PHL rats (from 49.3+/-1.4% to 43.2+/- 1.1, P < 0.05 and from 49.6+/-1.1 to 43.3+/-1.5%, P<0.05 respectively), while it remained consistently normal in EPO rats (48.9+/-1.2% to 48.9+/-1.50/%, P<0.05 vs other groups). Thereafter Htc did not change throughout the remaining period in all groups. At the end of the study, with respect to basal, resting blood pressure increased significantly by the same extent in CON (+ 13+/-2%) and EPO rats (+ 15+/-5%), while it remained constant in PHL rats. Notably, creatinine clearance significantly decreased in CON (-53+/-8% 8 vs basal) and EPO (-38+/-8% vs basal), while it did not change in PHL rats. Likewise the degree of proteinuria as well as renal morphologic alterations and glomerular hypertrophy/sclerosis was similar in CON and EPO rats, and was significantly more severe than in the phlebotomized group. The only difference detected between CON and EPO group was the greater mesangial hypercellularity in rHuEpo-treated rats. CONCLUSION: In uraemic rats, chronic treatment with rHuEpo aimed at normalization of Htc beginning the early stage of renal failure does not inevitably account for a rise in systemic blood pressure. In addition, neither erythropoietin per se nor the correction of haematocrit accelerates the progression of renal damage when blood pressure remains constant.