The AKT-mTOR pathway is activated in diabetic nephropathy. Renin-angiotensin system modulators exert beneficial effects on the diabetic kidney. We explored the action of losartan on AKT-mTOR phosphorylation in glomeruli and podocytes. Diabetes mellitus was induced to Sprague-Dawley rats by streptozotocin. Five months later, the rats were commenced on losartan and euthanized 2 months later. Kidneys were processed for immunofluorescence studies. Glomeruli were isolated for Western blot analysis. Diabetes increased activated forms of AKT and mTOR both in glomeruli and podocytes. In diabetic rats, losartan decreased phosphorylated/activated forms of AKT (Thr308) and mTOR (Ser2448) in glomeruli but decreased only activated mTOR in podocytes. However, in both glomeruli and podocytes of healthy animals, an inverse pattern was evident. In conclusion, a new body of evidence indicates the differential activation of AKT-mTOR in glomeruli and podocytes of healthy and diabetic animals in response to losartan.

Recent studies had demonstrated that serum and mesangial immunoglobulin A1 (IgA1) in patients with IgA nephropathy (IgAN) were polymeric and deglycosylated. The current study was to investigate the binding characteristics of monomeric and polymeric normal human IgA1 on mesangial cells and the influence of in vitro deglycosylation of IgA1 molecules. The normal human IgA1 was desialylated and degalactosylated with specific enzymes, respectively. The monomeric IgA1 (mIgA1) and polymeric IgA1 (pIgA1) were separated by Sephacryl S-300 chromatography. The binding capacities of the mIgA1 and pIgA1 to primary human mesangial cells (HMC) were evaluated by classical radioligand assay. Both the native mIgA1 and pIgA1 could bind to HMC in a dose-dependent and saturable manner. The maximal binding capacity of the native pIgA1 were significantly higher than that of the native mIgA1 (P < 0.05). However, the affinity of the native mIgA1 was almost 100 times higher than that of the native pIgA1. After deglycosylation, binding of the two deglycosylated mIgA1 to HMC could not be detected. However, the maximal binding capacities of the two deglycosylated pIgA1 to HMC were increased significantly compared with that of native pIgA1. The affinity of the two deglycosylated pIgA1 was similar to that of native pIgA1 (P > 0.05). The current study suggests differential binding characteristics of native monomeric and polymeric IgA1 on mesangial cells. Glycosylation of IgA1 molecules could significantly affect the binding of IgA1 on HMC.

IgA nephropathy (IgAN) is the most common glomerular disease and it is characterized by deposition of IgA1 molecules in mesangium. Recent studies had demonstrated that serum and mesangial IgA1 in IgAN were deglycosylated and IgA1 could bind to human mesangial cells (HMC) through a novel receptor. The aim of the current study is to investigate and compare the binding capacities of different in vitro deglycosylated IgA1 on human mesangial cells. Serum IgA1 was purified by jacalin affinity chromatography and then was desialylated (DesIgA1) and/or degalactosylated (Des/DeGalIgA1) with neuraminidase and/or beta-galactosidase. The efficacy of deglycosylations was assessed by Peanut agglutinin (PNA) and Vicia villosa (VV) lectin. The sizes of normal IgA1 and deglycosylated IgA1 were determined by Sephacryl S-300 chromatography and binding capacities to primary HMC were evaluated by radioligand binding assays. Normal IgA1 and deglycosylated IgA1 could bind to HMC in a dose-dependent, saturable manner. The maximal binding capacities and binding sites/cell of DesIgA1 and Des/DeGalIgA were significantly higher than that of normal IgA1. However, more aggregated IgA1 was found in DesIgA1 and Des/DeGalIgA1. Scatchard analysis revealed a similar Kd of normal IgA1 and deglycosylated IgA1. The current study suggested that the binding capacities of DesIgA1 and Des/DeGalIgA1 to HMC were significantly higher than that of normal IgA1, which at least in part was due to more macromolecular IgA1 in deglycoslated IgA1. However, there were no significant differences in the affinities of normal IgA1, DesIgA1 and Des/DeGalIgA1 with HMC. Deglycosylated IgA1 might play an important role in pathogenesis of IgAN.

During a subtraction study on gene expression in human kidney mesangial cells (HMCs), cDNA clones with sequence homology to paramyxovirus P, M and F genes were isolated. Subsequent investigation revealed that this particular HMC line was infected with a previously unknown paramyxovirus. Here, we report the isolation and genome characterization of this new virus, now named Beilong virus (BeV). The genome of BeV is 19,212 nucleotides (nt) in length and is the largest among all known members of the order Mononegavirales. The BeV genome contains eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. The SH and TM genes code for a small hydrophobic protein of 76 aa and a transmembrane protein of 254 aa, respectively. The BeV G gene, at 4527 nt, codes for an attachment protein of 734 aa and contains two additional open reading frames (ORFs) in the 3' half of the gene, coding for putative proteins of 299 and 394 aa in length. Although the exact origin of BeV is presently unknown, we provide evidence indicating that BeV was present in a rat mesangial cell line used in the same laboratory prior to the acquisition of the HMC line, suggesting a potential rodent origin for BeV.

IgA deposition in glomerular mesangium and the interaction with mesangial cells may well be the final common pathway to IgA nephropathy (IgAN). Altered hinge-region O-glycosylation of IgA1 from patients with IgAN may predispose to mesangial deposition and activation of the mesangial cell (MC) by IgA1, via a novel IgA1 receptor, and may be a key event in the pathogensis of IgAN. The aim of this study was to investigate the binding capacity and biological effects of IgA1, from both patients with IgAN and healthy controls, on human mesangial cells (HMC). Serum IgA1 was isolated with jacalin affinity chromatography, heated to aggregated form (aIgA1) and labelled with (125)I. Binding capacity of aIgA1 in vitro to cultured primary HMC was evaluated by a radioligand binding assay and the specificity of binding was determined by a competitive inhibition assay. Intracellular calcium release was studied by confocal analysis and phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis. Change of cell cycles was demonstrated by flow cytometry and HMC proliferation was evaluated by direct cell count. Expression of TGF-beta mRNA and production of supernatant fibronectin were tested by RT-PCR and indirect competitive ELISA, respectively. aIgA1 from both the patients with IgAN and normal controls bound to HMC in a dose-dependent, saturable manner, and was saturated at approximately 500 pmoles per 0.5 ml of aIgA1. aIgA1 from patients with IgAN, however, bound to HMC at a higher speed and Scatchard analysis revealed a Kd of (8.89 +/- 2.1) x 10(-8)m versus (4.3 +/- 1.2) x 10(-7)m for aIgA1 from healthy controls (P = 0.026). The binding was specific because it was only inhibited by unlabelled Mono-IgA1 (mIgA1) and not by serum albumin or IgG. aIgA1 from patients with IgAN could induce release of intracellular calcium, phosphorylation of ERK, DNA synthesis, proliferation of HMC, expression of TGF-betamRNA and secretion of fibronectin in HMC in a similar time-dependent manner as aIgA1 from healthy controls, but the effects were much stronger and the durations were much longer (P < 0.05, respectively). We conclude that aIgA1 from patients with IgAN has a higher binding capacity to HMC and stronger biological effects than aIgA1 from healthy controls. This suggests that direct interaction between IgA1 and HMC and subsequential pathophysiological responses may play an important role in the pathogenesis for IgAN.

IgA nephropathy (IgAN) is characterized by mesangial deposits of polymeric IgA (pIgA). The pathological consequences of IgA deposition are believed to center on direct interaction between IgA and the glomerular mesangial cell (MC). We have characterized a novel mesangial receptor that recognizes the Fc portion of IgA.

Five primary MC cultures were evaluated for IgA binding by flow cytometry, and specificity of binding was determined by competitive inhibition. Relative affinities of the receptor for all IgA isoforms were also determined, and binding of pIgA1 was compared to monomer. The identified Fc receptor was then compared with CD89, hitherto the only other Fcalpha receptor reported. CD89 protein and mRNA expression were detected by conventional and intracellular flow cytometry, sequencing of reverse transcription-polymerase chain reaction (RT-PCR) products, and Northern blotting.

All MCs constitutively expressed a receptor that bound IgA in an Fcalpha-dependent fashion. The receptor recognized secretory and serum IgA1 and IgA2 equally, but pIgA bound with much greater affinity than monomer. At no time were we able to detect CD89 synthesis, although three novel CD89-related mRNA transcripts were identified by RT-PCR.

We have clearly demonstrated that MCs consistently express an FcalphaR distinct from the myeloid FcalphaR CD89. This novel receptor binds pIgA with high affinity and may therefore mediate the mesangial injury that follows IgA deposition in IgAN. While immunogenically distinct, the mesangial Fcalpha receptor may share some molecular homology with CD89, as mRNA transcripts with partial identity to CD89 were found in all five MC cultures.

Laser-manipulated microdissection (LMM) is a method to cut out a single cell or limited tiny region from a specimen under microscopic observation by a laser beam. Laser pressure catapulting (LPC) is a method to push up and collect samples that were microdissected using a strong laser.

To induce experimental glomerulonephritis, anti-Thy1.1 monoclonal antibody (OX-7) was injected intravenously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mounted onto a 1.35 microm thin polyethylene membrane. Ten glomeruli were collected from 6 microm frozen sections of rat kidney by LMM and LPC. Isolated glomeruli were used to quantitate the expression of mRNA by real-time polymerase chain reaction (PCR).

Transforming growth factor-beta1 (TGF-beta1) mRNA was not detected in glomeruli isolated by the LMM and the LPC methods on day 0, although G3PDH mRNA was measurable in the same samples. On day 7 after the treatment with OX-7, the ratio of TGF-beta1/G3PDH mRNA was 1.89 +/- 0.96 (N = 6).

We established methods to isolate glomeruli from standard histochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of TGF-beta1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.

IgA nephropathy (IgAN) is characterized by deposition of polymers of IgA1 in the mesangium, accumulation of mesangial matrix and mesangial cell proliferation. Activation of the mesangial cell by IgA, via an IgA receptor, may be an initiating event in the pathology of IgAN.

We examined the ability of radiolabeled, normal serum IgA1 to bind human mesangial cells (HMC). Activation of HMC by monomeric (mIgA1) and heat aggregated IgA1 (AIgA1) was compared by Northern analysis of c-jun expression. The expression of FcalphaR1 (CD89) mRNA on our cultured mesangial cells was also assessed by Northern analysis, reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry.

125I-mIgA1 and 125I-AIgA1 bound to HMC in a dose-dependent, saturable manner with similar affinities. There were 1.2 x 10(6) binding sites per cell, with an affinity constant of 2.3 x 10(6) M(-1). AIgA1 induced c-jun expression in a time and dose-dependent manner (2.4-fold above baseline after 60 min exposure to AIgA1 200 microg/ml) while mIgA1 had no effect on c-jun expression. No message for CD 89 was detectable in quiescent or AIgA1 stimulated HMC by Northern analysis or RT-PCR using several primer sequences based on the sequence of U937 FcalphaR cDNA. Flow cytometry on the mesangial cells, using My 43, a monoclonal antibody to FcalphaR1 confirmed that CD 89 was not present on the cell.

These results demonstrate that HMC bind mIgA1 and AIgA1 with similar affinity. However, activation of HMC requires an aggregated form of IgA1. These processes are independent of FcalphaR1, suggesting the presence of a new IgA receptor on mesangial cells.

Angiotensin II (Ang II) has growth-stimulatory properties on different renal cell types. However, possible growth effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvasculature have not been formally investigated. Therefore, we isolated and characterized primary cultures of rat glomerular endothelial cells. We used a simple technique in which collagenase-treated glomeruli were sparsely plated in several 96-well culture plates and microscopically screened for cobblestone-like outgrowth. After two limiting dilutions, homogeneous cultures were obtained. Cells were characterized by positive staining for the endothelial markers factor VIII, CD 31, endothelial leukocyte adhesion molecule-1, and the lectin Bandeiraea simplificifolia. Ang II stimulated the synthesis and release of endothelin-1 in culture supernatants. Moreover, in contrast to syngeneic mesangial cells, glomerular endothelial cells expressed angiotensin-converting enzyme. Ang II stimulated a mild but significant proliferation of quiescent cells, as measured by [3H]thymidine incorporation and direct cell counting. This mitogenesis was transduced by losartan-blockade angiotensin type 1 receptors. Moreover, Ang II mediated phosphorylation of mitogen-activated protein kinase 2 and induction of transcripts for the immediate early gene Egr-1. Our results indicate that Ang II is a moderate mitogen for primary cultures of rat glomerular endothelial cells and activation of these metabolically active cells may play a role in the pathophysiology of several types of glomerulonephritis. Moreover, remodeling of glomerular endothelial cells by Ang II may be important in the progression of structural renal damage during the course of hypertensive injury.

Transmembrane signal transduction is the process whereby a ligand binds to the external surface of the cell membrane and elicits a physiological response specific for that ligand and cell type. It is now appreciated that numerous disease states represent disturbances in normal transmembrane signaling mechanisms. In the current paper, we focus our attention on the mesangial cell of the glomerular microcirculation as a prototypical model system for understanding normal and abnormal transmembrane signaling processes. Among the major receptor and effector mechanisms for transmembrane signal transduction in the mesangial cell, this paper emphasizes the phospholipase effector response to growth factors and vasoactive hormones. The post-translational and transcriptional pathways for regulation of phospholipase C and phospholipase A2 are described, including consideration of perturbations in these systems that characterize two disease models, namely: acute cyclosporine nephrotoxicity and early diabetic glomerulopathy.