Progressive glomerular sclerosis in experimental antiglomerular basement membrane glomerulonephritis

A model of chronic progressive glomerular sclerosis in experimental antiglomerular basement membrane (anti-GBM) glomerulonephritis was developed in Wistar rats. Wistar rats given the accelerated form of anti-GBM anti-body glomerulonephritis initially developed significant proteinuria and renal insufficiency associated primarily with a decrease in glomerular filtration rate (GFR) with normal renal clearance of para-aminohippuric acid and with markedly reduced filtration fraction. The glomerular functional abnormalities were associated with marked glomerular hypercellularity due to leukocytic infiltration as well as proliferation of intrinsic glomerular cells with crescent formation. Late in the course of the disease, by day 21, GFR had fallen further, associated with a parallel decrease in the clearance of para-aminohippuric acid and a normal filtration fraction. At this stage, glomerular hypercellularity had diminished and was replaced by glomerular sclerosis. The model appears to be a reproducible form of chronic glomerulosclerosis and demonstrates that the chronic phase of glomerular basement membrane (GBM) glomerulonephritis is distinctly different from that of the acute phase. It provides a controllable setting to study the glomerular sclerotic process independent of the initial inflammatory changes.     

Transplanted mesenchymal stem cells accelerate glomerular healing in experimental glomerulonephritis

Bone marrow-derived cells contribute to glomerular cell turnover and repair, but the cell types involved are unknown. Whether rat mesenchymal stem cells (MSC) can accelerate recovery from damage in rat mesangioproliferative anti-Thy1.1 glomerulonephritis was studied. After injection into the left renal artery on day 2 after disease induction, fluorescently labeled MSC were detected in 20 to 50% of glomeruli and rare intrarenal vessels but not in the tubulointerstitium, in contralateral kidneys, or in medium controls. In control experiments, injected mesangial cells were detected less frequently in glomeruli in comparison with injected MSC. In nephritic outbred Wistar rats, MSC injection led to an approximately 50% reduction of mesangiolysis on days 4 and 6 after disease induction, accompanied by three- to four-fold higher intraglomerular cell proliferation on day 4 and more rapid mesangial reconstitution as detected by alpha-smooth muscle actin expression. Injection of MSC into tail veins or intra-arterial injection of mesangial cells instead of MSC failed to reproduce any of these findings. In inbred Lewis rats, anti-Thy1.1 nephritis followed an aggravated course with transient acute renal failure. Acute renal failure was ameliorated by MSC injection into the left renal artery on day 2 after disease induction. Again, MSC led to more rapid recovery from mesangiolysis, increased glomerular cell proliferation, and reduction of proteinuria by 28%. Double immunostaining of 5-bromo-2'-deoxyuridine-labeled MSC for endothelial, mesangial, or monocyte/macrophage antigens showed that 85 to 95% of MSC that localized in glomeruli on day 6 failed to express these markers. In vitro, MSC secreted high amounts of vascular endothelial growth factor and TGF-beta1 but not PDGF-BB. In conclusion, even low numbers of MSC can markedly accelerate glomerular recovery from mesangiolytic damage possibly related to paracrine growth factor release and not to differentiation into resident glomerular cell types or monocytes/macrophages.     

Alpha8 integrin in glomerular mesangial cells and in experimental glomerulonephritis.

BACKGROUND: Mesangial cell (MC) proliferation and extracellular matrix accumulation are typical responses of renal glomeruli to injury. Extracellular matrix components are known to affect MC behavior, which is mediated primarily via integrin receptors of the beta1 family. In addition to alpha1, alpha3, alpha5, and alpha6 chains of beta1 integrins, recent studies have shown the alpha8 chain to be expressed in glomeruli and renal vasculature. alpha8beta1 can serve as a receptor for fibronectin, which is abundant in the mesangium. We investigated the glomerular expression pattern of the alpha8 chain in renal tissues of mouse, rat, and humans as well as in cultured MCs. In addition, the regulation of alpha8 expression in MCs was studied in culture and in nephritic rats. METHODS: The expression of alpha8 protein in kidney tissue and cultured MCs was investigated by immunohistochemistry, immunocytochemistry, and Western blotting. The effects of TGF-beta1 on alpha8 mRNA levels in MCs were studied by Northern blot analysis. In addition, time course studies of glomerular abundance and localization of alpha8 were performed in rats with mesangioproliferative anti-Thy1.1 nephritis. RESULTS: In tissue sections of normal human, rat, and mouse kidney, we found strong immunohistochemical staining for alpha8 in the mesangium and in the media of renal arterioles. Double staining for alpha8 and Thy1.1, a surface antigen of rat MCs, showed alpha8 to be specifically expressed in MCs but not in glomerular endothelial and epithelial cells. In anti-Thy1.1 nephritis of rats, the glomerular abundance of alpha8 protein was reduced in the early mesangiolytic phase but was increased greatly with subsequent MC proliferation, peaking at day 6 of disease. At later stages of this reversible form of nephritis, the number of MCs and the extent mesangial alpha8 staining declined to control levels. Cell culture experiments revealed that freshly plated MCs organize alpha8 into focal contacts within one hour after attachment to fibronectin and vitronectin substrata, showing colocalization with focal contact proteins vinculin and talin. Stimulation of MCs with transforming growth factor-beta1 led to increases of alpha8 mRNA and protein levels. CONCLUSIONS: These results show that in human, rat, and mouse glomeruli, alpha8 integrin is strongly and exclusively expressed in MCs. Gene expression of alpha8 is regulated in cultured MCs, and alpha8 protein abundance is regulated in vivo and in MC culture. It is currently unclear what functional properties this integrin receptor protein has with regard to MC anchorage to extracellular matrix and modulation of the MC phenotype in normal and diseased glomeruli. However, in view of its abundance in the mesangium, alpha8beta1 integrin could be an important MC receptor of matrix ligands and may play a role in the embryology, physiology, and pathophysiology of the glomerular capillary tuft.     

Adrenomedullin reduces mesangial cell number and glomerular inflammation in experimental mesangioproliferative glomerulonephritis

BACKGROUND: Adrenomedullin (ADM) is a vasodilator peptide that is abundantly expressed in the kidney. ADM has antiproliferative effects on glomerular mesangial cells (MC) in vitro. Whether or not treatment with ADM can reduce MC proliferation in vivo [i.e., in mesangioproliferative glomerulonephritis (GN)] is unknown. We tested the hypothesis that ADM substitution reduces MC proliferation in GN. METHODS: GN in rats was induced by injection of an anti-Thy-1.1 antibody. Rats received osmotic minipumps, which continuously delivered rat ADM (500 ng/hour, N = 11), or vehicle (N = 13) from day 3 to day 6 after GN induction. Rats were sacrificed 6 days after induction of GN. On kidney sections, cells staining positive for proliferating cell nuclear antigen, mesangial cells, monocytes, and apoptotic cells were counted. Parameters of inflammation and fibrosis were measured in renal cortex and sieved glomeruli by real-time polymerase chain reaction (PCR). RESULTS: Systolic blood pressure, diuresis, albuminuria, creatinine clearance, microaneurysm formation, and mesangial matrix expansion were not influenced by ADM infusion. However, ADM treatment significantly reduced the number of MC, showed a tendency to reduce total glomerular cell proliferation, and significantly increased apoptosis. ADM-treated GN animals showed significantly less glomerular monocyte infiltration. ADM treatment normalized transforming growth factor (TGF)-beta1 mRNA expression and reduced monocyte chemoattractant protein-1 (MCP-1), osteopontin, plasminogen activator inhibitor-1 (PAI-1), collagen I, and collagen III mRNA expression significantly. CONCLUSION: Exogenous ADM infusion reduces MC number and glomerular monocyte infiltration in the state of mesangial proliferation during acute experimental mesangioproliferative GN. These findings indicate that ADM can influence the course of mesangioproliferative GN.     

Lymphokine (MIF) production by glomerular T-lymphocytes in experimental glomerulonephritis

Glomerular T-lymphocyte infiltration has recently been demonstrated to precede glomerular macrophage influx in a pre-immunized model of anti-glomerular basement-membrane antibody-induced glomerulonephritis (antiGBM-GN). In the current study, the functional role of these glomerular T-lymphocytes in directing macrophage localization was sought by measuring their production of macrophage migration inhibition factor (MIF). MIF activity in supernatants from cultured isolated glomeruli was measured in a conventional capillary tube bioassay. Glomerular T-lymphocytes (OX19 positive cells) were maximal (1.95 +/- 0.19 cells/glomerular cross section, c/gcs) 24 hours after injection of antiGBM antibody into sensitized animals. Seventy-two hours after antibody injection, T-lymphocyte numbers were reduced (1.02 +/- 0.14 c/gcs) while macrophage accumulation was maximal (at 24 hrs 4.2 +/- 1.3 macrophages/glomerulus (m/g), at 72 hrs 19.8 +/- 3.7 m/g). MIF activity was only detected in supernatants from T-lymphocyte infiltrated glomeruli (12 hrs 40.81 +/- 4.32% migration inhibition, 24 hrs 45.11 +/- 4.11% migration inhibition, 48 hrs 38.24 +/- 3.53% migration inhibition, 72 hrs 20.86 +/- 3.85% migration inhibition, all P less than 0.05). Control glomeruli from normal animals, pre-immunized animals given normal sheep globulin, pre-immunized animals given anti-GBM antibody and Cyclosporin A, and non-pre-immunized animals given antiGBM antibody did not contain glomerular T-lymphocytes, and their supernatants contained no MIF activity. This data indicates that the glomerular T-lymphocytes in pre-immunized antiGBM-GN are sensitized cells which release MIF and thus may direct glomerular macrophage localization in this model of antibody-induced glomerulonephritis.     

Selective cyclooxygenase-2 inhibition impairs glomerular capillary healing in experimental glomerulonephritis

Selective cyclooxygenase-2 (COX-2) inhibitors have anti-inflammatory activity and reduce proteinuria in experimental membranous glomerulonephritis. Antiangiogenic properties of COX-2 inhibitors were recently reported. Whether these properties are relevant to the glomerular healing process in inflammatory glomerular diseases was investigated. For evaluation of the effects of selective COX-2 inhibitors on the glomerular healing process in a rat model of mesangioproliferative glomerulonephritis (induced by anti-Thy 1.1 antibody), a selective COX-2 inhibitor (rofecoxib or celecoxib) or vehicle was administered daily from day 1 after disease induction until euthanasia on day 6. Additional nephritic rats were treated with rofecoxib or vehicle from day 1 to day 10 and were monitored until day 28. Selective COX-2 inhibition led to significant increases in mesangiolysis (up to +71%) on days 2 and 6 and in albuminuria (up to 3.1-fold) on day 6. This augmentation of glomerular capillary damage was associated with rarefaction of glomerular endothelial cells, whereas the proliferation and activation of mesangial cells were not affected. No significant effects on the glomerular influx of polymorphonuclear neutrophils or the infiltration and proliferation of monocytes/macrophages at day 2 were noted. These effects were independent of systemic hemodynamic features, because rofecoxib did not affect systolic BP on day 2 or 5. Nephritic rats treated with rofecoxib for 10 d demonstrated persistent glomerular injury at day 28, as indicated by increased albuminuria (10-fold) and mesangial type IV collagen deposition (+24%). In normal rats, 5-d administration of rofecoxib failed to induce albuminuria or morphologic renal damage. In conclusion, selective COX-2 inhibitors impair glomerular capillary repair after mesangiolysis in rats with anti-Thy 1.1 glomerulonephritis. These data suggest that selective COX-2 inhibitors should be used with caution among patients with inflammatory endocapillary glomerular disorders.     

Glomerular extracellular matrix accumulation in experimental anti-GBM Ab glomerulonephritis

Thickening of the glomerular basement membrane (GBM) results from excessive accumulation of extracellular matrix (ECM) proteins following glomerular injury. We studied the temporal relationship between the expression of growth factors, ECM accumulation, ECM degrading proteinases, and their inhibitors in a rat model of anti-GBM antibody (Ab) glomerulonephritis (GN) by the RNase protection assay and immunohistochemistry. There were two- or fourfold increases in the expression of transforming growth factor-beta(1) (TGF-beta(1)) and platelet-derived growth factor (PDGF) A and B chain mRNAs 4 days after anti-GBM Ab administration. These changes were temporally associated with increased accumulation of alpha1(III) and alpha2(IV) collagens, fibronectin, and heparan sulfate proteoglycan along the GBM. The increase in matrix accumulation was associated with little or no increases in the proteinases, urokinase plasminogen activator (u-PA) and transin, respectively. There was a 1.6x increase in the u-PA/28s mRNA ratio on day 4 in rats with anti-GBM Ab GN, but this was not associated with an increase in u-PA biologic activity. By comparison, the mRNAs of the proteinase inhibitors, plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase (TIMP) were 5x greater than that of control rats on day 4. PAI-1 mRNA correlate with increased biologic activity. These data demonstrate a temporal association between TGF-beta(1) and PDGF expression and matrix accumulation within the GBM in anti-GBM Ab GN. In addition, it suggest that this matrix accumulation results from an imbalance between matrix synthesis and degradation.     

A case of idiopathic membranoproliferative glomerulonephritis with a transient glomerular deposition of nephritis-associated plasmin receptor antigen

The differential diagnosis of acute poststreptococcal glomerulonephritis (APSGN) and idiopathic membranoproliferative glomerulonephritis (MPGN) is sometimes difficult, as they share several key features in their laboratory and histological findings, especially during the acute phase of the diseases. We herein report an idiopathic case of MPGN in which the glomerular deposition of nephritis-associated plasmin receptor (NAPlr), a recently identified nephritic antigen for APSGN, was demonstrated. A 24-year-old postpartum woman developed nephrotic syndrome and hypocomplementemia. Although she showed no apparent findings of a prior infection, her serum titer of antistreptolysin O antibody was elevated. Renal biopsies were performed twice at intervals of 6 months, both of which showed findings fully consistent with those of MPGN. Of note, fluorescent immunostaining for NAPlr was positive in the glomeruli of the first biopsy but not in the second. Despite the use of a corticosteroid, hypocomplementemia persisted for more than 1 year. It was therefore suggested that a streptococcal infection may have influenced the development of glomerular injury in this idiopathic case of MPGN.     

DNAzyme for TGF-beta suppressed extracellular matrix accumulation in experimental glomerulonephritis

BACKGROUND: We developed an electroporation-mediated gene transfer method targeting glomerular mesangial cells. Injecting DNA solution via renal artery followed by electric pulses using tweezers-type electrodes could result in efficient transfection in mesangial cells. Therefore, this gene transfer system opened a feasible strategy to manipulate the function of several cytokines and growth factors in mesangial cells. Recently, a new generation of catalytic nucleic acid composed of DNA, named DNA enzyme (DNAzyme), has been developed. METHOD: We generated a DNAzyme (TGFDE) targeting transforming growth factor-beta1 (TGF-beta1), and examined the therapeutic effect of TGFDE in vitro and in vivo. RESULTS: In cultured rat mesangial cells, treatment with TGFDE blocked TGF-beta1 mRNA expression, and thereby suppressed type I collagen mRNA expression. Next, we introduced TGFDE or scrambled DNAzyme (TGFSCR) into anti-Thy-1 model of nephritic rats by electroporation 3 days after disease induction. Northern blot analysis and immunohistochemical staining demonstrated that glomerular message and protein expression of TGF-beta1, alpha-smooth muscle actin (alpha-SMA), and type I collagen were suppressed in TGFDE-transfected nephritic rats compared with untreated nephritic rats and TGFSCR-transfected rats on day 7. Consequently, we observed significant reduction in glomerular matrix score in TGFDE-transfected nephritic rats. CONCLUSION: Inhibition of TGF-beta1 expression by electroporation-mediated DNAzyme transfer might be useful for the therapy of glomerulonephritis.     

Calpain activation and secretion promote glomerular injury in experimental glomerulonephritis: evidence from calpastatin-transgenic mice

Glomerular injury and albuminuria in acute glomerulonephritis are related to the severity of inflammatory process. Calpain, a calcium-activated cysteine protease, has been shown to participate in the development of the inflammatory process. Therefore, for determination of the role of calpain in the pathophysiology of acute glomerulonephritis, transgenic mice that constitutively express high levels of calpastatin, a calpain-specific inhibitor protein, were generated. Wild-type mice that were subjected to anti-glomerular basement membrane nephritis exhibited elevated levels of calpain activity in kidney cortex at the heterologous phase of the disease. This was associated with the appearance in urine of calpain activity, which originated potentially from inflammatory cells, abnormal transglomerular passage of plasma proteins, and tubular secretion. In comparison with nephritic wild-type mice, nephritic calpastatin-transgenic mice exhibited limited activation of calpain in kidney cortex and limited secretion of calpain activity in urine. This was associated with less severe glomerular injury (including capillary thrombi and neutrophil activity) and proteinuria. There was a reduction in NF-kappaB activation, suggesting that calpain may participate in inflammatory lesions through NF-kappaB activation. There also was a reduction in nephrin disappearance from the surface of podocytes, indicating that calpain activity would enhance proteinuria by affecting nephrin expression. Exposure of cultured podocytes to calpain decreased nephrin expression, and, conversely, exposure of these cells to calpastatin prevented TNF-alpha from decreasing nephrin expression, demonstrating a role for the secreted form of calpain. Thus, both activation and secretion of calpains participate in the development of immune glomerular injury.     

Effects of retinoids on the TGF-beta system and extracellular matrix in experimental glomerulonephritis.

Transforming growth factor-beta1 (TGF-beta 1) overexpression plays a key role in the glomerular accumulation of extracellular matrix proteins in renal disease. Retinoids have previously been shown to significantly limit glomerular damage in rat experimental glomerulonephritis. Therefore, the effects of all-trans retinoic acid and isotretinoin on the components of the TGF-beta system and extracellular matrix proteins in anti-Thy1.1-nephritis (Thy-GN) were investigated. Vehicle-injected control rats were compared with rats treated with daily subcutaneous injections of 10 mg/kg body wt all-trans retinoic acid or 40 mg/kg body wt isotretinoin (n = 9 per group) either with a pretreatment (day -2 through 8) or posttreatment protocol (day +3 through 8), i.e., starting before or after induction of Thy-GN, respectively. Urinary TGF-beta 1 excretion was 60% lower in all-trans retinoic acid-treated animals with Thy-GN (P < 0.025). The increase of cortical TGF-beta 1 gene expression in Thy-GN rats was significantly attenuated with all-trans retinoic acid and even more with isotretinoin treatment as compared with untreated animals (P < 0.025). Cortical expression of TGF receptor II, but not receptor I gene expression, was significantly lower in animals treated with all-trans retinoic acid or isotretinoin (P < 0.05). In all-trans retinoic acid-treated animals with Thy-GN, the increase of glomerular TGF-beta 1 protein (P < 0.008) and TGF-beta 1 (P < 0.025) and TGF receptor II mRNA (P < 0.015) was significantly less. Immunohistochemistry revealed less glomerular staining for TGF-beta 1 and TGF receptor II in the presence of all-trans retinoic acid. TGF-beta 1 immunostaining was not restricted to monocytes and macrophages, as indicated by double-staining. Glomerular staining for collagen IV and collagen III was less in animals treated with isotretinoin (P < 0.02 for both) in contrast to all-trans retinoic acid, whereas fibronectin remained unchanged. It was concluded that the beneficial effects of retinoids on glomerular damage are presumably due to a marked reduction in renal TGF-beta 1 and TGF receptor II expression.     

Serum and glomerular IgG in poststreptococcal glomerulonephritis are correlated

Among 75 patients hospitalized for poststreptococcal acute glomerulonephritis, 33 (44%) had an elevated serum level of IgG. The IgA level was elevated in 11 of 39 patients (28%). Paradoxically, of 20 biopsied patients with elevated IgG levels, IgG was absent from the glomerular deposits in 11, while of 23 with normal IgG levels, IgG was absent from the glomerular deposits in only 2 (P <0.001). Also, patients with an elevated level more frequently had antistreptolysin O titers ≥833 Todd units (P <0.001). Elevated IgG did not correlate with severity of disease, with age of the patient, or with the serum albumin or C3 level. There appears to be a subset of patients with elevated serum IgG levels who with high frequency have IgG absent from their glomerular deposits. Thus, failure to form antibody to a glomerular-bound protein produced by the nephritogenic streptococcus, widely assumed to be the origin of the IgG in the glomerular deposits, is in some way significantly associated with elevated serum levels of IgG and of antibody to streptolysin O. Received September 23, 1997; received in revised form October 23, 1997; accepted November 19, 1997     

Identification of glomerular immune deposits in cryoglobulinemia glomerulonephritis

To provide further evidence of the nature of intraglomerular immune deposits in essential mixed cryoglobulinemia (EMC), we used two mouse monoclonal antibodies against cross-reactive idiotypes present on monoclonal rheumatoid factors (MoRFs) from patients with type II-EMC. MoAb Cc1 reacted with 9 of 16 circulating IgMk MoRFs tested, and MOAb Lc1 with four of the remaining. Using indirect immunofluorescence and immunoperoxidase techniques, we could identify the same cross-reactive idiotype of the serum MoRF in the renal biopsy specimens from 11 of 13 patients with EMC glomerulonephritis. Kidney specimens from the three patients, whose MoRF was not recognized by MoAbs Cc1 and Lc1, were negative. Two out of 30 control renal biopsies from patients with other forms of glomerulonephritis were shown to contain idiotype (Cc1 and Lc1) positive material. Both patients had serum polyclonal RF which could account for this finding. In conclusion, our results provide direct evidence that serum cryo-MoRF participate in the formation of glomerular immune deposits and, presumably, in the pathogenesis of renal damage in EMC glomerulonephritis.     

Oral administration of glomerular basement membrane prevents the development of experimental autoimmune glomerulonephritis in the WKY rat

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by a single injection of collagenase-solubilized rat glomerular basement membrane (GBM) in adjuvant. EAG is characterized by circulating and deposited anti-GBM antibodies, accompanied by focal necrotizing glomerulonephritis with crescent formation. The inhibitory effect of orally administered antigens has been reported in various animal models of autoimmunity but not in EAG in the rat. The effects of feeding rat GBM by gavage, at total doses of 0.5, 2.5, or 5 mg, before immunization were examined. A dose-dependent effect was observed on the development of EAG. A dose of 0.5 mg of GBM had no effect on disease, 2.5 mg resulted in a moderate reduction in the severity of nephritis but no change in anti-GBM antibody production, and 5 mg resulted in a marked reduction in circulating and deposited anti-GBM antibodies, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of infiltrating T cells and macrophages. Animals that were fed 5 mg of GBM showed a significant reduction in IgG2a but not IgG1, anti-GBM antibody levels, suggesting downregulation of Th1 responses. There was also a dose-dependent reduction in the proliferative responses of splenic T cells from treated animals to GBM antigen in vitro. These results clearly demonstrate that mucosal tolerance can be induced by oral administration of GBM antigen and that this approach is effective in preventing EAG.     

Endogenous Tim-1 (Kim-1) promotes T-cell responses and cell-mediated injury in experimental crescentic glomerulonephritis

The T-cell immunoglobulin mucin 1 (Tim-1) modulates CD4(+) T-cell responses and is also expressed by damaged proximal tubules in the kidney where it is known as kidney injury molecule-1 (Kim-1). We sought to define the role of endogenous Tim-1 in experimental T-cell-mediated glomerulonephritis induced by sheep anti-mouse glomerular basement membrane globulin acting as a planted foreign antigen. Tim-1 is expressed by infiltrating activated CD4(+) cells in this model, and we studied the effects of an inhibitory anti-Tim-1 antibody (RMT1-10) on immune responses and glomerular disease. Crescentic glomerulonephritis, proliferative injury, and leukocyte accumulation were attenuated following treatment with anti-Tim-1 antibodies, but interstitial foxp3(+) cell accumulation and interleukin-10 mRNA were increased. T-cell proliferation and apoptosis decreased in the immune system along with a selective reduction in Th1 and Th17 cellular responses both in the immune system and within the kidney. The urinary excretion and renal expression of Kim-1 was reduced by anti-Tim-1 antibodies reflecting diminished interstitial injury. The effects of anti-Tim-1 antibodies were not apparent in the early phase of renal injury, when the immune response to sheep globulin was developing. Thus, endogenous Tim-1 promotes Th1 and Th17 nephritogenic immune responses and its neutralization reduces renal injury while limiting inflammation in cell-mediated glomerulonephritis.