Low-protein diet suppresses serum insulin-like growth factor-1 and decelerates the progression of growth hormone-induced glomerulosclerosis.

A low-protein (LP) diet has been associated with amelioration of renal function in glomerulosclerosis (GS). However, the mechanisms involved are still unclear. We have used a mouse transgenic for bovine growth hormone (GH), which develops progressive GS and exhibits consistently elevated levels of circulating GH and insulin-like growth factor (IGF)-1, to study the effect of dietary protein restriction. LP (6% protein) and normal-protein (NP, 20% protein) diets were maintained for 30 weeks in mice with established GS of mild/moderate degree. The degree of GS was markedly attenuated in LP compared to NP mice. Quantitative analysis revealed a significantly lower GS index (1.4 +/- 0.9 in LP vs. 2.8 +/- 0.8 in NP) and glomerular volume (0.8 x 10(6) +/- 0.1 x 10(6) microm(3) in LP vs. 1.2 x 10(6) +/- 0.1 x 10(6) microm(3) in NP) in mice with restricted protein intake. These morphologic changes were accompanied by a significant reduction in renal expression of alpha(1) type-IV collagen (2.4-fold) and tenascin (1.4-fold) in LP mice. Serum IGF-1 decreased by 40% and showed a significant correlation with alpha(1) type-IV collagen expression with the LP diet. The present finding supports the use of the LP diet to decelerate the progression of GS and furthermore suggests that one of the mechanisms involved in this process is the GH/IGF-1 regulation by protein intake.

Inhibition of Na,K-ATPase activates PI3 kinase and inhibits apoptosis in LLC-PK1 cells.

In the present study we used LLC-PK1 cells, a porcine renal proximal tubular cell line, to investigate whether PI3 kinase activation was involved in the anti-apoptotic effect of ouabain, a specific inhibitor of Na,K-ATPase. Apoptosis was induced by actinomycin D (Act D, 5 microM) and assessed by appearance of hypodiploid nuclei and DNA fragmentation. Ouabain attenuated Act D-induced apoptotic response in a dose-dependent manner. Incubation in a low K(+) medium (0.1 mM) which is another way to decrease Na,K-ATPase activity also had anti-apoptotic effect. Both ouabain and low K(+) medium increased the PI3 kinase activity in p85 immunoprecipitates. Ouabain, as well as incubation in the low K(+) medium, also increased the phosphorylation of Akt. Inhibition of PI3 kinase by either wortmannin or LY294002 reversed the cytoprotective effect of ouabain. These data together indicate that inhibition of Na,K-ATPase activates PI3 kinase in LLC-PK1 cells which could then exert the cytoprotective effect.

Disulfiram inhibits TNF-alpha-induced cell death.

Disulfiram, a clinically employed alcohol deterrent, was recently discovered to inhibit caspase-3 and DNA fragmentation. Using LLC-PK1 cells and murine liver as models, we examined if the drug inhibited TNF-alpha-induced cell death. Disulfiram produced dose-dependent inhibition of TNF-alpha-induced cell death as well as caspase-3-like activity. Disulfiram retained 80% of its effect when added 4 h after TNF-alpha. Disulfiram protected the cells from cytokine-induced death for at least 6 days. The cells rescued by the drug preserved the ability to proliferate. The cells died spontaneously after exposure to TNF-alpha for just 70 min. Co-administration of 15 microM disulfiram and TNF-alpha for 70 min prior to their removal abolished TNF-alpha-induced killing, and this was associated with restoration of mitochondrial membrane potential and suppression of reactive oxygen species. Treatment of mice with TNF-alpha and D-galactosamine for 5 h markedly increased hepatic DNA fragmentation and caspase-3-like activity. Disulfiram at 0.6 mmol/kg abolished these effects. We conclude that disulfiram is a potent inhibitor of TNF-alpha-induced cell death in vitro. The underlying mechanisms include stabilization of mitochondrial membrane potential, suppression of reactive oxygen species, and inhibition of caspase-3-like activity. We further conclude that disulfiram inhibits DNA fragmentation in vivo in association with the blockade of caspase-3-like activity.

Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells.

Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3-like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha-induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF-alpha is mediated by caspase-3-like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3-like activity and reactive oxygen species.

Renal hemodynamic response to the creation of vascular access in patients with end-stage renal disease.

To evaluate the possibility that the placement of arteriovenous anastomosis (a/v a) may lead to the attenuation of glomerular hyperfiltration, we studied 5 nondiabetic patients before and after creation of vascular access for hemodialysis. Patients received no EPO and antihypertensive therapy was discontinued 24 h before each study. Cardiac output (CO) and a/v a flow rates were measured by Doppler echo, and GFR and ERPF by plasma decay curves of Tc99m DTPA and 131I-hippuran, respectively. Other parameters were calculated by standard formulas. Augmentation of CO and decrease in systemic vascular resistance occurred in all patients (p = 0.05), yet renal findings were less predictable since only three patients showed a decrease in renal vascular resistance and filtration fraction post a/v a. Thus, there is a discordant pattern of renal hemodynamic response to the creation of a/v a in end-stage renal disease and further studies are needed to better define the subset of patients who are prone to renal vasodilation after the placement of a/v a.

Radiocontrast removal by dialysis membranes.

To examine the effect of membrane characteristics on radiocontrast mass transfer, we studied in vitro clearances with cuprophane and polyacrylonitrile (PAN) dialyzers and with polysulfone hemofilter. A perfusate of saline at 37 degrees C, with 7.5 mmol/l (45 mg/dl) of urea and either Renografin (molecular weight 723) or Hexabrix (1405), was pumped through the blood path of dialyzers at 200 ml/min for 5 min. Each radiocontrast agent (RCA) and dialyzer was tested at 0, 150, and 250 or 300 mm Hg transmembrane pressure. In experiments with the use of hemofilter, clearances were tested at the perfusate flow of 50, 100 and 150 ml/min. RCAs were measured by fluorescent excitation analysis of iodine. Mean urea clearance was 16% higher in PAN than in cuprophane dialyzers. Clearance of RCAs was 1.5-3 times higher in PAN than cuprophane dialysers. With the latter, increases in transmembrane pressure resulted in a small amount of ultrafiltration (UF) and little increment in RCA clearance. With the former, increases in transmembrane pressure resulted in massive UF and remarkable increases in RCA clearance. Renografin clearance generally exceeded that of Hexabrix, which we attributed to Renografin's lower molecular weight. With the hemofilter, sieving coefficients were approximately 0.8 for each RCA. Yet, because of the lack of diffusive transport and a small surface area even at the highest perfusate flow rates, RCA clearance by the hemofilter was 20-50% less than that of cuprophane dialyzers. We conclude that PAN dialyzers are more efficient for RCA removal than cuprophane dialyzers or polysulfone hemofilters.

Splanchnic volume, not flow rate, determines peritoneal permeability.

To distinguish the effects of splanchnic blood flow rate from those of splanchnic volume on peritoneal transfer rates, measurements were made in rabbits before and after intraperitoneal exposure to sodium chromate. The sodium chromate induced reversible hepatic sinusoidal block with consequent portal venous congestion and stasis, which was demonstrable on histologic sections. Concurrently the ultrafiltration rate, and ultrafiltration coefficient each doubled after chromium even though the dialysate reabsorptive rate increased and the absorptive diffusion of glucose was at least as high as in control dialyses. Chromium induced significant increases in mass transfer coefficients of urea, potassium and phosphate and in protein clearance. These data suggest that splanchnic volume is an important determinant of peritoneal transfer functions and that the hepatic capillaries may contribute appreciably to transfer ordinarily ascribed to peritoneal capillaries alone.

Effects of histamine and its receptor antagonists on peritoneal permeability.

Peritoneal fluid and mass transfer rates were studied in rabbits undergoing control dialyses and dialyses with intraperitoneal histamine, or its receptor antagonists alone or in combination. These drugs had negligible effects on peritoneal ultrafiltration and small solute clearances. Histamine raised protein exudation from 1.6 to 2.9 mg/kg/min, an effect blocked by its antagonists which given alone did not lower protein loss. These data demonstrate the existence of histamine receptors in the peritoneal diffusion barrier and show that they do not control transport under baseline conditions, but can be blocked should abnormal histamine release occur. Increased peritoneal permeability with sterile peritonitis was unaffected by ranitidine, suggesting alternative mediators.

Urinary Charcot-Leyden crystals in the hypereosinophilic syndrome with acute renal failure.

A 48-year-old man with idiopathic hypereosinophilic syndrome (IHS) developed blast crisis along with a fulminant autoimmune hemolytic anemia. Hemoglobinuria and anuric acute renal failure (ARF) ensued. Urinalysis revealed countless Charcot-Leyden crysals (CLC). This is the only known report of Charcot-Leyden crystalluria. The CLC protein (lysophospholipase) should normally undergo glomerular filtration and catabolism by the tubules during reabsorption. Its abundant presence in the urine of our patient may reflect impairment of tubular reabsorption, saturation of the tubular reabsorptive process by excessive CLC load through residual functioning glomeruli, or a combination thereof. The extreme degree of hypereosinophilia suggests a massive load of CLC protein and acute tubular necrosis implies impaired tubular function, so both mechanisms should have been operative. At the autopsy, no eosinophilic infiltrates were present in the kidneys, which points against a local spillage of CLC protein into the tubules.

Prolonged intraperitoneal dwell decreases ultrafiltration coefficient in rabbits.

In rabbits undergoing peritoneal dialysis, hypertonic (6% dextrose) dialysis solution increased the net ultrafiltration rate (UF) from 233 to 462 microL/kg/min, which was not proportional to the increment in the osmotic gradient, so the ultrafiltration coefficient decreased. As intraperitoneal dwell of hypertonic dialysate was prolonged, the gross and net UFs and ultrafiltration coefficients decreased, and the UF per dextrose absorption declined. The decrement in UF was multifactorial, including a component of fluid and solute stagnation, increasing the distance over which osmotic forces must exert their effects. Excessively hypertonic dialysis fluid should be used only briefly to achieve ultrafiltration efficiently and to avoid the high dextrose loading.

Minimal-change nephropathy associated with pancreatic carcinoma.

A 67-year-old man presented with nephrotic syndrome and polymyalgia rheumatica. A renal biopsy revealed minimal-change nephropathy. The proteinuria and rheumatologic findings responded to prednisone therapy. The patient presented three months later with biliary tract obstruction secondary to pancreatic adenocarcinoma metastatic to the liver. The glomerulopathy and polymyalgia rheumatica in this case seemed to be components of the paraneoplastic syndrome. The response of both entities to prednisone therapy supports the hypothesis that they are caused by derangements in cell-mediated immunity. The fact that the tumor progressed despite resolution of the nephrosis and polymyalgia rheumatica suggests that cell-mediated immunity in general is altered by the tumor and not that the carcinoma liberates a factor that directly damages the kidney.

The role of the capillary wall in restricting diffusion of macromolecules. A study of peritoneal clearance of dextrans.

In 5 nephrectomized rabbits the peritoneal clearance of neutral dextrans from plasma to dialysate decreased from 7.8 to 3.3 microliters/kg/min as molecular mass increased from 17,000 to 43,000 daltons, and was relatively constant at 2.8 microliters/kg/min from 49,000 to 97,000 daltons in accord with prior studies. The clearance from dialysate to plasma was measured by determining the distribution volume, which averaged 72 ml/kg, and the plasma concentration 5 h after intraperitoneal instillation. Inward clearances ranged from 11.4 to 19.9 microliter/kg/min, did not correlate well with solute size and were significantly higher than outward clearances. The data suggest that while the capillary wall is the major barrier to macromolecule transfer, absorption can bypass vascular capillaries and occur via the lymphatics. It is suggested that lymphatic flow rate from the peritoneum exceeds 16 microliter/kg/min.

Contrasting effects of amphotericin B and the solvent sodium desoxycholate on peritoneal transport.

To distinguish amphotericin B effects on peritoneal transport from those of the solvent, sodium desoxycholate, dialyses in intact rabbits with either substance added intraperitoneally were compared to controls. Powered amphotericin B added to instilled dialysis fluid increased peritoneal ultrafiltration from 0.31 to 0.44 ml/kg/min (p less than 0.02), but did not affect mass transport (e.g. urea clearance changed from 0.86 to 1.04 ml/kg/min). In contrast, 10 mg of desoxycholate induced peritoneal irritation and raised clearances of urea (0.76-1.34 ml/kg/min), potassium, phosphate and dextrose, but did not affect ultrafiltration. Intraperitoneally, 1 mg/kg of desoxycholate changed clearances inconsistently, but lowered the ultrafiltration rate from 0.33 to 0.21 ml/kg/min. The dialysate-plasma dextrose gradient dissipated faster with 10 mg/kg of desoxycholate. Amphotericin B tended to raise ultrafiltration per osmotic gradient and mass transport of sodium. Selective increase in fluid flux results from amphotericin B, not its solvent.

The mechanism of dextrose-enhanced peritoneal mass transport rates.

The mechanism whereby hypertonic dextrose affects peritoneal transport was investigated in a short-term model of peritoneal dialysis using alert intact rabbits. During control (1.5% dextrose) dialyses osmotic ultrafiltration was 0.28 mg/kg/min, the clearance of potassium was 0.98, urea 0.54, phosphate 0.32, and dextrose (reverse) 0.21 ml/kg/min. With 4.25% dextrose, the ultrafiltration rate increased to 0.73 ml/kg/min (P less than 0.02), but solute transport did not increase despite the added convective flux. The posthypertonic exchanges did not differ from control despite the effect of residual dialysate contaminating this peritoneal lavage. By indicator dilution residual volume averaged 12% of total dialysate volume. Acute volume expansion by intravenous dextrose after desoxycorticosterone acetate (DOCA) pretreatment increased the ultrafiltration coefficient, potassium and urea clearances significantly, and DOCA alone was ineffective. It is suggested that in uremic humans hypertonic dextrose dialysis increases peritoneal mass transport rates because the absorbed dextrose causes extracellular volume expansion that cannot be eliminated promptly. No evidence of a direct effect of dextrose augmenting peritoneal permeability was detected.

Mercury-induced autoimmune glomerulonephritis in inbred rats. I. Kinetics and species specificity of autoimmune responses.

The nephropathy observed in rats after administration of mercuric chloride can be used to clarify the mechanisms underlying renal autoimmunity induced by chemicals. As a necessary preliminary step in the study of this animal model, we have investigated the kinetics and species-specificity of autoimmune responses to renal antigens. By a recently developed enzyme-linked immunosorbent assay (ELISA), circulating autoantibodies to the glomerular basement membrane of the kidney (anti-GBM) have been detected within 8 days after the initiation of mercuric chloride treatment. Anti-GBM antibodies reach a peak by 15 days and then decrease rapidly in the following 2 weeks. Extensive cross-reactions between rat and human GBM antigens have been detected by ELISA, indicating a high degree of conservation of some renal autoantigens and suggesting certain similarities between the autoimmune response induced in rats by mercuric chloride and that observed in human glomerulonephritis caused by anti-GBM. Dose-response studies have been performed to ascertain whether anti-GBM responses are correlated with massive kidney damage and release of renal antigens. We have noted that a wide range of levels of mercuric chloride are capable of stimulating the production of anti-GBM and that animals receiving this chemical in as low a concentration as 0.02 mg/100 g body weight (i.e. a dose ten times lower than those causing massive nephrotoxic effects) still have anti-GBM specifically bound to their kidneys. Thus, it is possible that the administration of mercury compounds to BN rats results in kidney autoimmunity not only because of the release of renal autoantigens, but also through the activation of specific lymphocytes and/or disruption of regulatory networks. Finally, we have observed that both BN and MAXX rats produce anti-GBM after mercuric chloride treatment, while M520 rats do not. Since the MAXX strain was initially obtained from a cross of BN and Lewis rats and shares antigens of the major histocompatibility complex with the BN strain, our findings stress the importance of genetic factors in chemical-induced autoimmunity and suggest that a similar situation may occur in human subjects exposed to environmental chemicals.

Mercury-induced autoimmune glomerulonephritis in inbred rats. II. Immunohistopathology, histopathology and effects of prostaglandin administration.

The repeated administration of mercuric chloride to BN rats induces the production of anti-GBM. In the present paper, we describe the immunohistopathology and histopathology of the kidneys from mercuricchloride-treated rats. Direct immunofluorescence demonstrated bright linear deposits of immunoglobulins at the level of the GBM of the kidney. Light microscopy failed to reveal substantial glomerular changes, but electron microscopy demonstrated a spectrum of ultrastructural alterations of the glomeruli (including the detachment of endothelial cells from the GBM and the presence of electron-opaque deposits). In the aggregate, these findings are suggestive of membranous glomerulonephritis. We also investigated whether treatment with low doses of PG had any effect on the course of this experimental model of autoimmune renal disease. Two groups of mercuric-chloride-treated BN rats received different doses of DMPGE2. This resulted in significantly lower levels of circulating autoantibodies to the GBM, as well as a decrease in the amounts of rat immunoglobulins bound to the kidneys and an increase in proteinuria. On the other hand, there were no major differences in renal histopathology between rats treated with DMPGE2 and controls.

Amphotericin selectively increases peritoneal ultrafiltration.

Because amphotericin B is known to affect transport rates across biologic membranes, the effects of this agent on transport parameters in an animal model of peritoneal dialysis were investigated. When amphotericin B in doses ranging from 0.5 to 25 mg/kg was instilled intraperitoneally with commercial dialysis solution, diffusive clearances of phosphate and urea did not differ from control values measured in the same animals, and only a modest increase in potassium clearance was detected. Ultrafiltration due to the osmotic gradient induced by the dextrose content of the dialysis solution increased significantly to 0.31 mL/kg/min with amphotericin B, compared with control values of 0.18 mL/kg/min. The drug did not affect dextrose transport and the osmotic gradient did not differ in the two groups. Hence, the ultrafiltration coefficient was higher with amphotericin B (14 microL/kg/min/mosm), than during control dialyses (6 microL/kg/min/mosm). Increased water flux was detected at the lowest dose and there was no dose relationship over the range studied. Amphotericin B may be the type of agent that will be clinically useful in patients with reduced peritoneal ultrafiltration capacity, and safer analogues should be explored.

Mesangial proliferative glomerulonephritis with IgM deposits. Clinicopathologic analysis and evidence for morphologic transitions.

To determine the natural history of mesangial proliferative glomerulonephritis (MesPGN) with IgM deposits and its relationship to minimal change disease (MC) and focal segmental glomerulosclerosis (FGS), we studied the clinical characteristics and outcome in 20 patients with MesPGN, 8 with MC, and 10 with FGS. IgM deposits were present in glomeruli of all MesPGN patients. Progression to FGS was documented in 2 patients with MesPGN, 1 of whom developed renal failure. Transition from MC to MesPGN occurred in 1 patient. 2 MC patients developed FGS, with decline in renal function in 1 of them. These data suggest the possibility of histologic transition from MC to FGS directly or through the stage of MesPGN.