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Protective roles of thioredoxin, a redox-regulating protein, in renal ischemia/reperfusion injury. Background Thioredoxin (TRX) is a small protein with redox-regulating functions. Although TRX is known to be induced in response to various forms of oxidative stress, including ischemia/reperfusion injury, the induction and the specific role of this protein in the kidney have not been fully investigated. Methods Renal ischemia/reperfusion was induced by the clipping and release of renal arteries in C57BL/6 and human thioredoxin-overexpressing transgenic (hTRX-Tg) mice. TRX protein was detected by immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). TRX mRNA was detected by in situ hybridization and Northern blotting. Renal functions were evaluated by measuring the levels of blood urea nitrogen and serum creatinine in these mice. Results With ischemia/reperfusion, endogenous murine TRX was rapidly depleted from the cytosol in the cortical proximal tubuli and detected in the urinary lumen, whereas it was spread diffusely in all segments of the tubular epithelial cells in sham-operated mice. The urinary excretion of TRX increased transiently after ischemia/reperfusion and recovered to the control level in 72 hours. In the medullary thick ascending limb (mTAL), however, TRX was specifically retained in the cytosol. A similar distribution change of transgenic hTRX was observed in the kidney of hTRX-Tg. These hTRX-Tg mice were more resistant to the injury to the mTAL and functional deterioration caused by ischemia/reperfusion, compared with wild-type mice. Conclusion The present findings suggest that TRX is retained in mTAL and secreted from proximal tubuli into urine during renal ischemia/reperfusion. The mTAL-specific retention of TRX may have a protective effect against renal ischemia/reperfusion injury.

Coagulation process proceeds on cultured human mesangial cells via expression of factor V.BackgroundIn a previous clinicopathological study, we observed mesangial factor V expression accompanied by the intact form of cross-linked fibrin deposition in the active type of IgA nephropathy. The conversion of prothrombin to thrombin by factor Xa is potently accelerated more than 104-fold by the presence of factor V, which is a membrane-bound cofactor. Another membrane-bound cofactor, tissue factor, is known to play an initiating role in the coagulation cascade and to be synthesized in mesangial cells (MCs) by the stimulation of tumor necrosis factor-α (TNF-α). However, the synthesis of factor V, which plays on the terminating stage of prothrombin activation, has not been reported previously in MCs by in vitro study. Our current study tested the coagulation process via expression of factor V by the stimulation of proinflammatory cytokine, TNF-α, in cultured human MCs.MethodsTo evaluate factor V protein expression, immunoperoxidase staining with densitometric evaluation and Western blot analysis were conducted after stimulation of TNF-α. To test factor V activity, stimulated MCs were incubated in combination with factor Xa, prothrombin, fibrinogen and factor XIII, and fibrin production on MCs was assessed after immunoperoxidase staining on the cell surface. In a blocking test using an antibody against factor V, suppression of fibrin production was evaluated to clarify the role of factor V activity. For the evaluation of factor V mRNA expression in cultured human MCs, in situ hybridization and Northern blot analysis were performed.ResultsFactor V protein expression in MCs after TNF-α stimulation increased both time- and dose-dependently. As a marker of factor V activity with exogenous factor Xa, fibrin production on TNF-α–stimulated MCs was increased in a time-dependent manner and was inhibited by the addition of anti-factor V antibody. Factor V mRNA was identified in MCs by in situ hybridization and showed an increase after stimulation with TNF-α on Northern blot analysis.ConclusionsOur data suggest that the coagulation process proceeds on MCs as the result of increased expression of endogenous factor V activity on its cell surface in cooperation with exogenous factor Xa.

Direct inhibitory effects of simvastatin on matrix accumulation in cultured murine mesangial cells Background 3-Hydroxy-3-methylglutarylcoenzymeA(HMG-CoA) reductase inhibitors have been demonstrated to suppress glomerular injuries in various renal diseases. These agents inhibit in vitro proliferation of several cell types, including mesangial cells. This effect indicates the ability to ameliorate mesangioproliferative lesions, independent of the improvement of hypercholesterolemia. On the other hand, it is not clear whether HMG-CoA reductase inhibitors directly regulate extracellular matrix (ECM) accumulation from mesangial cells. Methods In this study, to examine the in vitro effects of simvastatin, an HMG-CoA reductase inhibitor, on mRNA expressions of matrix proteins, growth factors, and matrix turnover proteins, we incubated cultured murine mesangial cells stimulated by fetal calf serum (FCS) with or without simvastatin for 24 hours, and Northern analysis was performed. Results Simvastatin showed a slightly suppressive effect on mRNA expression of type IV collagen and fibronectin and a slightly up-regulative effect on that of type I collagen, whereas mRNA expression of type III collagen was markedly up-regulated. mRNA expression of platelet-derived growth factor (PDGF)-B chain and PDGF receptor β-subunit was suppressed, whereas that of transforming growth factor-β (TGF-β) was not affected by simvastatin. Concerning matrix turnover proteins, simvastatin markedly reduced mRNA expression of plasminogen activator inhibitor-1 (PAI-1) without affecting the expression of tissue-type plasminogen activator (tPA). Conclusion These results suggest type-specific modulation of matrix protein production independent of TGF-β and the suppressive effects of autocrine PDGF by administration of HMG-CoA reductase inhibitors in mesangial cells. Moreover, the beneficial effects of these agents on matrix protein accumulation may be through promoting ECM degradation derived from PAI-1 suppression.

Mesangiolytic glomerulopathy in severe congestive heart failure. To study the glomerular morphological abnormalities in congestive heart failure (CHF), we analyzed 27 autopsy cases without other causes of renal disease. Their mean age was 59 years, and they showed mild prerenal azotemia. They had generally been treated with digitalis and diuretics, and a few of them with captopril or nifedipine. The abnormal glomerular findings of enlargement, hyperemia, and mesangial thickening were observed at high frequencies (61%, 64%, and 57%, respectively). They characteristically showed mesangiolysis (ML) by the findings of microaneurysms (81%) and mesangial degeneration (70%) such as loose reticular matrix and poor matrix area. In addition, glomerular infiltration of mononuclear leukocytes including macrophages was noted in 70% of the cases. Glomerular enlargement was not correlated with the grade of hyperemia, but it was correlated with the grade of ML index of % glomeruli with microaneurysms (F = 7.22, p < 0.004). There was an inverse relationship between the grades of mesangial thickening and of the ML index (P < 0.005). The number of glomerular leukocytes was positively correlated with mean glomerular size (P < 0.002) and with the ML index (P < 0.03). Notably, the glomerular macrophage-positive cases showed a prominently higher mean ML index than the negative cases (P < 0.005). There was an inverse correlation between the mean glomerular size and the partial oxygen pressure in arterial blood (PaO2; P < 0.01), and a positive correlation between the mean glomerular size and hematocrit (Hct) levels (P < 0.02). The cases positive for mesangiolytic mesangial degeneration showed significantly lower PaO2 values than the cases negative for this lesion (P < 0.04). In the analysis of the various causes of CHF, the patients with congenital cardiac anomalies showed mean levels of the lowest PaO2 (P < 0.02) and the highest Hct (P < 0.03) and histologically the largest mean glomerular size (P < 0.04). There was no difference in the ML index and the glomerular leukocyte number among the subgroups classified by the causes. These results indicate that ML associated with glomerular enlargement is the major glomerular abnormality characteristic in patients with severe CHF and suggest that glomerular infiltration of leukocytes, especially of macrophages, should play an important role in the progression of both ML and glomerulomegaly. The contributions of persistent hypoxia and up-regulated angiotensin II as the causative factors of these glomerular abnormalities in congestive heart failure are discussed.

Chromosomal mapping of hyperserum IgA and glomerular IgA deposition in a high IgA (HIGA) strain of DdY mice. Background The high IgA (HIGA) strain of ddY mice is an inbred model of IgA nephropathy (IgAN), established by selective mating of outbred ddY mice. HIGA mice show high levels of serum IgA and glomerulonephritis with mesangial IgA deposition. To identify the genetic loci responsible for hyperserum IgA and glomerular IgA deposition in this strain, quantitative trait loci analysis was carried out. Methods By crossing HIGA with BALB/c mice, 244 F2 generations were produced. Serum IgA levels and glomerular IgA deposition were examined at 40 weeks of age. Genetic markers were typed at 105 microsatellites and the quantitative trait loci of hyperserum IgA and glomerular IgA deposition were confirmed using Map Manager QTX software. Results Two significant quantitative trait loci of hyperserum IgA were identified on chromosome 2 [logarithm of odds (LOD) = 5.01] and chromsome 4 (LOD = 4.45), and a suggestive quantitative trait locus of hyperserum IgA was located on chromosome 1 (LOD = 3.49). On chromosome 15, a significant quantitative trait locus of glomerular IgA deposition was identified (LOD = 4.40) without the hyperserum IgA locus. Serum IgA level was weakly correlated with the intensity of glomerular IgA in 244 F2 mice; however, the quantitative trait loci of hyperserum IgA were not significantly associated with glomerular IgA deposition. Conclusion These findings indicate that, in HIGA mice, glomerular IgA deposition is mainly regulated by a quantitative trait locus on chromosome 15, and hyperserum IgA synergistically but weakly affect glomerular IgA deposition. The immune disturbance similar to IgAN was revealed to be under multigenic control in HIGA mice.

Mesangial factor V expression colocalized with fibrin deposition in IgA nephropathy.BackgroundFactor V in its active form (Va) plays a key role at the termination of the intrinsic coagulation pathway, serving as a membrane-bound cofactor for the conversion of prothrombin to thrombin by factor Xa. Cross-linked fibrin (XFb) is often observed in mesangial areas in active types of human glomerulonephritis. In this study, to clarify contribution of factor V in intramesangial coagulation, mesangial factor V expression and its relationship to mesangial proliferation and fibrin deposition in IgA nephropathy (IgAN) were investigated.MethodsTwenty-two patients with IgAN were studied. XFb was detected in renal biopsy specimens using anti–d-dimer antibody combined with plasmin exposure, and factor V was detected with rabbit antibody against human factor V. Double-labeling immunohistochemistry was used to investigate the relationship of the glomerular distribution of factor V to XFb. The relationship of factor V staining to the activity index or XFb deposition was evaluated. The expression of factor V mRNA was assessed by in situ hybridization in relationship to the antigen staining of α-smooth muscle actin (α-SMA). The ultrastructural distribution of factor V in glomeruli was studied by immunoelectron microscopy.ResultsXFb and factor V were observed in the mesangium and along capillary loops in seven and nine specimens, respectively. Factor V had intense, frequent expression in the proliferating and necrotizing areas, showing a significant relationship to XFb (P < 0.05). Furthermore, XFb deposition and factor V expression were markedly correlated with disease activity (P = 0.005 and P = 0.008, respectively). By double-labeling experiments, XFb and factor V were often seen colocalized in mesangial areas of the glomeruli, which showed necrotizing lesions and/or intense cellular proliferation. By in situ hybridization, factor V mRNA was detected mainly in the mesangial cells, which were positive for α-SMA, and partly in the endothelial cells. By immunoelectron microscopy, factor V presence was confirmed in the mesangium and endothelium.ConclusionThe present findings suggest that factor V is strongly expressed in mesangial cells in active IgAN accompanied with mesangial proliferation and may exert procoagulant activity, leading to intramesangial coagulation.

Role of coagulation factor Xa and protease-activated receptor 2 in human mesangial cell proliferation. Background Fibrin deposition and mesangial cell proliferation are frequently observed in the active type of mesangioproliferative glomerulonephritis. Coagulation factors, such as factor V and factor Xa are colocalized with fibrin in the mesangial areas in active type of IgA nephropathy with mesangial cell proliferation. In this study, therefore, we studied the role of factor Xa and its receptor, protease-activated receptor 2 (PAR2) in mesangial cell proliferation and fibrin deposition, and examined ant-proliferative effects of a specific factor Xa inhibitor, DX-9065a, in cultured human mesangial cells. Methods To examine the effect of DX-9065a on the factor Xa–induced proliferation of cultured human mesangial cells, we measured thymidine incorporation and cell numbers. We also examined the effect of DX-9065a on extracellular regulated kinase (ERK) activation and fibrin production induced by factor Xa in human mesangial cells. Results Factor Xa increased [ 3 H]-thymidine incorporation and cell numbers in a dose-dependent manner in mesangial cells, which was inhibited by DX-9065a. DX-9065a also suppressed factor Xa–triggered fibrin deposition on mesangial cell surface. Factor Xa induced the activation of ERK in mesangial cells and this activation was also completely inhibited by DX-9065a, but not inhibited by PAR1 antagonist. Factor Xa–induced cell proliferation and ERK activation were inhibited by PD98059. Conclusion There results suggest that factor Xa can induce mesangial cell proliferation through the activation of ERK via PAR2 in mesangial cells and that PAR2 may play a crucial role in the cell proliferation induced by factor Xa. Our results implicate that DX-9065a may be a promising agent to regulate proliferation of mesangial cellss and inhibit the coagulation process in mesangium.

Rapid normalization of interleukin-8 production after low-density lipoprotein-apheresis in steroid-resistant nephrotic syndrome. Background Low-density lipoprotein apheresis (LDL-A) treatment combined with steroids demonstrated significant improvement of nephrotic proteinuria in steroid- or immunosuppressive-resistant patients from focal and segmental glomerulo-sclerosis (FGS). The mechanisms of the effect of LDL-A in nephrotic syndrome (NS) are unknown, but a reduction in inflammatory cytokines and chemokines secreted from macro-phages has been supposed. Methods Serum levels of interleukin (IL)-8, tumor necrosis factor-a (TNF-a), and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay in 27 patients with NS [13 with FGS and 14 with minimal change nephrotic syndrome (MCNS)] before and after LDL-A and in 13 age-matched, healthy controls. We also selected three FGS patients who were resistant to steroid therapy for at least one month and who had undergone six LDL-A procedures. The effects of steroids and LDL-A on the production of IL-8, TNF-a, and MCP-1 by peripheral blood mononuclear cells (PBMCs) were also determined in some patients. Results In NS, the serum levels of IL-8 and TNF-a, but not MCP-1, were significantly higher than in healthy controls. After LDL-A, IL-8 and TNF-a tended to decrease. IL-8 production by lipopolysaccharide (LPS)-stimulated PBMC, mainly adherent cells, was significantly reduced in both the steroid-resistant FGS group and nontreated NS group compared with controls, but TNF-a production was reduced in the only FGS group. After LDL-A, only IL-8 production recovered to the control group level. Conclusion Significant amelioration of IL-8 production independent of any effect of steroids on LPS-stimulated PBMCs may reflect a beneficial effect of LDL-A in normalizing the function of circulating monocytes in steroid-resistant FGS.