Combined effects of moderately elevated blood glucose and locally produced TGF-beta1 on glomerular morphology and renal collagen production.

BACKGROUND: There is a correlation between renal graft rejection and blood glucose (BG) levels. Furthermore, diabetic patients may develop non-diabetic renal diseases, which in some circumstances progress rapidly. Since transforming growth factor-beta1 (TGF-beta) levels are elevated in many renal diseases, the accelerated progression may be due to interactions between glucose and locally produced TGF-beta1. Therefore, we investigated the effect of mild hyperglycaemia on glomerular morphology and collagen production in TGF-beta1 transgenic mice. METHODS: To achieve BG concentrations of approximately 15 mmol/l in TGF-beta1 transgenic and non-transgenic mice, we used multiple streptozotocin (STZ) injections, and after 8 weeks, we measured the changes in glomerular morphology and total collagen content. We also analysed extracellular matrix (ECM) and protease mRNA levels using real-time polymerase chain reaction (PCR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK) expression by immunohistochemistry. RESULTS: Mild hyperglycaemia alone had no effect on glomerular structure or ECM deposition. Over-expression of TGF-beta1 increased basement membrane thickness (BMT) and the mesangial volume fraction. Furthermore, it augmented ECM, Matrix metalloproteinase-2 (MMP), MMP-9, plasminogen activator inhibitor-1 (PAI) and tissue inhibitor of metalloproteinase-1 (TIMP) gene expression and pERK1/2 immunostaining. Elevated BG in combination with TGF-beta1 resulted in enlargement of glomerular volume, total mesangial volume and renal collagen content. Moreover, high BG exaggerated TGF-beta1-induced changes in the BMT, MMP-2 and TIMP-1 expression and pERK1/2 staining. CONCLUSION: Even moderate elevations in BG accelerate the progression of those kidney diseases in which TGF-beta1 is involved. This emphasizes the importance of strict BG control in renal transplant patients and diabetic patients with renal malfunctions unrelated to diabetes.     

Glomerular matrix accumulation is linked to inhibition of the plasmin protease system.

TGF-beta plays a pivotal role in the pathological accumulation of extracellular matrix in experimental glomerulonephritis. Increased TGF-beta expression leads to increased synthesis and deposition of extracellular matrix components while administration of anti-serum to TGF-beta suppresses the major manifestations of the disease. We hypothesized that TGF-beta might also enhance matrix accumulation by decreasing matrix turnover via effects on protease/protease inhibitor balance. Plasmin is a potent protease capable of degrading a variety of matrix molecules. Plasmin generation from plasminogen is regulated by plasminogen activator(s) (PA) and plasminogen activator inhibitor(s) (PAI). In this study PA activity was markedly reduced and PAI-1 synthesis dramatically increased when TGF-beta was added to normal glomeruli. Diseased glomeruli also showed decreased PA activity, increased PAI-1 synthesis and increased PAI-1 deposition into matrix. Administration of anti-TGF-beta serum to glomerulonephritic rats blocked the expected increase in glomerular PAI-1 deposition. Thus changes in the PA/PAI balance favoring accumulation of matrix are induced by TGF-beta in normal glomeruli and are present in nephritic glomeruli when endogenous TGF-beta production is high. Our findings implicate the plasmin protease system in tissue repair following acute glomerular injury and suggest another mechanism by which TGF-beta enhances the matrix accumulation characteristic of many glomerular diseases.     

血管紧张素1a型受体基因敲除小鼠肾脏局部肾素-血管紧张素系统的变化及其对糖尿病肾小球硬化的影响

目的 探讨血管紧张素1a型受体（AT1aR）基因敲除小鼠肾脏局部肾素-血管紧张素系统（RAS）的组分改变对糖尿病肾病（DN）肾小球硬化的影响及其可能机制。 方法 AT1aR基因敲除小鼠和野生型小鼠腹腔注射链脲佐菌素（STZ，300 mg/kg）诱导糖尿病模型12周后，取肾脏组织作冰冻组织切片，用激光捕获微切割技术分离肾小球，提取RNA。用实时定量PCR的方法检测肾小球内AT1aR、血管紧张素1b型受体（AT1bR）、血管紧张素2型受体（AT2R）、血管紧张素原、血管紧张素转化酶（ACE）、肾素、醛固酮合成酶（CYP11B2）的mRNA表达。PAS染色观察肾脏病理变化。免疫组化检测转化生长因子β1（TGF-β1）、1型纤溶酶原激活物抑制物(PAI-1)、单核细胞趋化因子1(MCP-1)和肾素的表达。比较不同基因型小鼠肾小球细胞外基质和各细胞因子的表达变化。 结果 与野生型小鼠相比，AT1aR基因敲除小鼠肾小球内AT1bR、血管紧张素原、肾素、CYP11B2的表达明显上调（P < 0.05），AT2R表达下调，ACE无明显改变；AT1aR基因敲除小鼠肾小球细胞外基质明显增加（P < 0.05），TGF-β1、PAI-1、MCP-1和肾素的表达均明显增加（P < 0.05）。 结论 AT1aR基因敲除并不能使糖尿病小鼠肾脏病变改善。RAS组分的表达改变（AT1bR的上调和AT2 的下调，肾素的上调和CYP11B2的上调）参与糖尿病肾小球病变过程。     

TGF-beta type II receptor deficiency prevents renal injury via decrease in ERK activity in crescentic glomerulonephritis

The role of transforming growth factor-beta (TGF-beta) receptor complex in the pathogenesis of crescentic glomerulonephritis (GN) is not clear. To test the hypothesis that TGF-beta signaling plays a crucial role in the development and progression of crescentic GN by inducing the activation of extracellular signal-regulated kinase (ERK) and expression of its target genes, anti-glomerular basement membrane (GBM) GN was induced in TGF-beta type II receptor (TGF-betaIIR) gene heterozygous (TGF-betaIIR(+/-)) C57BL/6J mice and wild-type animals. GN was initiated in preimmunized mice by administration of rabbit anti-mouse GBM serum. TGF-betaIIR deficiency was significantly associated with decreased renal damage at days 14, 21, and 28 after induction of GN: renal function impairment, proteinuria, proportion of crescents, glomerular accumulation of periodic acid-Schiff-positive material, relative cortical interstitial volume, as well as renal cortical phosphorylation of ERK and plasminogen activator inhibitor type I (PAI-1) and alpha2(I) collagen mRNA levels were significantly decreased in TGF-betaIIR(+/-) mice compared with wild-type animals. These results provide the first direct evidence that TGF-betaIIR deficiency protects against renal injury in crescentic GN, possibly by inhibiting the sustained activation of ERK and PAI-1 and alpha2(I) collagen gene expression. Thus, TGF-beta signaling appears to play an important role in the development and progression of crescentic GN by inducing the ERK activity, and PAI-1 and alpha2(I) mRNA expression.     

Plasminogen activator inhibitor-1 is a significant determinant of renal injury in experimental crescentic glomerulonephritis

Crescentic glomerulonephritis is characterized by glomerular fibrin deposition, and experimental crescentic glomerulonephritis has been shown to be fibrin-dependent. Net fibrin deposition is a balance between activation of the coagulation system causing glomerular fibrin deposition and fibrin removal by the plasminogen-plasmin (fibrinolytic) system. Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis by inhibiting plasminogen activators and has effects on leukocyte recruitment and matrix deposition. To test the hypothesis that the presence of PAI-1 and its levels were a determinant of injury in crescentic glomerulonephritis, accelerated anti-glomerular basement membrane glomerulonephritis was induced in mice genetically deficient in PAI-1 (PAI-1 -/-), PAI-1 heterozygotes (PAI-1 +/-), and mice engineered to overexpress PAI-1 (PAI-1 tg). Compared with strain-matched genetically normal animals, PAI-1 -/- mice with glomerulonephritis developed fewer glomerular crescents, less glomerular fibrin deposition, fewer infiltrating leukocytes, and less renal collagen accumulation at day 14 of disease. The reduction in disease persisted at day 28, when injury had become more established. In contrast, mice overexpressing the PAI-1 gene (PAI-1 tg), that have basal plasma and renal PAI-1 levels several times, normal developed increased glomerular crescent formation, more glomerular fibrin deposition, increased numbers of infiltrating leukocytes, and more renal collagen at both time points. These studies demonstrate that PAI-1 is a determinant of glomerular fibrin deposition and renal injury in crescentic glomerulonephritis.     

Plasminogen activator inhibitor-1 and diabetic nephropathy

SUMMARY:   Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Decreased ECM degradation as well as increased ECM synthesis plays an important role in ECM remodeling that favours tissue fibrosis. Plasminogen activator (PA)/plasmin/PA inhibitor (PAI) system is involved in ECM degradation and PAI-1 plays a critical role in ECM remodeling in the kidney. Normal human kidneys do not express PAI-1 but PAI-1 is overexpressed in pathologic conditions associated with renal fibrosis including diabetic nephropathy. Reactive oxygen species mediate PAI-1 up-regulation in renal cells cultured under high glucose, hypoxia, and TGF-β1. Recent studies utilizing PAI-1 deficient mice suggest that PAI-1 induce ECM deposition in diabetic kidney through increased ECM synthesis by TGF-β1 up-regulation as well as through decreased ECM degradation by suppression of plasmin and MMP-2 activity.     

Role for transforming growth factor-beta1 in alport renal disease progression

BACKGROUND: Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. METHODS: Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. RESULTS: The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. CONCLUSIONS: The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.     

Transforming growth factor-β3 affects plasminogen activator inhibitor-1 expression in fetal mice and modulates fibroblast-mediated collagen gel contraction

For over two decades, the precise role of transforming growth factor-beta (TGF-beta) isoforms in scarless healing of mammalian fetal skin wounds has generated much interest. Although their exact role remains to be established, it has been suggested that high TGF-beta3 activity may correlate with a scarless phenotype. Previously, we showed that plasminogen activator inhibitor-1 (PAI-1), a known TGF-beta downstream molecule and marker of fibrosis, is also developmentally regulated during fetal skin development. In this study, the relationship between TGF-beta3 and PAI-1 was investigated using embryonic day 14.5 TGF-beta3 knockout (ko) mice. The results showed increased PAI-1 expression in the epidermis and dermis of ko mice, using an ex vivo limb-wounding study. Furthermore, increased PAI-1 expression and activity was seen in embryo extracts and conditioned media of ko dermal fibroblasts. When TGF-beta3 knockout fibroblasts were placed into three-dimensional collagen matrices, they were found to have decreased collagen gel contraction, suggesting altered cell-matrix interaction. These findings provide a further avenue for the interactive role of TGF-beta3 and PAI-1 during fetal scarless repair.     

Amelioration of diabetic nephropathy in SPARC-null mice

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular protein that inhibits mesangial cell proliferation and also affects production of extracellular matrix (ECM) by regulating transforming growth factor-beta1 (TGF-beta1) and type I collagen in mesangial cells. This study is an investigation of the role of SPARC in streptozotocin (STZ)-induced diabetic nephropathy (DN) of 6-mo duration in wild type (WT) and SPARC-null mice. SPARC expression was evaluated by immunohistochemistry (IHC) and by in situ hybridization (ISH). Deposition of type I and IV collagen and laminin was evaluated by IHC, and TGF-beta 1 mRNA was assessed by ISH. Renal function studies revealed no significant difference in BUN between diabetic SPARC-null mice and diabetic WT mice, whereas a significant increase in albumin excretion was detected in diabetic WT relative to diabetic SPARC-null mice. Diabetic WT animals exhibited increased levels of SPARC mRNA and protein in glomerular epithelial cells and in interstitial cells, in comparison with nondiabetic WT mice. Neither SPARC mRNA nor protein was detected in SPARC-null mice. Morphometry revealed a significant increase in the percentage of the glomerular tufts occupied by ECM in diabetic WT compared with nondiabetic WT mice, although there was no difference in the mean glomerular tuft area among groups. In contrast, diabetic SPARC-null mice did not show a significant difference in the percentage of the glomerular tufts occupied by ECM relative to nondiabetic null mice. Tubulointerstitial fibrosis was ameliorated in diabetic SPARC-null mice compared with diabetic WT animals. Further characterization of diabetic SPARC-null mice revealed diminished glomerular deposition of type IV collagen and laminin, and diminished interstitial deposition of type I and type IV collagen correlated with decreases in TGF-beta 1 mRNA compared with WT diabetic mice. These observations suggest that SPARC contributes to glomerulosclerosis and tubulointerstitial damage in response to hyperglycemia through increasing TGF-beta 1 expression in this model of chronic DN.     

Heterozygous mice for TGF-betaIIR gene are resistant to the progression of streptozotocin-induced diabetic nephropathy

BACKGROUND: Transforming growth factor-beta (TGF-beta) receptor complex and its downstream Smad signaling intermediates constitute an extracellular matrix (ECM) accumulation pathway. METHODS: In the present study, we examined whether decreased expression of the TGF-beta type II receptor (TGF-betaIIR) in TGF-betaIIR gene heterozygous (TGF-betaIIR+/-) (HT) mice could inhibit the Smad signaling pathway and subsequent progression of renal lesions when streptozotocin (STZ) diabetes is induced. RESULTS: At the end of the 28-week experiment after STZ injections, wild-type diabetic mice showed severe glomerular hypertrophy and mesangial matrix accumulation occasionally featuring nodular glomerulosclerosis. In contrast, mean glomerular area and mesangial volume density were significantly decreased in the HT diabetic mice as compared with the wild-type diabetic mice. Immunostaining for phosphorylated Smad2/Smad3 and TGF-betaIIR in the glomerular cells was also significantly reduced in the HT diabetic mice. Southwestern histochemistry using digoxigenin-labeled CAGA sequence probes showed that localization of labeled probes to the nuclei of glomerular cells in the HT diabetic mice was significantly less frequent than that in the wild-type diabetic animals. Northern blot analysis showed that alpha1(IV) collagen mRNA levels were significantly reduced in the kidney tissue of HT diabetic mice as compared with the wild-type diabetic mice. CONCLUSION: These results suggest that decreased expression of TGF-betaIIR in the HT diabetic mice can inhibit the progression of diabetic renal injury by inhibiting the downstream Smad signaling pathway and subsequent ECM gene expression. Thus, TGF-betaIIR appears to play an important role in the progression of diabetic nephropathy by mediating intracellular Smad signaling.     

Protease-activated receptor 1 and plasminogen activator inhibitor 1 expression in chronic allograft nephropathy: the role of coagulation and fibrinolysis in renal graft fibrosis.

BACKGROUND: Chronic allograft nephropathy (CAN), the major cause of renal graft failure, frequently displays extensive interstitial fibrin deposition. Little is known in regard to the cause of the altered coagulation/fibrinolysis balance and its relevance in the pathogenesis of CAN. Thrombin, present within the fibrin clots, can interact with a specific receptor, protease-activated receptor 1 (PAR-1), and modulate a variety of cell functions. On the other hand, the derangement of the fibrinolytic system may directly affect extracellular matrix (ECM) degradation. METHODS: In the present study, we investigated, by in situ hybridization, PAR-1 gene expression and the mRNA levels for tissue factor and plasminogen activator inhibitor 1 (PAI-1), two key regulatory molecules of coagulation and fibrinolysis, in 16 CAN biopsies and in 10 normal human kidney grafts. The thrombin-induced transforming growth factor beta (TGF-beta) gene and protein expression in proximal tubular cells (PTC) was investigated by Northern blotting and ELISA, respectively. RESULTS: Fibrin deposits, absent in normal grafts, were observed in the interstitial space and arterial wall of CAN. Tissue factor gene expression was not increased either at the vascular or at the interstitial level in CAN. On the contrary, PAI-1 gene expression, barely detectable in control tissue, was strikingly increased in CAN, with a distribution resembling the pattern of fibrin deposition. Note that PAI-1 gene expression was directly correlated with the degree of interstitial fibrosis. In addition, fibrin deposits were strictly associated with a marked increase of PAR-1 gene expression in endothelial cells and PTC. The tubular expression of PAR-1 was significantly higher in Banff grade II-III than in grade I. In vitro, incubation of PTC with thrombin caused a significant up-regulation of TGF-beta gene expression, followed by an increased TGF-beta release into the supernatant. Interestingly, urine from CAN patients contained significantly higher levels of TGF-beta. CONCLUSIONS: Fibrin deposits in CAN may result from the increased expression of PAI-1 and the subsequent inhibition of fibrinolysis. The reduced fibrinolysis may cause, in turn, a decreased ECM turnover. Finally, thrombin, preserved in the active form within the fibrin clots, may interact with PAR-1 highly expressed on PTC and induce an up-regulation of ECM deposition in a TGF-beta-dependent manner.     

Growth factor expression in a murine model of cryoglobulinemia

BACKGROUND: Increased expression of growth factors including platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are thought to play pivotal roles during mesangial expansion and glomerulosclerosis. Thymic stromal lymphopoietin (TSLP) transgenic mice develop mixed cryoglobulinemia and a membranoproliferative glomerulonephritis (MPGN). Here we describe the renal expression of isoforms of PDGF and TGF-beta in relation to changes in extracellular matrix (ECM) components and markers of cell proliferation and activation in this model. METHODS: A total of 123 mice, including 61 TSLP transgenic mice and 62 wild-type controls, were sacrificed at defined intervals. PDGF-A chain, -B chain, PDGF alpha- and beta-receptor (beta-R) and TGF-beta1 mRNA were analyzed by in situ hybridization. Expression of alpha smooth muscle actin (alphaSMA), collagen type I, collagen type IV, laminin, and a marker of proliferating cells (PCNA) were assessed by immunohistochemistry. Slides also were studied by combined immunohistochemistry and in situ hybridization with an antibody that recognizes monocytes/macrophage and with riboprobes that detect PDGF B-chain, PDGF beta-R or TGF-beta1 mRNA. RESULTS: Increased numbers of proliferating glomerular cells appeared early in the disease course, associated with de novo expression of alphaSMA. Expression of PDGF B-chain and beta-R mRNA was increased in the mesangium and in parietal epithelial cells of TSLP transgenic mice and correlated with the number of PCNA positive cells. Increased TGF-beta1 mRNA expression paralleled the deposition of type IV collagen. A significant proportion of Mac-2 positive macrophages expressed TGF-beta1 mRNA, while only a small percentage of glomerular macrophages expressed PDGF B-chain mRNA. No PDGF beta-R mRNA expression by macrophages was detected. CONCLUSION: TSLP transgenic mice develop a membranoproliferative glomerulonephritis in which glomerular cell proliferation and matrix deposition are associated with an increased expression of PDGF B-chain, PDGF beta-R and TGF-beta1. These findings extend the paradigms covering these growth factors established in the rat Thy 1 model of mesangiolysis and repairs to a murine model of progressive glomerulonephritis closely resembling human MPGN.     

Selective lymphocyte inhibition by FTY720 slows the progressive course of chronic anti-thy 1 glomerulosclerosis

BACKGROUND: Progression is a hallmark of chronic renal disease and histologically characterized by fibrosis and inflammation of the tubulointerstitial compartment. To define the role of lymphocytes in this process, the novel lymphocyte-specific inhibitor FTY720 was administered to rats with anti-thy 1-induced chronic progressive glomerulosclerosis. In this model, the initial and short-term inflammatory glomerular injury progresses self-perpetuatedly toward tubulointerstitial fibrosis by not primarily immune-mediated, intrarenal mechanisms. METHODS: Chronic progressive glomerulosclerosis was induced by murine anti-thy 1 antibody injection into uninephrectomized rats. Treatment with FTY720 (0.3 mg/kg body weight) was started 7 days after disease induction. Proteinuria was measured every 4 weeks. In week 20, the following parameters were determined: blood lymphocyte number, kidney function, both glomerular and tubulointerstitial histologic matrix accumulation, protein expression of transforming growth factor-beta1 (TGF-beta1), fibronectin, and plasminogen activator inhibitor-1 (PAI-1) as well as infiltration with macrophages and lymphocytes. RESULTS: Treatment with FTY720 lowered blood lymphocyte count and renal lymphocyte infiltration highly significantly. In comparison to the untreated chronic progressive glomerulosclerosis animals, the lymphocyte depletion achieved significantly limited the progression of the disease, as shown by lowered proteinuria, tubulointerstitial matrix expansion, and TGF-beta1, fibronectin, and PAI-1 expression, as well as improved renal function. Glomerular matrix protein expression and accumulation was moderately lowered by FTY720. Glomerular macrophage infiltration was not, tubulointerstitial macrophage infiltration was moderately, but not significantly, decreased by FTY720 treatment. CONCLUSION: Lymphocyte depletion by FTY720 limits the progression of anti-thy 1-induced glomerulosclerosis toward chronic tubulointerstitial fibrosis and renal insufficiency. The data suggest that lymphocytes actively participate in the progression of chronic experimental kidney disease, and that FTY720 may be a novel approach to slow the progressive course of human chronic renal diseases.     

Renal expression of fibrotic matrix proteins and of transforming growth factor-beta (TGF-beta) isoforms in TGF-beta transgenic mice

Renal pathology in mice that are transgenic for the murine albumin enhancer/promoter linked to a full-length porcine transforming growth factor-beta1 (TGF-beta1) gene has been described previously. In these mice, transgene expression is limited to the liver and the plasma level of TGF-beta is increased. The earliest renal pathologic change is glomerulosclerosis, at 3 wk of age, and this is followed by tubulointerstitial fibrosis. In this study, it was hypothesized that circulating TGF-beta1 increases renal extracellular matrix accumulation and activates local TGF-beta gene expression. Immunostaining at 5 wk revealed increased amounts of collagen I and III within the mesangium, glomerular capillary loops, and interstitium, while the amount of collagen IV was normal. Similarly, Northern analysis showed increased expression of mRNA encoding collagen I and III, as well as biglycan and decorin, while the expression of collagen IV was unchanged. These changes began as early as 1 wk of age, a time before the appearance of glomerulosclerosis. To evaluate matrix degradation, collagenase IV activity was evaluated by gelatin zymography and an increase in matrix metalloproteinase-2 was found. Finally, the production of tissue inhibitors of metalloproteinase was evaluated. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA was increased 18-fold, while TIMP-2 and TIMP-3 were unchanged. In 2-wk-old transgenic kidney, local expression of TGF-beta1, beta2, and beta3 protein was similar to wild-type mice. In 5-wk-old transgenic mice, TGF-beta1 and beta2 protein was present in increased amounts within glomeruli, and renal TGF-beta1 mRNA was increased threefold. It is concluded that elevated levels of circulating TGF-beta1 may act on the kidney to increase matrix protein production and decrease matrix remodeling. Only after glomerulosclerosis is established does local glomerular overproduction of TGF-beta become manifest.     

Lovastatin inhibits transforming growth factor-beta1 expression in diabetic rat glomeruli and cultured rat mesangial cells

Diabetic nephropathy is a leading cause of end-stage renal disease and is characterized by excessive deposition of extracellular matrix (ECM) proteins in the glomeruli. Transforming growth factor-beta (TGF-beta) is the major mediator of excessive accumulation of ECM proteins in diabetic nephropathy through upregulation of genes encoding ECM proteins as well as downregulation of genes for ECM-degrading enzymes. It has been shown that lovastatin, an inhibitor of 3-hydroxy3-methylglutaryl CoA reductase, delays the onset and progression of different models of experimental nephropathy. To evaluate the effect of lovastatin on the development and progression of diabetic nephropathy, streptozotocin-induced diabetic rats were studied for 12 mo. In untreated diabetic rats, there were significant increases in blood glucose, urine albumin excretion, kidney weight, glomerular volume, and TGF-beta1 mRNA expression in the glomeruli compared with normal control rats treated with citrate buffer only. Treatment with lovastatin in diabetic rats significantly suppressed the increase in urine albumin excretion, kidney weight, glomerular volume, and TGF-beta1 mRNA expression despite high blood glucose levels. To elucidate the mechanisms of the renal effects of lovastatin, rat mesangial cells were cultured under control (5.5 mM) or high (30 mM) glucose with lovastatin alone, mevalonate alone, or with both. Under high glucose, TGF-beta1 and fibronectin mRNA and proteins were upregulated. These high glucose-induced changes were suppressed by lovastatin (10 micro/M) and nearly completely restored by mevalonate (100 microM). These results suggest that lovastatin has a direct cellular effect independent of a cholesterol-lowering effect and delays the onset and progression of diabetic nephropathy, at least in part, through suppression of glomerular expression of TGF-beta1.     

MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-beta in rat mesangial cells

BACKGROUND: Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-beta (TGF-beta)-induced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-beta-induced rat PAI-1 expression in rat mesangial cells. METHODS: TGF-beta1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay. RESULTS: Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-beta1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-beta1, and the disruption of this AP-1-like sequence suppressed basal and TGF-beta1-induced promoter activation. TGF-beta1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-beta1-induced rat PAI-1 promoter activity. Moreover, interferon-gamma (IFN-gamma) inhibited TGF-beta1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway. CONCLUSION: These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-beta1-induced PAI-1 expression is unique to rat mesangial cells.