Genetic dissection of the complex pathological manifestations of collagen disease in MRL/lpr mice

An MRL strain of mice bearing a Fas-deletion mutant gene, lpr, MRL/MpJ-lpr/lpr (MRL/lpr) develops collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren’s syndrome, respectively. Development of such lesions seems dependent on host genetic background since the congenic C3H/HeJ-lpr/lpr (C3H/lpr) mice rarely develop them. To identify the gene loci affecting each lesion, a genetic dissection of these complex pathological manifestations was carried out. First, histopathological features in MRL/lpr, C3H/lpr, (MRL/lpr × C3H/lpr) F1 intercross, and MRL/lpr × (MRL/lpr × C3H/lpr) F1 backcross mice were analyzed. Genomic DNA of the backcross mice were subjected to association studies by Chi-squared analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. As a result, gene loci recessively associated with each lesion were mapped on different chromosomal positions. We concluded that each of these lesions in MRL/lpr mice is under the control of a different set of genes, suggesting that the complex pathological manifestations of collagen disease result from polygenic inheritance.     

Interleukin-10 inhibits experimental mesangial proliferative glomerulonephritis

Conflicting reports exist regarding the effects of interleukin-10 (IL-10) on mesangial cells. There have been reports of both proliferative and antiproliferative effects, and both proinflammatory and anti-inflammatory effects of IL-10 on mesangial cells. However, the potential for IL-10 to affect glomerulonephritis characterized by mesangial proliferation is not known. To test the hypothesis that IL-10 would limit experimental mesangial proliferative glomerulonephritis, IL-10 was administered to rats in which mesangial proliferative glomerulonephritis was induced by administration of anti-Thy 1 antibody. Compared to control treated rats, IL-10 treated rats showed less proliferation, with fewer cells in glomeruli. Glomerular cellular proliferation was reduced, assessed by the numbers of cells within glomeruli expressing either proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine. Glomerular macrophage influx (but not the proportion of glomerular macrophages that were PCNA positive) was reduced by IL-10 administration. There was no significant reduction in glomerular alpha-smooth muscle actin staining. IL-10 treatment resulted in reduced renal IL-1beta mRNA expression and reduced glomerular ICAM-1 expression, but renal expression of MCP-1 and osteopontin mRNA was unaltered. This study demonstrates that in experimental mesangial proliferative glomerulonephritis IL-10 diminishes inflammatory cell recruitment and mesangial cell proliferation. The effects of IL-10 in inhibiting mesangial cell proliferation are likely to be due to a combination of direct effects of IL-10 on mesangial cells and effects mediated by macrophages.     

Possible role of autoantibodies against nephrin in an experimental model of chronic graft-versus-host disease

Nephrin, a product of the NPHS1 gene, is a component of the slit diaphragms that are found between glomerular foot processes and is a crucial element for glomerular filtration barrier. Recently, nephrin has been focused in a number of studies of proteinuria development including various types of acquired glomerular diseases including minimal change nephrotic syndrome and membranous nephropathy. However, the precise role of nephrin in such acquired glomerular diseases is still unknown. To analyse the role of nephrin further, two kinds of anti-nephrin antibodies were raised in the rabbits and applied to an experimental mouse model of chronic graft-versus-host disease, in which (C57BL/10 x DBA/2) F1 mice developed clinically apparent severe proteinuria with significant glomerular lesions 7 weeks after parental DBA/2 cell transfer. Antibody-sandwich ELISA detected anti-nephrin antibodies during week 2 to week 6, with the peak at week 2 or week 4. Colocalization of nephrin and IgG on week 4, week 6, and week 8 was revealed by confocal microscopic analysis, suggesting that in situ immune complex formation with nephrin in glomerular lesion. Taken together, it seems to be suggested nephrin and its autoantibody have a certain role in the development of glomerular lesion in our model mice.     

Tissue-type plasminogen activator and its inhibitor in human glomerulonephritis.

We carried out an immunohistochemical study of tissue-type plasminogen activator (PA) and urokinase-type PA, and their inhibitors, PA inhibitor-1 and PA inhibitor-2, using renal biopsy specimens obtained from 86 patients with various forms of glomerulonephritis. The controls were four normal renal tissue specimens. On immunofluorescent observation, granular staining for tissue-type PA was found to be distributed along the glomerular capillary walls. The fluorescence was weak in the normal renal tissue and occasionally intense in the tissues of patients with IgA nephritis, minimal change nephrotic syndrome, and lupus nephritis. PA inhibitor-1 was abundant in the glomerular epithelial cells and scarce in the mesangial area and glomerular capillary lumens of the normal renal tissues. This was confirmed by immunoelectron microscopy using gold staining. The fluorescence of PA inhibitor-1 was weaker in some specimens of nephritic tissues than in the normal renal tissues. Urokinase-type PA and PA inhibitor-2 were negative within the glomeruli in all the specimens. In the glomerulonephritic tissues which were fibrin deposition-positive, tissue-type PA expression in the glomeruli tended to be strong. An association between fibrin deposition and PA inhibitor-1 staining was not clear. These data suggest that expression of tissue-type PA in the glomeruli increases in association with fibrin deposition.     

Fc receptor-mediated accumulation of macrophages in crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody administration in WKY rats

Anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats is characterized by glomerular accumulation of CD8(+) T cells and monocytes/macrophages, followed by crescent formation. The mechanism of leukocyte accumulation after antibody binding to GBM is still unclear. To unveil an involvement of Fcgamma receptors (FcgammaR) in leukocytes recruitment we examined the expression of FcgammaR in glomeruli and the effects of the administration of F(ab')(2) fragment of anti-GBM antibody or FcgammaR blocking on the initiation and progression of this model. A gradual increase of FcgammaR mRNA expression in glomeruli during the time course of disease suggested their significance in the development of glomerulonephritis. Glomerular lesions and proteinuria were induced only in rats injected with intact IgG of anti-GBM antibody, but not with the F(ab')(2) fragment. In vivo blocking of FcgammaR by administering heat-aggregated IgG led to the decrease of mRNA expression for all types of FcgammaR (types 1, 2 and 3) and a significant amelioration of glomerulonephritis manifestations. By flow cytometry and immunohistochemistry FcgammaR2-expressing cells in glomeruli were identified as macrophages, but not CD8(+) T cells. The expression of FcgammaR1 and 3 was significantly decreased, and that of FcgammaR2 became undetectable in CD8(+) T cell-depleted rats. Thus, CD8(+) T cells may stimulate FcgammaR expression on macrophages, contributing to their glomerular accumulation and injury. These studies provide direct evidence for a crucial involvement of IgG Fc-FcgammaR interaction in glomerular recruitment of macrophages and following induction of anti-GBM glomerulonephritis in WKY rats.     

Basement membrane charge in human glomerular disease

Fixed anionic charges in the mammalian glomerulus, on both the basement membrane and the epithelial cell foot processes, are believed to form an important part of the glomerular filtration barrier. There is good evidence that their loss causes proteinuria. The charges can be visualized ultrastructurally using cationic dyes, but the requirement of these techniques for perfusion or immersion of fresh tissue has largely confined such studies to experimental models. We have extended the widely used polyethyleneimine technique, to study the charge of glomerular basement membranes in human tissue reprocessed out of paraffin blocks up to 10 years old. We studied selected cases of glomerular disease, where the diagnosis was not in any doubt. In the majority of diseases studied, a continuous charge layer persisted despite severe abnormalities of the basement membrane. Two exceptions were found. In amyloidosis, accumulation of fibrils was associated with a considerable decrease or loss of stainable basement membrane charge. In S.L.E., numerous small defects in the charge layer were noted. The persistence of charge is contrary to reported findings in several animal models of glomerular disease, including puromycin nephrosis, Heymann nephritis and streptozotocin diabetes. Although this method is not subject to precise quantitative analysis, we conclude that in the majority of cases, proteinuria in man is not caused by an extensive loss of glomerular basement membrane anionic charge.     

Serum creatinine concentration and renal interstitial volume

Summary Renal biopsies of 44 patients with endocapillary acute glomerulonephritis (gn) and 64 patients with moderately severe mesangioproliferative gn were investigated morphometrically (point-counting-method, tubulometry).In both gn's statistically significant positive correlations between relative interstitial volume and the concentration of serum creatinine at the time of biopsy were found.Despite severe glomerular lesions the serum creatinine concentration is not increased in most cases of endocapillary acute gn, providing the relative interstitial volume is not increased by more than 15%.Increased serum creatinine concentration without a markedly enlarged interstitium was found in 11 cases of endocapillary acute gn with clinically and morphologically proven acute renal failure. In these cases the glomerular function is probably impaired by the Thurau-mechanism.In all other patients, especially in those with moderately severe mesangioproliferative gn, the serum creatinine concentration rises with an enlargement of relative interstitial volume. This reduction of renal function may be explained by a decrease to the total cross-sectional area of postglomerular vessels, caused by interstitial fibrosis. That may possibly lead to diminished renal blood flow and glomerular filtration with an increase of the serum creatinine concentration.Supported by the Deutsche Forschungsgemeinschaft     

Role of group A streptococcal IgG-binding proteins in triggering experimental glomerulonephritis in the rabbit

Our previous studies have indicated that the IgG-binding M-family proteins (IgGBP) of group A streptococci may be involved in eliciting experimental acute poststreptococcal glomerulonephritis (APSGN) in the rabbit. These surface proteins were also found to trigger production of anti-IgG, which might conceivably act to enhance renal deposition of immune complexes (IC). In the present study, a clinical isolate of serotype M22 (strain AL168), an isogenic double mutant deficient for both the IgGBPs Mrp and Emm, as well as mutants deficient in only one of the proteins were tested for capacity to induce glomerulonephritis. Streptococci to be used for injecting rabbits were heat-killed. Surface-bound IgG was removed by 1 M KSCN and cells were then repeatedly washed in PBS before use. Rabbits were injected intravenously with 109 cells three times a week for 8 weeks and, following one month of rest, for another 6 weeks. Deposits of IgG and C3 as well as induced chemokines TNF-alpha, IL-1beta and IL-6 were traced in cryostat sections using specific antibodies and appropriate peroxidase-labelled anti-antibodies. In four rabbits immunized with the double mutant strain, no deposits were found, and as examined by TEM, only subtle and transient renal changes were observed. In contrast, the original strain AL168 induced pronounced inflammatory and degenerative glomerular changes in all four rabbits injected, and deposits of TNF-alpha, IL-1beta and IL-6 were found in mesangial and endothelial cells. Similar deposits and glomerular changes were seen in all eight rabbits injected with the mrp-emm+ mutant and in four out of seven animals receiving the mrp+emm- mutant. There was a highly significant correlation between high levels of circulating anti-IgG and development of APSGN. These results confirm an important role of streptococcal IgGBP in triggering experimental APSGN as earlier proposed by our group.     

四种祛风类中药治疗原发性肾小球肾炎的临床研究

目的 :评价口服火把花根、祖师麻、清风藤碱和雷公藤多甙单独与联合用药治疗原发性肾小球肾炎的效果。方法 :选择 6 5 6例经单味或两味祛风类中药治疗的原发性肾小球肾炎患者 ,观察不同治疗患者的临床表现、尿蛋白定量、尿常规、肝肾功能等指标变化。结果 :单味药治疗肾炎的缓解率为 2 4.0 %～ 44 .5 % ,两味药联合治疗肾炎的缓解率为 5 1.9%～ 5 9.2 % ;清风藤碱治疗的各种临床表现的肾炎缓解率均低于其他治疗组 ,单味药火把花根、祖师麻和雷公藤多甙对单纯性蛋白尿的缓解率为 5 0 .0 %～ 6 9.7% ,对单纯性血尿的缓解率为 36 .0 %～ 5 0 .0 % ,对蛋白尿合并血尿的缓解率为 19.5 %～ 2 2 .7% ,联合用药对各种表现的缓解率均高于单味药组 ,对单味药未缓解的部分病例两味药治疗仍有一定效果 ,复发率低于单味药组 ;联合用药组消化道反应、肾功能损害、停经和白细胞减少发生率与单味药组均无明显差别 ,转氨酶增高发生率增加 ,但谷丙转氨酶 >10 0 U /L者少见。结论 :四种祛风类单味药对肾小球肾炎均有一定疗效 ,联合治疗能够显著增加对肾小球肾炎的疗效     

Excretion of epidermal growth factor-like material in acute Henoch-Schönlein purpura nephritis

In Henoch-Schönlein purpura nephritis (HSPN), glomeruli may develop cellular crescents composed of infiltrating monocytes and proliferating renal epithelia. In this study, we demonstrate that peripheral human monocytes can release an epidermal growth factor (EGF)-like substance detectable by a radioreceptor assay, which recognizes both EGF and transforming growth factor-alpha (TGF-alpha), but not with a radioimmunoassay, which recognizes only EGF. Furthermore, we report that urine from pediatric patients during the acute phase of HSPN contains a similar EGF-like species in addition to the endogenous EGF which is normally present. The EGF-like material was not present in urine from nine healthy children or from six children with acute post-streptococcal glomerulonephritis. The extent of crescent formation in our patients is uncertain, since renal biopsy was performed in only one case. However, we speculate that the urinary material resembling TGF-alpha which appears during the acute phase of HSPN may derive from monocytes infiltrating the kidney.