Experimental Glomerulopathy Research Articles

The cytoprotective role of a low-molecular-weight heparin fragment studied in an experimental model of glomerulotoxicity

Abnormal glomerular glycosaminoglycan metabolism is involved in the onset of the morphological and functional aberrations of glomerulopathies. In the present study, a heparin derivative, low-molecular-weight heparin, was tested for its ability to afford renoprotection in an established model of experimental glomerulopathy. Two groups of male albino rats of the Wistar strain (140±10 g) received a single intravenous injection of adriamycin (7.5 mg/kg) to induce glomerulopathy, and one of them received low-molecular-weight heparin (Certoparin Sodium, Troparin®; 300 μg/day/rat s.c.) treatment, commencing on day 8, for a week. Urinary protein/creatinine ratio, serum albumin, urea, uric acid and creatinine clearance were evaluated. Renal cell injury was assessed in terms of renal tissue lactate dehydrogenase, aminotransferases (aspartate and alanine transaminases) and alkaline phosphatase activities, as well as renal antioxidant status (superoxide dismutase, catalase and glutathione peroxidase, reduced glutathione, vitamins E and C). The kidney tissue was subjected to histopathologic examination. Low-molecular-weight heparin significantly reduced proteinuria and improved creatinine clearance and serum albumin levels in the rats with glomerulopathy. The significant rise in serum uric acid in the rats with glomerulopathy was reversed by low-molecular-weight heparin. Altered tissue enzyme activities in response to injury, oxidative stress challenged renal antioxidant system and abnormal renal histology were observed in the untreated nephrotic rats, while low-molecular-weight heparin treatment protected the nephrotic rats against these changes. Thus, in this study, low-molecular-weight heparin was evaluated for its role in combating glomerular injury, on the basis of some salient biochemical parameters, oxidative injury indices and histologic picture. The ability of low-molecular-weight heparin to restore glomerular anatamo-functional features in this nephrotoxic condition illuminates its multi-faceted renoprotective role.

Podocyte process effacement in vivo.

Foot process effacement is the most characteristic change in podocyte structure under a wide variety of human and experimental glomerulopathies with heavy proteinuria. It consists of simplification and even total disappearance of the interdigitating foot process pattern, resulting in the formation of a diffuse cytoplasmic sheet along the glomerular basement membrane. Although abundant evidence related to structural changes in podocyte foot processes has been reported, cellular or molecular mechanisms that occur within podocytes during the development of foot process effacement remain unclear. This review summarizes recent advances concerning structural and functional aspects of foot process effacement in vivo. Following a description of the general morphology of foot process effacement, the role of the cytoskeleton and its related proteins in the effacement are discussed. Finally, the relevance of foot process effacement in glomerular function is considered.

Changes in blood pressure (BP) in experimental glomerulopathy by polysaccharides

Changes in blood pressure (BP) in experimental glomerulopathy by polysaccharides

Expression of Agrin, Dystroglycan, and Utrophin in Normal Renal Tissue and in Experimental Glomerulopathies

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

Localization by Immunoelectron Microscopy of Schistosoma mansoni Antigens in the Glomerulus of the Hamster (Mesocricetus auratus) Kidney

Adult worm antigen (AWA) and soluble egg antigen (SEA) were localized ultrastructurally by immunoelectron microscopy using two monoclonal antibodies in the glomeruli of hamsters infected with Schistosoma mansoni cercariae or injected with S. mansoni eggs. AWA was detected in all cercaria-infected groups from the 30th day on and was present mainly in cytoplasm of mesangial cells, mesangial matrix, and glomerular basement membrane, either as isolated gold particles or in small electron-dense deposits of probable immune origin. AWA was encountered also on the inner side of the glomerular basal membrane, close to endothelial cells, and in the foot processes of the glomerular epithelial cells. SEA was detected at similar sites, apparently in lesser amounts, in uninfected hamsters inoculated with S. mansoni eggs into the jugular vein. Schistosomal antigens are apparently processed mainly by mesangial cells which are considered to be critical in the pathogenesis of S. mansoni associated glomerulopathy. Mesangioproliferative glomerulonephritis, immunoglobulin (IgG and IgM), and C₃ deposits were observed in hamsters in which AWA and SEA were visualized. During early phases of the infection and in hamsters in which granulomatous pneumonitis was induced by S. mansoni eggs, glomeruli were unchanged or showed a slight mesangial proliferation. Our findings suggests that egg antigens also contribute to the pathogenesis of experimental glomerulopathy in the hamster.

Antiproteinuric effect of ciclosporin A in adriamycin nephropathy in rats.

Ciclosporin A (CsA) can reduce proteinuria in various forms of human and experimental glomerulopathies. This antiproteinuric effect can be the result of a decrease of immunological damage, a decrease in the glomerular filtration rate (GFR), or a change in the permselective properties of the glomerular capillary wall. In this study we investigated the effect of CsA on Adriamycin-induced nephropathy in rats. A single intravenous injection of Adriamycin (5 mg/kg body weight) induced a severe nephrotic syndrome with a massive albuminuria (+/- 400 mg/24 h from 3 weeks onwards) and a hypoalbuminemia (+/- 7 mg/ml after 5 weeks). The IgG/albumin selectivity index was 0.16 +/- 0.05, indicating a preferential loss of albumin. A 5-day treatment with CsA reduced the albumin excretion by almost 50% (from 336 +/- 91 to 178 +/- 58 mg/24 h; p = 0.002) and induced an increase in the serum albumin level (from 7.1 +/- 4.1 to 12.8 +/- 3.2 mg/ml; p = 0.002) in contrast to the vehicle olive oil (OO). CsA also decreased the GFR by 40% (from 0.74 +/- 0.11 to 0.41 +/- 0.11 mg/ml/100 g body weight; p = 0.002). Albuminuria corrected for the GFR (fractional excretion of albumin, FE(alb)) was still significantly lower in CsA-treated than in OO-treated animals (FE(alb) CsA: 1.35 +/- 0.88, FE(alb) OO: 3.17 +/- 2.29%; p = 0.0005). This suggests that other factors are also involved in the reduction of albuminuria. To exclude that CsA has an effect on the tubular reabsorption of albumin, we evaluated the blockade of the tubular reabsorption by lysine and found no difference in albuminuria between the CsA- and OO-treated groups. These experiments suggest that the antiproteinuric effect of CsA is not (only) due to a decrease in the GFR, but also to a decrease of the enhanced permeability of the glomerular capillary wall for albumin.

Vitamin E ameliorates renal injury in an experimental model of immunoglobulin A nephropathy.

IgA nephropathy is one of the most common forms of glomerular disease. Nearly 25% of affected patients progress to end-stage renal disease over a 20-25-y follow-up period. IgA-containing immune complexes stimulate oxygen-free radical production by mesangial cells in vitro. The excessive oxidant stress may mediate glomerular injury in this disorder. Therefore, we studied whether dietary supplementation with the antioxidant agent, vitamin E, attenuates renal disease in an experimental model of incipient IgA nephropathy with mild kidney inflammation. IgA nephropathy was induced in male Lewis rats by oral immunization with 0.1% bovine gamma-globulin (BGG)-containing drinking water for 8 wk. At the completion of this period, animals received BGG, 1 mg/dose i.v., on three successive days. Experimental rats (n = 10) received a specially formulated diet containing 100 IU of vitamin E/kg of chow, whereas control animals (n = 10) were fed chow containing 30 IU of vitamin/kg of chow. The BGG immunization regimen induced mesangial IgA deposition in all rats. Vitamin E supplementation resulted in a nearly 5-fold increase in the serum vitamin E concentration. Vitamin E-treated rats gained more weight and had a lower incidence of hematuria, 20% versus 80% (p < 0.03). Moreover, proteinuria was decreased by 50%, and reduced renal plasma flow was restored to normal, compared with untreated rats with IgA nephropathy. Glomerular hypertrophy occurred in animals with IgA nephropathy, but less so in those receiving vitamin E supplementation. Renal cortical malondialdehyde content was reduced from 1.55 +/- 0.10 to 1.22 +/- 0.09 nmol/mg of protein (p < 0.01) in rats fed the vitamin E-enriched diet. Finally, renal transforming growth factor-beta 1 gene expression was reduced by 34% in rats with IgA nephropathy receiving vitamin E treatment (p < 0.05). We conclude that experimental IgA nephropathy is associated with increased renal oxidant injury. Dietary treatment with the antioxidant agent, vitamin E, attenuated renal functional and structural changes in this experimental glomerulopathy. These studies support the importance of clinical trials for the evaluation of the efficacy of antioxidant therapy in patients with IgA nephropathy.

Effect of Prostaglandin E2 and Hyaluronan on Mesangial Cell Proliferation: A Potential Contribution to Glomerular Hypercellularity in Diabetes

Proliferation of mesangial cells is a feature of several forms of human and experimental glomerulopathy, including that seen in diabetes. The nonsulfated glycosaminoglycan hyaluronan participates in the regulation of pericellular matrix assembly and is a mitogen in some cell types. We have shown previously that hyaluronan production is increased in the glomerulus in a glucose- and prostaglandin-dependent manner. We have investigated the effect of diabetes and of addition of hyaluronan and prostaglandin E2 (PGE2) on the uptake of [3H]thymidine by glomerular core preparations enriched in mesangial cells. When compared with nondiabetic controls, it was shown that [3H]thymidine uptake was significantly increased in glomerular core preparations from streptozotocin-induced diabetic rats (to 169 +/- 5%, P < 0.001). In glomerular cores from both experimental groups, hyaluronan (50-250 ng/ml) or PGE2 (10(-12) to 10(-8) mol/l) increased the uptake of [3H]thymidine. Further, mesangial cells from nondiabetic control glomerular cores, when maintained in culture in early passage, responded with increased [3H]thymidine uptake to raised glucose (5.6-25 mmol/l) and to added hyaluronan and PGE2. We propose that prostaglandin and hyaluronan production in response to a raised glucose environment in diabetes can contribute to mesangial hypercellularity.

Cellular Immunity in Interstitial Nephropathy

Inflammatory tubulointerstitial nephritis (TIN), either as a primary or as a secondary event, plays an essential role in the development of all forms of chronic renal failure. Experimental models of TIN should help in understanding TIN in humans. As in experimental glomerulopathies, the target antigen can be a kidney structural antigen, from the tubular basement membrane (TBM) or not, or a foreign antigen. While some models are due to deposits of free antibodies or circulating immune complexes, many others involve cell-mediated immunity. This last aspect explains the importance and the originality of experimental TIN.

The role of platelet‐activating factor (PAF) in experimental glomerular injury

Platelet-activating factor (PAF) is a potent autacoid that participates in inflammation and other pathophysiological processes. In this review we deal with recent evidence suggesting that PAF is a mediator that is released early during glomerular injury. PAF can be synthesized in the glomerulus by infiltrating intrinsic glomerular cells. Normal glomeruli produce PAF upon stimulation, and glomerular PAF synthesis is increased in a variety of experimental glomerulopathies. The local infusion of PAF into the renal artery of isolated blood-free kidneys induces proteinuria. PAF attracts and activates inflammatory cells. Glomerular mesangial, endothelial and epithelial cells are also targets for PAF. Therapy with specific PAF receptor antagonists has prevented or reduced proteinuria and improved glomerular inflammation in several experimental models of proliferative glomerulonephritis and minimal change nephrosis. However, the beneficial effect of administration of PAF antagonists once proteinuria is fully developed has been minimal. PAF may also play a role in the recruitment of inflammatory interstitial cells.

Experimental glomerulopathy induced by monocrotaline in rats

Experimental glomerulopathy induced by monocrotaline in rats

Ultrastructural Immunohistochemistry of Glomerulonephritisin Needle Biopsy

In two patients with glomerulonephritis (GN) IgG and C 3 were visualized in ultrastructure by means of HRP-conjugated antisera. The first patient had an acute postinfectious extra-intracapillary GN lasting for about two months with granular fluorescence of anti-IgG, -C 3, and -C 1q. Electron microscopy revealed widespread endothelial defects, a well-pronounced polymorphonuclear stasis, and typical perimembranous "humps". These deposits reacted with HRP-anti-C 3 but the ultrastructural proof of IgG was negative. A weak and sporadic reaction of both these conjugates was seen in the swollen mesangial matrix while intraluminal plugs of coagulated plasma and extracapillary exudates yielded a dense coarse reaction product. In the second patient (allograft, three years after transplantation) the membranous and proliferative probably recurrent GN with nephrotic syndrome showed massive perimembranous deposits in the late involution stage. Granular fluorescence of the main Ig classes and of C 3 was sporadic or absent. In the ultrastructural immunoenzyme assay, too, the residues of deposits failed to react with HRP-anti-IgG or -C 3 and the mesangial matrix harboured only sporadic foci of faint positivity; however, dense product was again seen in capillary plasmatic "microthrombi". The discrepancy between immunofluorescence microscopy and immunoenzyme histochemistry, noted also by others in experimental glomerulopathies, may reflect the instability and dynamic properties of immune deposits with an early loss of antibody reactivity and a more protracted though not persistent local activation of complement. In light microscopy, fluorescent granules may correspond not only to the sites of immune deposition but also to accidental intracapillary plasma precipitates.

The Specificity of Nephritogenic Antibodies: I. Evidence on Anti-T-Cell Specificity in Nephritogenic Antibodies Detected by Cytotoxicity and MIF Assays

Heterologous antibodies directed to brushborder antigens of rat kidney tubules are nephritogenic in that an immediate immune complex glomerulopathy occurs after injection of these antibodies into normal rats. Since previous observations suggested the presence of anti-T-cell specificity within these antibodies, we now compared the specificities of these antibodies with those of heterologous anti-rat thymocyte antibodies, using immunfluorescence (IF), cytotoxicity and migration inhibition (MIF) assays. The results suggest that anti-brushborder antiserum contains anti-thymocyte specificities, while antithymocyte antiserum contains antibrushborder specificity. These specificities could be removed selectively from both of the antisera by absorption with insoluble tissue extracts containing the appropriate antigens, i.e. thymocyte antigens or brushborder antigens respectively, indicating that two specificities are involved rather than only cross-reacting antibodies. Since it is unlikely for several other reasons that this feature is simply the result of an immunologic cross-reaction due to antibodies raised by immunization with purified kidney tissue antigens, this dual specificity of anti-brushborder antibody might play a role in the pathogenesis of experimental glomerulopathy.

Molecular weight analysis of proteinuria in experimental glomerulopathies.

The quantity of urinary proteins and their molecular weight composition was analyzed in different experimental glomerulopathies using the SDS-PAA-electrophoresis. Masugi nephritis, heterologous and autologous immune complex nephritis as well as D-penicillamine induced glomerulonephritis were studied in rabbits, guinea pigs and rats. The procedure allows (1) to distinguish physiological from low grade glomerular proteinuria by their respective characteristic patterns in early disease stages (2) to follow up the disease course of individual animals without sacrifice and (3) to discriminate species specificity of physiological urinary proteins. It is recommended to use this technique of urinary protein analysis in experimental conditions, where mild glomerular damage is expected.