Quantitative studies of in situ immune complex glomerulonephritis in the rat induced by planted, cationized antigen.

Cationized human IgG can bind to the rat glomerular basement membrane (GBM), act as planted antigen, and induce in situ immune complex formation accompanied by severe glomerulonephritis. Perfusion of highly cationized human IgG (isoelectric point {more than} 9.5) via the left renal artery resulted in preferential localization within the perfused kidney (up to 56 percent of dose injected); after intravenous administration, only 4 percent was bound to the kidneys. The planted antigen was localized along the glomerular capillary walls and was accessible for antibody administered intravenously 1 h after perfusion, when virtually no antigen remained in the circulation. Persistence of cationized human IgG in the perfused kidney was markedly prolonged when complexed with antibody; one-half the cationized human IgG was still present after 12 d. There was a difference in the disappearance rates of antigen and antibody, as cationized human IgG was removed faster from the kidney than the antibody, the binding of which remained almost unchanged during the first week. Renal perfusion of a minimum of 20 mug of cationized human IgG, followed by intravenous injection of antibody, regularly induced severe glomerulonephritis with a proteinuria of at least 100 mg/24 h. The degree and the persistence of proteinuria induced depended on the dose of cationized human IgG perfused. Experiments using radiolabeled antigen and antibody showed that after renal perfusion of 20 mug cationized human IgG, 11.1 mug was kidney bound at the time of antibody injection. At the onset of proteinuria, 4.0 mug of antigen and 31.9 mug of anti-human IgG antibody were present in the perfused kidney. Immunofluorescence revealed immune deposits consisting of cationized human IgG and rabbit IgG (anti-human IgG) along the GBM. The staining pattern was linear (confluent) during the first 2 d and became granular during the course of the disease. Electronmicroscopically, a prominent finding was the accumulation of dense deposits, mainly in the subepithelial space and beneath the slit pores.

Attachment of polymorphonuclear leukocytes to glomeruli with immune deposits.

A method for quantitative evaluation of a biological activity of immune complexes deposited in glomeruli is described. The activity reflects the activation of complement and is represented by the number of PMN attached to a glomerulus. It is possible to compare data from different individuals or different phases of glomerulonephritis. The complement component concerned is considered to be C3, activated through the classical or alternate pathway.

Selective planting of cationized, haptenized ovalbumin on the rat tubular basement membrane.

We developed an experimental protocol for planting exogenous antigens with different molecular weights and charges on the constituents of the renal tubulointerstitium. The cationized antigens were injected selectively into the left renal arteries of Wistar rats. Antigen localization was documented by immunohistochemistry on frozen sections. Cationized bovine serum albumin (BSA; 68 kDa, isoelectric point = 9.5) localized almost exclusively along the glomerular capillary wall. After application of highly cationic polyethyleneimine, cationized BSA given subsequently was found in a linear distribution along the glomerular capillary wall and along the peritubular capillaries. The fate of highly cationized ovalbumin conjugated with trinitrophenol (TNP-OA), subjected to gel filtration to obtain monomers (42 kDa, isoelectric point > 10) differed; it was deposited in a linear pattern on the tubular basement membrane (TBM) and Bowman's capsule, and remained up to 36 h after injection. Noncationized, monomeric TNP-OA (42 kDa, isolectnic point = 4.6) showed fine granular deposition in the tubular epithelium exclusively. These findings indicate that the barrier of the glomerular BM acts selectively on antigens with different molecular weights. They either settle on the peritubular capillaries, after passing the glomerular, or reach the urinary space, after which they are reabsorbed by the tubular epithelial cells to reach the TBM.

Ultrastructural alterations of glomerular anionic sites in idiopathic membranous glomerulonephritis.

The relation of ultrastructural alterations of glomerular anionic sites to morphological changes of the glomerular basement membrane (GBM) was studied in biopsy specimens from patients with idiopathic membranous glomerulonephritis (MGN) using polyethyleneimine (PEI) as a cationic probe. The subepithelial deposits did not fix PEI, and only a very few anionic sites were found between subepithelial deposits and adjacent epithelial cells. Thus, anionic sites of the lamina rara externa (LRE) were markedly decreased in number and interrupted by subepithelial deposits in the earlier stages (stage I and II). As the deposits were incorporated into the GBM in the later stage (stage III), the anionic sites of the LRE were restored on the epithelial cell surface coat (ESC) covering the deposits, but foot process effacement persisted during the course of the disease. The anionic sites of the lamina rara interna (LRI) appeared much less altered in all stages of MGN. These findings suggest that subepithelial deposition of immune reactants is associated with focal loss and disorganization of anionic sites on the GBM, resulting in a defect of glomerular-barrier function.

Ultrastructural alterations of glomerular anionic sites in IgA nephropathy.

The ultrastructural alterations of glomerular anionic sites were studied in biopsy specimens from 34 patients with IgA nephropathy using polyethyleneimine (PEI). Prominent common findings in the glomeruli of the patients were few PEI particles in electron dense deposits in the mesangial and subepithelial area and marked reduction in glomerular anionic sites covered with deposits. The anionic sites of the glomerular basement membrane (GBM) and epithelial cell surface coat (ESC) appeared unaltered in the patients with hematuria and/or mild proteinuria. But in patients with proteinuria in the nephrotic range, focally discrete loss of anionic sites in the lamina rara externa (LRE) was seen and the number of anionic sites of the ESC were decreased with retraction of the foot processes. The anionic sites of the lamina rara interna showed much less change in these patients. Subepithelial deposits were often seen concomitantly with focal loss of anionic sites in the LRE at the site of the deposits, but subendothelial deposits had little influence on the anionic sites of the neighboring GBM. The anionic sites of GBM that showed focal thinning with small GBM projections were appreciably decreased in number, but those in split GBM were not decreased. These results suggest that either loss of the negative charge on the glomerular capillary wall associated with subepithelial immune deposition or morphological changes of the GBM contribute to the progression of proteinuria in IgA nephropathy.

Production of transforming growth factor-beta by cultured rat mesangial cells.

The purpose of this study was to examine whether transforming growth factor-beta (TGF beta) acts as an autocrine cytokine in cultured mesangial cells. Measangial cell conditioned media (CM) were prepared and tested for their effect on mesangial cell proliferation. CM showed a concentration dependent inhibition on mesangial cell proliferation and the activity was enhanced by treating conditioned media with acid. Gel filtration analysis showed peak inhibitory activity to reside in fractions with an estimated molecular weight range of 16-30 KD. The activity was partially blocked by anti-TGF beta antibody, but not nonimmune control IgG. The presence of TGF beta was confirmed using the mink lung epithelial cell assay. Furthermore, the addition of anti-TGF beta antibody directly into culture media significantly enhanced mesangial cell proliferation. These results demonstrate that measangial cell produce both active and latent forms of TGF beta, which functions as an autocrine growth inhibitor for mesangial cells.

Bone marrow cell infusion ameliorates progressive glomerulosclerosis in an experimental rat model.

Bone marrow (BM) cells contribute to the maintenance and repair of several compartments of the kidney, including the endothelium, interstitium, epithelium, and the mesangium. The aim of this study was to explore the therapeutic use of bone marrow-derived cells (BMDC) that can differentiate into endothelial and mesangial cells in a model of progressive glomerulosclerosis. To investigate the involvement of BMDC in glomerular repair, progressive glomerulosclerosis was induced in enhanced green fluorescent protein BM chimeric rats by a one-shot injection of anti-Thy-1.1 monoclonal antibody, followed by unilateral nephrectomy. Subsequently, these rats were treated with either a BM cell infusion or phosphate-buffered saline. Renal function, intravital glomerular hemodynamics, and histological alterations were examined 12 weeks after anti-Thy-1.1 monoclonal antibody injection. Inflammatory infiltration of macrophages in the kidneys was evaluated by immunofluorescence of ED-1. We also determined whether BMDC contributed to repair and regeneration of endothelial and mesangial cells by immunofluorescence monitoring. As a result, BM cell infusion improved renal function and glomerular hemodynamics, and histological alterations with reduced glomerular infiltration of macrophages, leading to dramatically reduced mortality in this model of progressive glomerulosclerosis. We also demonstrated that, in the BM cell infusion group, more BMDC contributed to repair and regeneration of endothelial and mesangial cells than in the untreated group. The present study provides us with a conceptual basis for the development of therapeutic stem cell strategies aimed at enhancing recovery from progressive glomerulosclerosis.

Local delivery of angiotensin receptor blocker into the kidney ameliorates progression of experimental glomerulonephritis.

Intrarenally synthesized angiotensin II (Ang II) may be involved in the progression of glomerulonephritis, leading to irreversible glomerulosclerosis. There is increasing evidence that systemic angiotensin receptor blocker (ARB) treatment has beneficial effect on the prognosis of progressive glomerulonephritis and diabetic nephropathy. However, the cellular and molecular mechanisms behind this therapeutic effect of ARB remain unclear. In this study, we used a novel strategy of local ARB delivery via type-1 collagen sponge, to treat progressive glomerulonephritis that would result in irreversible glomerulosclerosis in our previously established rat model. At days 9 and 14 after disease induction, mild proteinuria, 20.7+/-4.7 and 10+/-1.3 mg/day, was found. Local ARB treatment reduced proteinuria significantly to 3.19+/-3.2 and 5.25+/-0.95 mg/day (P < 0.01), respectively. Scoring of glomerular matrix expansion and sclerotic index revealed that local ARB treatment significantly ameliorated glomerular pathology. Ang II type 1 receptor mRNA expression was remarkably enhanced in the Ang II group and ARB treatment reversed this effect at 14 days. Local delivery of ARB significantly improved glomerular blood flow levels, compared to the untreated disease control group, from 710+/-18.25 to 859.44+/-22.86 microm/s, respectively. Local delivery of ARB into the kidney affected local RAS and thus improved the renal injury and function in the potentially progressive glomerulosclerosis of rat model.

Turbulence of glomerular hemodynamics involved in progressive glomerulosclerosis.

There is increasing evidence that changes of glomerular hemodynamics or glomerular growth responses may promote the development of glomerulosclerosis. Major problems retarding research progress include lack of suitable experimental animal models, with the exception of the ablation model, and the need for in vivo real-time analysis of glomerular hemodynamics. This study examined the sequence of pathological changes from the viewpoints of microcirculation and histopathology, from the acute stage to the chronic course and the final stage of glomerulosclerosis, using the confocal laser scanning microscope system. There is a marked difference in prognosis between sham-operated (two-kidney) and nephrectomized (one-kidney) rats after injection with anti-Thy-1 antibody. The former reversibly returns to normal and the latter irreversibly go to progressive sclerosis, respectively. The turning point determining the progression of glomerulosclerosis in both groups seemed to be the period from 7 to 14 days after disease induction, when disturbance of local intraglomerular blood flow continued in the one-kidney groups. In conclusion, this study provides the first hemodynamic-based evidence showing that disturbance of intraglomerular microcirculation is a critical marker for progressive glomerulosclerosis.

Masugi nephritis in T cell-depleted state. Influence of adult thymectomy and anti-thymocyte serum.

The effects of surgical removal of the thymus or the administration of antiserum to thymus-derived lymphocytes on the development of Masugi nephritis were investigated in Wistar rats. While thymectomy at weaning (ATx) had no significant effect on the humoral antibody response to injected rabbit IgG, repeated injections of rabbit anti-rat thymocyte serum (ATS) suppressed it remarkably. Both treatments did not show morphological evidences that glomerular inflammation and injury were suppressed in the autologous phase of Masugi nephritis. Glomerular lesions in rats receiving the nephrotoxic serum (NTS) one month after ATx (ATx-1+NTS) appeared to be severer in hypercellularity, mitotic counts, the amount of deposited fibrin-related substances and crescent formation than those in other nephritic groups. Morphological study of ATS+NTS rats revealed that the glomerular changes were nearly equal to those of NTS-injected control rats, in spite of markedly suppressed humoral response to injected rabbit IgG and the absence of host IgG along the glomerular capillary walls.

Sensitive analysis of apoptosis using confocal laser scan microscopy.

Application of the TUNEL method and immunostaining cell-specific markers to a whole isolated glomerulus in combination with confocal laser scan microscopy can be used to analyze cell turnover including apoptosis within glomeruli. Furthermore, the technologies can be used to deepen our understanding of glomerular cell biology and pathophysiology at the cellular and molecular levels.

Studies on the 'linear pattern' in renal glomeruli demonstrated with immunofluorescence.

We have experimentally induced the 'linear pattern' in immunofluorescence: the linear deposition of endogenous immunoglobulin (Ig) along the glomerular basement membrane (GBM) in rats injected with both protamine and nephrotoxic serum. This Ig was demonstrated to have no specific antibody activity against GBM or rabbit serum. Our findings could be of value in the analysis of the mechanism and pathological meaning of the 'linear pattern' of endogenous Ig in immunofluorescence.

PDGF regulates gap junction communication and connexin43 phosphorylation by PI 3-kinase in mesangial cells.

BACKGROUND: Gap junctional intercellular communication (GJIC) plays an important role in the regulation of cell growth, migration, and differentiation. Ultrastructural and histochemical studies indicate the existence of a high density of gap junctions among mesangial cells (MCs), but little is known about their regulation. Because of the close link between growth and GJIC, we examined how platelet-derived growth factor (PDGF) may affect GJIC in cultured MCs. METHODS: MCs were exposed to PDGF in the presence or absence of phosphatidylinositol 3' kinase (PI3K) inhibitors, and GJIC was evaluated by the transfer of Lucifer yellow. The gap junction protein connexin43 (Cx43) was examined by immunohistochemistry, immunoprecipitation, and Western blot. RESULTS: The addition of PDGF into MC culture caused a rapid and transient inhibition of GJIC, with maximal inhibition (80%) occurring 15 minutes after PDGF exposure and returning to control levels after 90 minutes. This action of PDGF could be largely prevented by pretreatment of MCs with the PI3K inhibitor LY294002. Immunochemical staining showed that PDGF did not alter the localization and distribution of Cx43. Immunoprecipitation studies demonstrated that PDGF induced a rapid and transient increase of tyrosine phosphorylation of Cx43 protein, which was dose dependent and in accordance with the time course of the disruption of GJIC. PDGF also elicited activation of extracellular signal-regulated kinase (ERK). Using two structurally unrelated PI3K inhibitors, wortmanin and LY294002, both tyrosine phosphorylation of Cx43 and activation of ERK stimulated by PDGF were largely blocked. CONCLUSION: These results suggest that PDGF abrogates GJIC function in MCs via the PI3K-dependent signaling pathway. Disruption of GJIC by PDGF could be one mechanism by which PDGF modulates MC behavior. Participation of PI3K in the regulation of GJIC demonstrates the complex coordination of molecular events that accompany MC mitogenesis.

Quantitative study of mesangial injury with proteinuria induced by monoclonal antibody 1-22-3.

Murine MoAb 1-22-3 has already been reported to bind to the mesangial cell surface and to cause transient proteinuria and mesangial morphological changes characterized by mesangiolysis, subsequent mesangial cell proliferation and mesangial matrix increase by a single i.v. injection. In this study, MoAb-induced glomerulopathy was quantitatively analysed. No correlation between the severity of mesangial morphological changes and the degree of proteinuria was detected (r = 0.190). The minimum dose injected to induce abnormal proteinuria was 25 micrograms. This dose corresponded to 1.79 micrograms/2 kidneys 30 min after MoAb injection. The highest average level of proteinuria was observed in rats injected with 500 micrograms of MoAb, and less proteinuria was observed in rats injected with 10.0, 5.0 and 2.0 mg. Although the amounts of kidney-fixing MoAb and the subsequent deposition of rat C3 in the high-dose-injected group were larger than in the 500 micrograms injected group, the numbers of infiltrating inflammatory cells were the same in both groups. No correlations between the degrees of such mediators and proteinuria were observed.

Active in situ immune complex glomerulonephritis using the hapten-carrier system: role of epitope density in cationic antigens.

The role of epitope density in cationic antigen was investigated in an active model of in situ immune complex glomerulonephritis (ICGN) using the hapten-carrier system. Trinitrophenol (TNP) was conjugated with variable density to cationic human immunoglobulin (C-HIgG) to yield TNP6.2-C-HIgG (low-valency antigen) and TNP31.3-C-HIgG (high-valency antigen). In rats preimmunized with TNP17.3-bovine serum albumin (BSA), endocapillary proliferative GN with proteinuria developed in rats receiving high-valency antigen. In contrast, no significant abnormalities in renal histology or urinalysis were observed when a low-valency antigen was injected. These results indicate that glomerular injury produced by hapten-specific immune reaction is affected by the number of haptenic groups conjugated to the carrier molecule (epitope density) in active in situ ICGN.

Hapten-specific cellular immune response producing glomerular injury.

A new model of experimental glomerulonephritis (GN) in which a cell-mediated (delayed-type) reaction predominated was established. Cationized TNP-BSA conjugates were planted in the kidney of rats by perfusion via the renal artery. Rats which had been sensitized with the TNP hapten 7 days previously revealed marked exudative and proliferative changes in their glomeruli, accompanied by transient proteinuria. A cellular influx was seen without deposition of any autologous antibody. The resulting data are compatible with the idea that cell-mediated immune reactions can participate in tissue damage in glomerular disease.