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Hemodynamic stress in concert with metabolic pathways that are activated by hyperglycemia, glycated proteins, and oxidative stress induce a host of growth factors in the kidney. The fibrogenic cytokine transforming growth factor-beta (TGF-beta), through its Smad3 signaling pathway, is the etiologic agent of renal hypertrophy and the accumulation of mesangial extracellular matrix components in diabetes. Neutralizing anti-TGF-beta antibodies, antisense TGF-beta1 oligodeoxynucleotides or knocking off the Smad3 gene prevent and/or reverse the hypertrophic and profibrotic effects of the diabetic state in mice. However, there is limited evidence to support a role for TGF-beta in the development of albuminuria. Podocyte-derived vascular endothelial growth factor (VEGF), a permeability and angiogenic factor whose expression is also increased in animal models of diabetic kidney disease, appears to act in a novel autocrine signaling mode to induce the podocytopathy of diabetes, especially the genesis of albuminuria. Future strategies for therapy of diabetic nephropathy may therefore need to involve interception of both the TGF-beta and the VEGF signaling pathways to counter the matrix accumulation and to improve the albuminuria. Interception of the renin-angiotensin system may achieve this goal but other novel strategies will need to be developed that would be more efficacious. However, a note of caution should be raised not to lower the heightened activities of these two signaling pathways much below normal levels because a basal activity for each is essential for the optimal homeostasis of glomerular cells.

Transforming growth factor (TGF)-beta plays a critical role in diabetic nephropathy. To isolate the contribution of one of the signaling pathways of TGF-beta, the Smad3 gene in the mouse was knocked out at exons 2 and 3, and the effect was studied in streptozotocin (STZ)-induced diabetes over a period of 6 wk. TGF-beta activity was increased in the diabetic mice but was not able to signal via Smad3 in the knockout (KO) mice. As expected in the wild type, the kidneys of the STZ-diabetic mice showed both structural and functional defects that are characteristic of diabetic renal involvement. In the Smad3-KO mice, however, the defects that were improved were renal hypertrophy, mesangial matrix expansion, fibronectin overproduction, glomerular basement membrane thickening, plasma creatinine, and the blood urea nitrogen. The parameters not significantly altered by the Smad3-KO were albuminuria, reduction in podocyte slit pore density, and the increase in vascular endothelial growth factor abundance and activity. It seems that the absence of Smad3 modifies the natural course of murine diabetic nephropathy, providing renal functional protection and preventing structural lesions relating to kidney hypertrophy and matrix accumulation, even though albuminuria and changes in podocyte morphology persist. In conclusion, the effects of the Smad3-KO mirror the effects of anti-TGF-beta therapy in diabetes, suggesting that the chief component of TGF-beta signaling that is relevant to kidney disease is the Smad3 pathway.

Podocyte-derived vascular endothelial growth factor (VEGF) is upregulated in diabetes and may contribute to albuminuria. Although believed to act upon the glomerular endothelium, VEGF may have pronounced effects on the podocyte itself. The functionality of this VEGF autocrine loop was investigated in conditionally immortalized mouse podocytes. Exogenous VEGF(164) increased the production of alpha3(IV) collagen, an integral component of the glomerular basement membrane (GBM); this effect was completely prevented by SU5416, a pan-VEGF receptor inhibitor. The VEGF inhibitor also partially prevented the stimulation of alpha3(IV) collagen by transforming growth factor (TGF)-beta1, establishing a novel role for endogenous VEGF. However, VEGF did not influence the production of another novel chain of collagen IV, alpha5(IV) collagen, and SU5416 failed to reverse the known inhibitory effect of TGF-beta1 on alpha5(IV) collagen production. Cultured mouse podocytes possess at least the VEGFR-1 receptor, confirmed by RT-PCR, immunoblotting, and immunocytochemistry. By these techniques, however, VEGFR-2 is absent. VEGF signaling proceeds via autophosphorylation of VEGFR-1 and activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Thus, podocyte-derived VEGF operates in an autocrine loop, likely through VEGFR-1 and PI3K, to stimulate alpha3(IV) collagen production. The TGF-beta1-stimulated endogenous VEGF may have significant implications for podocyte dysfunction in diabetic glomerulopathy, manifesting as GBM thickening and altered macromolecular permeability.

Background: To firmly establish the role of the transforming growth factor-beta1 (TGF-beta1) isoform in the pathophysiology of diabetic tubulointerstitial hypertrophy and fibrosis, we examined how the total absence of TGF-beta1 would alter the effect of high glucose on cellular hypertrophy and matrix expression in tubuloepithelial cells cultured from TGF-beta1 null mice., Methods: Primary tubule cell cultures, obtained from kidneys of TGF-beta1 knockout mice and their wild-type littermates, were treated with exogenous TGF-beta1 or high glucose. The TGF-beta system was characterized at the ligand and receptor levels using Northern and Western blotting. Cellular hypertrophy and growth were assessed by thymidine incorporation, cell counting, leucine incorporation, and protein content. Fibronectin expression was assessed by Northern analysis and enzyme-linked immunosorbent assay (ELISA)., Results: Knockout cells did not express TGF-beta1 but did express TGF-beta2, TGF-beta3, and TGF-beta type I and type II receptors. Exogenous TGF-beta1 down-regulated the ligand-binding type II receptor but up-regulated type I receptor expression. Knockout cells proliferated more rapidly than wild-type cells, but restoring TGF-beta1 to knockout cells slowed their proliferation. In wild-type cells, high glucose caused cellular hypertrophy, evidenced by greater leucine incorporation and protein content along with decreased thymidine incorporation. High glucose also increased fibronectin message and protein. However, in knockout cells, high glucose failed to induce hypertrophy and was severely limited in its capacity to stimulate fibronectin., Conclusion: In tubular epithelial cells, TGF-beta1 mediates the hypertrophic and fibronectin-stimulatory effects of high glucose, confirming the role of the TGF-beta1 isoform in the pathogenesis of diabetic tubular hypertrophy and fibronectin overexpression.

The critical role of hyperglycemia in the genesis of diabetic nephropathy has been established by cell culture studies, experimental animal models, and clinical trials. Certain cytokines and growth factors have been identified as likely mediators of the effects of high ambient glucose on the kidney, but prominent among these is TGF-beta, a prototypical hypertrophic and fibrogenic cytokine. Overexpression of TGF-beta has been demonstrated in the glomerular and tubulointerstitial compartments of experimental diabetic animals. The TGF-beta receptor signaling system is also triggered, as evidenced by upregulation of the TGF-beta type II receptor and activation of the downstream Smad signaling pathway. Treatment of diabetic mice with neutralizing anti-TGF-beta antibodies prevents the development of renal hypertrophy, mesangial matrix expansion, and the decline in renal function. Antibody therapy also reverses the established lesions of diabetic glomerulopathy. These studies argue strongly in support of the hypothesis that overactivity of the TGF-beta system in the kidney is a crucial mediator of diabetic renal hypertrophy and mesangial matrix expansion.

The manifestations of diabetic nephropathy may be a consequence of the actions of certain cytokines and growth factors. Prominent among these is transforming growth factor beta (TGF-beta) because it promotes renal cell hypertrophy and stimulates extracellular matrix accumulation, the 2 hallmarks of diabetic renal disease. In tissue culture studies, cellular hypertrophy and matrix production are stimulated by high glucose concentrations in the culture media. High glucose, in turn, appears to act through the TGF-beta system because high glucose increases TGF-beta expression, and the hypertrophic and matrix-stimulatory effects of high glucose are prevented by anti-TGF-beta therapy. In experimental diabetes mellitus, several reports describe overexpression of TGF-beta or TGF-beta type II receptor in the glomerular and tubulointerstitial compartments. As might be expected, the intrarenal TGF-beta system is triggered, evidenced by activity of the downstream Smad signaling pathway. Treatment of diabetic animals with a neutralizing anti-TGF-beta antibody prevents the development of mesangial matrix expansion and the progressive decline in renal function. This antibody therapy also reverses the established lesions of diabetic glomerulopathy. Finally, the renal TGF-beta system is significantly up-regulated in human diabetic nephropathy. Although the kidney of a nondiabetic subject extracts TGF-beta1 from the blood, the kidney of a diabetic patient actually elaborates TGF-beta1 protein into the circulation. Along the same line, an increased level of TGF-beta in the urine is associated with worse clinical outcomes. In concert with TGF-beta, other metabolic mediators such as connective tissue growth factor and reactive oxygen species promote the accumulation of excess matrix. This fibrotic build-up also occurs in the tubulointerstitium, probably as the result of heightened TGF-beta activity that stimulates tubular epithelial and interstitial fibroblast cells to overproduce matrix. The data presented here strongly support the consensus that the TGF-beta system mediates the renal hypertrophy, glomerulosclerosis, and tubulointerstitial fibrosis of diabetic kidney disease.

Treatment with a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody can prevent the development of diabetic nephropathy in the db/db mouse, a model of type 2 diabetes. However, it is unknown whether anti-TGF-beta therapy can reverse the histological lesions of diabetic glomerulopathy once they are established. Diabetic db/db mice and their non-diabetic db/m littermates were allowed to grow until 16 weeks of age, by which time the db/db mice had developed glomerular basement membrane (GBM) thickening and mesangial matrix expansion. The mice were then treated with an irrelevant control IgG or a panselective, neutralizing anti-TGF-beta antibody for eight more weeks. Compared with control db/m mice, the db/db mice treated with IgG had developed increased GBM width (16.64+/-0.80 nm vs. 21.55+/-0.78 nm, P<0.05) and increased mesangial matrix fraction (4.01+/-0.81% of total glomerular area vs. 9.55+/-1.04%, P<0.05). However, the db/db mice treated with anti-TGF-beta antibody showed amelioration of GBM thickening (18.40+/-0.72 nm, P<0.05 vs. db/db-IgG) and mesangial matrix accumulation (6.32+/-1.79%, P<0.05 vs. db/db-IgG). Our results demonstrate that inhibiting renal TGF-beta activity can partially reverse the GBM thickening and mesangial matrix expansion in this mouse model of type 2 diabetes. Anti-TGF-beta regimens would be useful in the treatment of diabetic nephropathy.

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.

Unlabelled: Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes., Background: The podocyte takes center stage in the pathogenesis of glomerular basement membrane (GBM) thickening and proteinuria in diabetic glomerulopathy. In part, GBM thickening may occur when the podocyte synthesizes increased amounts of collagen IV. Proteinuria may develop if the podocyte secretes excessive amounts of vascular endothelial growth factor (VEGF), which may increase the glomerular permeability to macromolecules. The augmented production of collagen IV and VEGF may be caused by metabolic mediators of diabetes such as hyperglycemia and transforming growth factor-beta (TGF-beta)., Methods: The effects of high glucose and exogenous TGF-beta1 were examined on a mouse podocyte cell line that retains its differentiated phenotype. The gene expression and protein production of certain alpha chains of collagen IV, the major isoforms of VEGF, and components of the TGF-beta system were assayed. An inhibitor of TGF-beta signaling was used to determine whether some of the high glucose effects might be mediated by the TGF-beta system., Results: Compared with normal glucose (5.5 mmol/L), high glucose (HG, 25 mmol/L) for 14 days stimulated [3H]-proline incorporation, a measure of collagen production, by 1.8-fold, and exogenous TGF-beta1 (2 ng/mL) for 24 hours stimulated proline incorporation by 2.4-fold. Northern analysis showed that exposure to HG for 14 days increased the mRNA level of alpha1(IV) collagen by 51% and alpha5(IV) by 90%, whereas treatment with TGF-beta1 (2 ng/mL) for 24 hours decreased the mRNA level of alpha1(IV) by 36% and alpha5(IV) by 40%. Consistent with these effects on mRNA expression, Western blotting showed that HG increased alpha1(IV) protein by 44% and alpha5(IV) by 28%, while TGF-beta1 decreased alpha1(IV) protein by 29% and alpha5(IV) by 7%. In contrast to their opposing actions on alpha1 and alpha5(IV), both HG and exogenous TGF-beta1 increased alpha3(IV) collagen and VEGF, with TGF-beta1 having the greater effect. An inhibitor of the TGF-beta type I receptor (ALK5) was able to prevent the stimulation of alpha3(IV) and VEGF proteins by HG. Unlike in other renal cell types, HG did not increase TGF-beta1 mRNA or protein in the podocyte, but HG did induce the expression of the ligand-binding TGF-beta type II receptor (TbetaRII). Because HG had up-regulated TbetaRII after two weeks, the addition of physiological-dose TGF-beta1 (0.010 ng/mL) for 24 hours stimulated the production of alpha3(IV) and VEGF proteins to a greater extent in high than in normal glucose. Up-regulation of TbetaRII in the podocyte was corroborated by immunohistochemistry of the kidney cortex in the db/db mouse, a model of type 2 diabetes., Conclusions: High glucose and exogenous TGF-beta1 exert disparate effects on the expression of alpha1 and alpha5(IV) collagen. However, high glucose and TGF-beta1 coordinately induce the production of alpha3(IV) collagen and VEGF in the podocyte. The HG-induced increases in alpha3(IV) collagen and VEGF proteins are mediated by the TGF-beta system. By increasing the expression of TbetaRII, high glucose may augment the response of the podocyte to ambient levels of TGF-beta1.

Background: Glycated albumin has been causally linked to the pathobiology of diabetic renal disease through its ability to stimulate the expression of transforming growth factor-beta1 (TGF-beta1), activate protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), and promote production of extracellular matrix proteins in cultured glomerular cells. Whether glycated albumin modulates glomerular TGF-beta1 expression in vivo is not known. To address this issue, we assessed glomerular TGF-beta1 expression and pathology in response to reducing the burden of glycated albumin in vivo., Methods: We measured serum glycated albumin, urine protein, glomerular TGF-beta1 expression and morphometry, and collagen IV and fibronectin mRNA in db/m and db/db controls and in db/db mice treated for eight weeks with a synthetic compound that inhibits the condensation of glucose with albumin., Results: In situ hybridization studies showed markedly increased glomerular TGF-beta1 mRNA in control db/db mice, which was significantly reduced in db/db mice treated for eight weeks with test compound. The treatment protocol, which normalized serum glycated albumin, concomitantly reduced the elevated protein excretion and the renal overexpression of mRNAs encoding fibronectin and collagen IV, and significantly decreased the mesangial matrix expansion, observed in db/db control animals., Conclusions: These findings, to our knowledge, provide the first evidence that glomerular overexpression of TGF-beta1 in diabetes derives at least in part from elevated glycated albumin concentrations, and can be partially suppressed by inhibiting the formation of this glycated protein. The results further suggest that glycated albumin has an important nephropathogenic role in diabetes that is operative, and can be therapeutically addressed, independent of glycemic status.

As reports of mice genetically deficient for TGF-beta1 demonstrated aberrant renal class II MHC expression, we investigated inducible class II MHC expression on renal tubular epithelial cells derived from TGF-beta1 knockout (-/-) and wild-type (+/+) mice. IFN-gamma markedly upregulated class II MHC (I-A(b)) expression in both (-/-) and (+/+) tubular epithelial cells. Coincubation studies of (+/+) and (-/-) tubular epithelial cells with IFN-gamma+LPS, or pretreatment of these cells with TGF-beta1, revealed inhibition of IFN-gamma-induced I-A(b) mRNA and cell surface expression that occurred via a decrease in class II transactivator gene expression in both (+/+) and (-/-) tubular epithelial cells. In addition, ICAM-1 was constitutively expressed on both (+/+) and (-/-) tubular epithelial cells and was upregulated by IFN-gamma or IFN-gamma+LPS. ICAM-1 expression in (+/+) and (-/-) tubular epithelial cells, however, was decreased by TGF-beta1. Parallel analysis evaluating B7-1 expression detected low levels of B7-1 in unstimulated (+/+) and (-/-) tubular epithelial cells that were increased by IFN-gamma, LPS, and IFN-gamma+LPS. IFN-gamma+LPS-mediated upregulation of B7-1 was also blocked by pretreatment with TGF-beta1. Cytokine analysis detected significantly higher levels of TNF-alpha and MIP-1alpha mRNA in all treated (-/-) preparations than in (+/+) tubular epithelial cell controls. These studies demonstrate normal patterns of class II MHC, ICAM-1, and B7 expression in TGF-beta1 (-/-) tubular epithelial cells in response to IFN-gamma, LPS, and TGF-beta1. Upregulated cytokine expression at baseline and in response to proinflammatory mediators is apparent in (-/-) tubular epithelial cells, however, and suggests that dysregulation of cytokine expression in inflammatory responses may be a primary event in multifocal inflammation observed in TGF-beta1-deficient animals., (Copyright 2002 S. Karger AG, Basel)

Progressive renal injury in diabetes mellitus leads to major morbidity and mortality. The manifestations of diabetic nephropathy may be a consequence of the actions of certain cytokines and growth factors. Prominent among these is transforming growth factor-beta (TGF-beta) because it promotes renal cell hypertrophy and stimulates extracellular matrix accumulation, the two hallmarks of diabetic renal disease. In cell culture, high ambient glucose increases TGF-beta mRNA and protein in proximal tubular, glomerular epithelial, and mesangial cells. Neutralizing anti-TGF-beta antibodies prevent the hypertrophic and matrix stimulatory effects of high glucose in these cells. In experimental and human diabetes mellitus, several reports describe overexpression of TGF-beta in the glomeruli and tubulointerstitium. We demonstrate that short-term treatment of diabetic mice with neutralizing monoclonal antibodies against TGF-beta significantly reduces kidney weight and glomerular hypertrophy and attenuates the increase in extracellular matrix mRNAs. Long-term treatment of diabetic mice further improves the renal pathology and also ameliorates the functional abnormalities of diabetic nephropathy. Finally, we provide evidence that the renal TGF-beta system is significantly up-regulated in human diabetes. The kidney of a diabetic patient actually elaborates TGF-beta1 protein into the circulation whereas the kidney of a non-diabetic subject extracts TGF-beta1 from the circulation. The data we review here strongly support the hypothesis that elevated production or activity of the TGF-beta system mediates diabetic renal hypertrophy and extracellular matrix expansion.

Activation of the renal transforming growth factor-beta (TGF-beta) system likely mediates the excess production of extracellular matrix in the diabetic kidney. To establish the role of the TGF-beta system in type 2 diabetic nephropathy, we examined the intrarenal localization and expression of the TGF-beta1 isoform, the TGF-beta type II receptor, and the Smad signaling pathway in the 16-week-old db/db mouse, a genetic model of type 2 diabetes that exhibits mesangial matrix expansion, glomerular basement membrane thickening, and renal insufficiency that closely resemble the human disease. Compared with its nondiabetic db/m littermate, the db/db mouse showed significantly increased TGF-beta1 mRNA expression by in situ hybridization in both glomerular and tubular compartments. Likewise, TGF-beta1 protein, by immunohistochemical staining, was increased in both renal compartments, but the fractional expression of TGF-beta1 protein was less than that of the mRNA in the glomerulus. In situ hybridization and immunohistochemical staining for the TGF-beta type II receptor revealed concordant and significant increases of both mRNA and protein in the glomerular and tubular compartments of diabetic animals. Finally, immunohistochemistry showed preferential accumulation of Smad3 in the nuclei of glomerular and tubular cells in diabetes. The complementary technique of Southwestern histochemistry using a labeled Smad-binding element demonstrated increased binding of nuclear proteins to Smad-binding element, indicating active signaling downstream of the TGF-beta stimulus. We therefore propose that the TGF-beta system is up-regulated at the ligand, receptor, and signaling levels throughout the renal cortex in this animal model of type 2 diabetes. Our findings suggest that the profibrotic effects of TGF-beta may underlie the progression to glomerulosclerosis and tubulointerstitial fibrosis that characterize diabetic nephropathy.

Background: The activation of protein kinase C (PKC) and transforming growth factor-beta (TGF-beta) in glomerular mesangial cells has been linked to mesangial matrix expansion in diabetic nephropathy. The role of these mediators in affecting the changes associated with diabetes in the biology of glomerular endothelial cells (GEnCs), which synthesize components of the glomerular basement membrane, is not known. We postulated that the PKC and TGF-beta systems promote the increased endothelial cell synthesis of glomerular basement membrane that is evoked by Amadori-modified glycated albumin, which is present in elevated concentrations in diabetes., Methods: We examined the effects of PKC inhibition on collagen IV and TGF-beta1 production by mouse GEnCs incubated with glycated albumin and the influence of glycated albumin on PKC activity, TGF-beta 1 production, and proliferation by these cells., Results: In physiologic (5.5 mmol/L) glucose concentrations, glycated albumin caused an increase in type IV collagen production that was totally prevented by a general PKC inhibitor GF 109203X (GFX), but only partly prevented by a neutralizing anti-TGF-beta antibody. Glycated albumin increased the steady-state level of TGF-beta 1 mRNA and stimulated the production of TGF-beta 1 protein, which was also prevented by the PKC inhibitor GFX. Of note, glycated albumin significantly stimulated PKC activity, as measured by the phosphorylation of a PKC-specific substrate. Cell proliferation, measured by [(3)H]-thymidine incorporation and cell counting, was decreased in the presence of glycated albumin. This effect was completely prevented by GFX and partially reversed by anti-TGF-beta antibody. Exogenous TGF-beta 1 inhibited cell proliferation to a degree similar to that of glycated albumin., Conclusions: PKC signaling and consequent TGF-beta 1 activation participate in the glycated albumin-induced stimulation of basement membrane collagen production by GEnC. By reducing the proliferative capacity, which is likely mediated by PKC and partly by TGF-beta, glycated albumin impedes the ability of the glomerular capillary endothelium to act as a first line of defense against deleterious circulating factors in the diabetic state.

Transforming growth factor beta (TGF-beta) plays an important role in the development of tubulointerstitial fibrosis in chronic renal disease. We were interested whether interference with oxygen radicals may modulate TGF-beta expression. Unexpectedly, we discovered that diphenylene iodine (DIP), an inhibitor of NADP(H) oxidase, induces a robust increase in TGF-beta transcript expression in cultured mouse proximal tubular cells (MCT cells). A similar increase was seen with EUK-8, a synthetic salen-manganese complex with high oxyradical scavenger activities. This induction of TGF-beta1 mRNA was paralleled by increasing protein expression. Transient transfection of MCT cells with a reporter construct in which murine TGF-beta1 enhancer/promoter elements were cloned in front of the luciferase gene, revealed that DIP, EUK-8, and Tiron all stimulated transcription of the TGF-beta1 gene whereas exogenous H2O2 suppressed transcription. Antisense oligonucleotides against p22phox, but not sense oligonucleotides, also increased transcriptional activity of TGF-beta1. Mutagenesis of Sp1 binding sites in the mouse TGF-beta1 enhancer/promoter abolished the stimulatory effect of the antioxidants. Gel shift experiments revealed that DIP as well as EUK-8 activated binding of nuclear proteins to Sp1 consensus sequence. Our data provide evidence that TGF-beta1 transcription is negatively regulated in MCT cells under basal conditions by NADP(H) oxidase-mediated oxygen radicals. Thus, antioxidant therapy may increase local synthesis of TGF-beta1 in the tubulointerstitium.

In summary, metabolic, hemodynamic, and genetic factors are all important in the development and progression of diabetic nephropathy (33). Recent studies using cell culture techniques and experimental animal models have provided important insight into the role of hyperglycemia in this disease. The nature of the factors directly arising as a consequence of hyperglycemia and the steps involved in diabetic complications are not completely understood. The characteristic lesions of diabetic nephropathy may be intimately related to the effects of high ambient glucose on intracellular signaling events, various growth factors/cytokines, and nonenzymatic glycation of proteins (36). The growth factor TGF-beta has emerged as a key participant in the cascade of events which leads to kidney sclerosis. Increased TGF-beta expression in the kidney in diabetes mellitus mediates the renal actions of high ambient glucose to promote cellular hypertrophy and stimulate extracellular matrix biosynthesis. Neutralizing the actions of TGF-beta with highly specific monoclonal antibodies or with application of antisense technology can effectively prevent the induction of the kidney lesions of diabetes in mice independent of any changes in blood glucose levels. Such maneuvers are crucial for establishing proof-of-concept and Koch's postulates (17), but they are still far removed from clinical applicability. Nevertheless, it is hoped that further studies to elucidate the efficacy of novel interventions to intercept the activity of the renal TGF-beta system will prove useful for effectively halting the progression of diabetic nephropathy.

Background: Reactive oxygen species (ROS) are excessively produced in pathologic states, including many renal diseases. Transforming growth factor-beta (TGF-beta) may mediate renal fibrotic injury, and ROS may act through the TGF-beta pathway to exert a profibrotic effect., Methods: The expression of TGF-beta1 and extracellular matrix (ECM) components were assessed in cultured human mesangial cells (HMCs) incubated with glucose oxidase (GO), an enzyme that continuously generates hydrogen peroxide from glucose. A neutralizing anti-TGF-beta antibody was added to test the hypothesis that hydrogen peroxide acts through activation of the TGF-beta pathway to stimulate ECM expression., Results: Northern blot analysis revealed significantly increased steady-state levels of TGF-beta1 and ECM proteins (collagen types I, III, and IV, and fibronectin) by approximately twofold. While no significant effect on mRNA stability after treatment with GO was observed, other studies employing promoter-reporter assays, competitive-quantitative reverse transcription-polymerase chain reaction, mink lung epithelial cell proliferation assay, and TGF-beta1 enzyme-linked immunosorbent assay all demonstrated significant stimulation by GO (>1.5-fold) of TGF-beta1 promoter activity, mRNA level, bioactivity, and protein production, respectively. Catalase pretreatment prevented the GO-induced stimulation of TGF-beta1 mRNA. When incubations were performed with a panselective neutralizing anti-TGF-beta antibody, the GO-stimulated expression of ECM molecules was prevented., Conclusions: GO-induced hydrogen peroxide production induces TGF-beta1 synthesis and thereby increases ECM gene expression in cultured HMCs. These cellular responses may underlie the development and progression of renal diseases characterized by oxidative stress.

High ambient glucose exerts its injurious effects on renal cells through nonenzymatic and enzymatic pathways, including altered signal transduction and upregulation of the transforming growth factor-beta (TGF-beta) system. Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) cascade, is activated in mesangial cells cultured in high glucose and in glomeruli of diabetic rats. However, the biologic consequences of ERK activation in the kidney have not been investigated. To clarify the role of ERK activation, mouse mesangial cells were exposed to normal (5.5 mM) or high (25 mM) glucose with or without addition of PD98059, a specific inhibitor of MAPK/ERK kinase (MEK), an upstream kinase activator of ERK. Cells that were exposed to high glucose exhibited significant increases in ERK activity, TGF-beta1 expression (total protein, mRNA levels, and promoter activity), [(3)H]-proline uptake, and alpha1(I) collagen and fibronectin mRNA levels. Treatment with PD98059 (up to 25 microM) significantly inhibited these parameters. In contrast, 25 microM PD98059 had no significant effect on any of the parameters measured in cells that were exposed to normal glucose. Overexpression of MAPK phosphatase CL 100 prevented TGF-beta1 promoter activation by high glucose, confirming the involvement of the MEK-ERK pathway in response to high glucose. The conclusion is that activation of ERK in mesangial cells is responsible for high-glucose-induced stimulation of TGF-beta1 and contributes to the increased extracellular matrix expression.

Background: The recently discovered arachidonic acid derivatives, isoprostanes, are increased in pathological conditions associated with oxidative stress, such as diabetes. No role has yet been described for isoprostanes during the development of diabetic nephropathy. Cell culture in high ambient glucose has been used as a model in elucidating cellular mechanisms underlying diabetic nephropathy. Among the growth factors involved in the effect of high glucose, transforming growth factor-beta (TGF-beta) has been described as playing a key role in the development of nephropathy., Methods: Streptozotocin-induced diabetic rats were supplemented in their diet with the antioxidant vitamin E (1000 U/kg diet). Blood and urine samples were taken to determine renal function and isoprostane concentration, as determined by gas chromatography/mass spectrometry. Glomerular mesangial and endothelial cells were cultured in high ambient glucose to determine the synthesis of isoprostanes and the role of isoprostanes in high glucose-induced synthesis of TGF-beta., Results: Streptozotocin-induced diabetic rats had marked increases in plasma levels and urinary excretion rates of F(2)-isoprostanes. Dietary supplementation with vitamin E normalized (plasma) and reduced (urine) isoprostane levels and, surprisingly, improved proteinuria and blood urea nitrogen (BUN) levels. High ambient glucose increased F(2)-isoprostane synthesis in glomerular endothelial and mesangial cells in culture. Incubation of glomerular cells with F(2)-isoprostanes stimulated the production of TGF-beta., Conclusions: Increased F(2)-isoprostane synthesis during diabetes appears to be responsible in part for the increase in renal TGF-beta, a well-known mediator of diabetic nephropathy.

Objectives: Patients with posterior urethral valves (PUV) are at significant risk for progression to end-stage renal disease, despite early correction of the obstruction. Experimental models of urinary obstruction demonstrate increased renal expression of the profibrotic inflammatory mediator, transforming growth factor-beta(1) (TGF-beta(1)). Urinary TGF-beta(1) excretion is elevated in certain glomerular diseases, but has not been well studied in patients with obstructive lesions. The objective of this study was to examine urinary TGF-beta(1) excretion in children with PUV., Methods: Fourteen patients with PUV, aged 3.2 to 14.5 years, with estimated glomerular filtration rates (GFRs) of 12.8 to 139 mL/min/1.73 m(2) were enrolled. Sixteen normal subjects (9 male, 7 female), aged 4.3 to 20.5 years, served as controls. Total urinary TGF-beta(1) concentration was assayed by enzyme-linked immunoabsorbent assay, and expressed as a ratio to urinary creatinine concentration., Results: Urinary TGF-beta(1) excretion was significantly greater in patients with PUV (range 0 to 0.063, median 0.019 ng/mg urine creatinine) compared with that of healthy controls (range 0 to 0.022, median 0.005 ng/mg urine creatinine) (P <0.01). There was no correlation between urinary TGF-beta(1) excretion and estimated GFR, past urinary diversion surgery, or bladder wall thickening. Among healthy controls, urinary TGF-beta(1) was not correlated with age or gender., Conclusions: Results from this study suggest that TGF-beta(1) may contribute to progressive renal insufficiency in patients with PUV. Further studies are indicated to determine if agents that affect TGF-beta(1) expression, such as angiotensin-converting enzyme inhibitors, can slow the progression of renal disease in PUV.

Emerging evidence suggests that transforming growth factor-beta (TGF-beta) is an important mediator of diabetic nephropathy. We showed previously that short-term treatment with a neutralizing monoclonal anti-TGF-beta antibody (alphaT) in streptozotocin-diabetic mice prevents early changes of renal hypertrophy and increased matrix mRNA. To establish that overactivity of the renal TGF-beta system mediates the functional and structural changes of the more advanced stages of nephropathy, we tested whether chronic administration of alphaT prevents renal insufficiency and glomerulosclerosis in the db/db mouse, a model of type 2 diabetes that develops overt nephropathy. Diabetic db/db mice and nondiabetic db/m littermates were treated intraperitoneally with alphaT or control IgG, 300 microgram three times per week for 8 wk. Treatment with alphaT, but not with IgG, significantly decreased the plasma TGF-beta1 concentration without decreasing the plasma glucose concentration. The IgG-treated db/db mice developed albuminuria, renal insufficiency, and glomerular mesangial matrix expansion associated with increased renal mRNAs encoding alpha1(IV) collagen and fibronectin. On the other hand, treatment with alphaT completely prevented the increase in plasma creatinine concentration, the decrease in urinary creatinine clearance, and the expansion of mesangial matrix in db/db mice. The increase in renal matrix mRNAs was substantially attenuated, but the excretion of urinary albumin factored for creatinine clearance was not significantly affected by alphaT treatment. We conclude that chronic inhibition of the biologic actions of TGF-beta with a neutralizing monoclonal antibody in db/db mice prevents the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes.

Inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) relies on their ability to bind complementary mRNA sequences and prevent translation. The proximal tubule is a suitable target for ODN therapy in vivo because circulating ODNs accumulate in the proximal tubule in high concentrations. Because increased proximal tubular transforming growth factor- beta1 (TGF-beta1) expression may mediate diabetic renal hypertrophy, we investigated the effects of antisense TGF-beta1 ODN on the high-glucose-induced proximal tubular epithelial cell hypertrophy in tissue culture and on diabetic renal hypertrophy in vivo. Mouse proximal tubular cells grown in 25 mM D-glucose and exposed to sense ODN as control (1 microM) exhibited increased (3)[H]leucine incorporation by 120% and total TGF-beta1 protein by 50% vs. culture in 5.5 mM D-glucose. Antisense ODN significantly decreased the high-glucose-stimulated TGF-beta1 secretion and leucine incorporation. Continuous infusion for 10 days of ODN (100 microg/day) was achieved via osmotic minipumps in diabetic and nondiabetic mice. Sense ODN-treated streptozotocin-diabetic mice had 15.3% increase in kidney weight, 70% increase in alpha1(IV) collagen and 46% increase in fibronectin mRNA levels compared with nondiabetic mice. Treatment of diabetic mice with antisense ODN partially but significantly decreased kidney TGF-beta1 protein levels and attenuated the increase in kidney weight and the alpha1(IV) collagen and fibronectin mRNAs. In conclusion, therapy with antisense TGF-beta1 ODN decreases TGF-beta1 production and attenuates high-glucose-induced proximal tubular cell hypertrophy in vitro and partially prevents the increase in kidney weight and extracellular matrix expression in diabetic mice.

Recent experimental work implicates transforming growth factor-beta (TGF-beta) as an aetiologic mediator of diabetic nephropathy and the ubiquitous glucose transporter GLUT1 as an important permissive factor for the tissue injury caused by hyperglycaemia. High ambient glucose increases GLUT1 expression and glucose transport activity when compared with physiologic glucose concentrations. Treatment of rat mesangial cells with TGF-beta up-regulates GLUT1 mRNA and protein levels and significantly increases glucose uptake. Addition of neutralizing anti-TGF-beta antibody prevents the stimulatory effects of high glucose on GLUT1 expression. Cultured rat mesangial cells transduced with the human GLUT1 gene and thus overexpressing the GLUT1 protein show marked increase in glucose uptake and the synthesis of extracellular matrix molecules, even when grown in normal ambient glucose concentrations. Thus, TGF-beta and GLUT1, two proteins that are up-regulated in glomerular mesangial cells in a hyperglycaemic milieu, can influence the expression of one another. It is therefore fair to conclude that, with successful interruption of the TGF-beta-GLUT1 axis, the beneficial effects of strict glucose control on the development of diabetic nephropathy could likely be augmented.

The renoprotective effect of captopril on progression of diabetic nephropathy was demonstrated by the Collaborative Study Group Captopril Trial and might be independent of blood pressure. Because angiotensin II is known to stimulate the prosclerotic cytokine, transforming growth factor-beta (TGF-beta), we postulated that the renoprotective effect may be due to inhibition of TGF-beta1 production. TGF-beta1 levels were measured in serum at baseline and 6 months from patients in the captopril trial. TGF-beta1 analyses were performed on all available patient sera. Analysis was performed between the percent change in TGF-beta1 levels during the first 6 months versus the percent change in glomerular filtration rate (GFR) in the subsequent 2 years. TGF-beta1 levels increased by 11% (P = 0. 003) in the placebo group (n = 24), whereas there was a decrease of 14% (P = 0.01) in the captopril group (n = 34). There was an inverse correlation between the percent change in TGF-beta1 levels during the first 6 months and the percent change in GFR over the ensuing 2-year period in patients from both the placebo (r = -0.55, P = 0. 005) and captopril groups (r = -0.45, P = 0.008). In patients with initial GFR below 75 mL/min, there was an even stronger correlation in percent change in TGF-beta1 levels and percent change in GFR in both placebo (n = 9, r = -0.69, P = 0.03) and captopril groups (n = 21, r = -0.73, P = 0.0001). Our data suggest that captopril decreases TGF-beta1 levels in diabetic nephropathy and that changes in TGF-beta1 levels may predict the course of diabetic nephropathy.

Background: Leptin inhibits food intake and increases energy expenditure. Although the kidney expresses abundant transcripts of the short form of the leptin receptor (Ob-Ra), a role for this hormone in renal function remains unclear. Because individuals with massive obesity who may exhibit increased leptin serum concentrations develop renal glomerulosclerosis, we studied whether leptin can influence renal growth and profibrogenic processes., Methods: The effects of recombinant leptin on proliferation and synthesis of transforming growth factor-beta1 (TGF-beta1) was investigated in cultured glomerular endothelial cells of the rat (GERs) and syngeneic mesangial cells. Furthermore, leptin receptor expression and potential signal transduction pathways were evaluated in GERs. In addition, leptin was also infused for different time periods (72 hr and 3 weeks) into naive rats., Results: Recombinant mouse leptin induced proliferation of GERs, but not of syngeneic mesangial cells. Coincubation with angiotensin II and leptin exerts additive proliferative effects in GERs. An antileptin-receptor antibody totally abolished this proliferation but did not influence serum-induced proliferation. GER expressed high affinity receptors of the Ob-Ra type (Kd, 4 nM; Bmax, 9700 receptors/cell). Leptin also stimulated phosphorylation of STAT1alpha, and kinase inhibitors attenuated proliferation, suggesting a pivotal role of phosphorylation in this process. Incubation of GERs with leptin also induced mRNA expression of TGF-beta1 and enhanced secretion of this profibrogenic cytokine. Short-term leptin infusion (72 hr) into naive rats induced a significant proliferation, mainly restricted to glomerular endothelial cells, and enhanced glomerular TGF-beta1 mRNA levels. In rats continuously infused for three weeks with leptin, glomerular TGF-beta1 expression was still enhanced, and an additional increase in glomerular collagen type IV mRNA and protein expression was detected. These animals revealed an increase in proteinuria compared with control-infused rats., Conclusion: Our findings are the first in vitro and in vivo demonstration that leptin is a renal growth and profibrogenic factor. These results may be an important contribution to our understanding of how leptin can contribute to renal damage, characterized by endocapillary proliferation and subsequent development of glomerulosclerosis, in pathophysiological situations with high circulating levels such as in diabetics or obese individuals. Although the effects of leptin itself are moderate, growth-promoting and profibrogenic effects may be enhanced in concert with other factors such as angiotensin II.

Renal tubular epithelial cells and interstitial fibroblasts are active participants in tubulointerstitial fibrosis, the best correlate of decreased glomerular filtration in diabetic nephropathy. It was reported previously that high ambient glucose stimulates transforming growth factor-beta (TGF-beta) mRNA and bioactivity, promotes cellular hypertrophy, and increases collagen synthesis in proximal tubular cells. This study evaluates the effects of high glucose and TGF-beta on the behavior of murine renal cortical fibroblasts (TFB) in culture. High glucose (450 mg/dl) significantly increased [3H]-thymidine incorporation (by 60 to 80% after 24 to 72 h) and cell number, without significantly increasing cell death when compared with normal glucose (100 mg/dl). There also was a transient increase in the mRNA of the c-myc and egr-1 early-response genes. Exogenous TGF-beta1 was promitogenic rather than antiproliferative in contrast to other renal cell types. Northern blot analysis demonstrated constitutive expression of TGF-beta1, -beta2, and -beta3 transcripts. Exposure to high glucose increased all three TGF-beta isoforms in a time-dependent manner. High glucose as well as exogenous TGF-beta1 also increased [3H]-proline incorporation, alpha2(I) collagen mRNA, and type I collagen protein (measured by immunoassay). Treatment with a neutralizing pan-selective monoclonal anti-TGF-beta antibody markedly attenuated the stimulation by high ambient glucose of thymidine incorporation, TGF-beta1 mRNA, and type I collagen mRNA and protein levels. It is concluded that high ambient glucose and exogenous TGF-beta1 share similar actions on renal fibroblasts. Moreover, the stimulation of cell proliferation and collagen type I synthesis in these cells by high ambient glucose are mediated by activation of an autocrine TGF-beta system.

Background: Transforming growth factor-beta (TGF-beta) is an important hypertrophic and prosclerotic cytokine in the pathogenesis of diabetic nephropathy. The mechanisms of regulation of the TGF-beta system by high ambient glucose in kidney cells are incompletely defined. This study examined the mechanisms of regulation of TGF-beta1 expression by high glucose in murine mesangial cells (MMCs) in culture., Methods: MMCs were cultured in either normal (100 mg/dl) or high (450 mg/dl) D-glucose concentration. Total TGF-beta1 protein secretion and bioactivity, mRNA expression and stability, and gene transcription rate were measured; promoter-reporter chloramphenicol acetyltransferase (CAT) assays and electrophoretic mobility shift assay (EMSA) were performed to investigate the presence of putative glucose-response elements., Results: Raising the ambient D-glucose concentration for 72 hours increased TGF-beta1 bioactivity in cell culture medium by 47% and total TGF-beta1 secretion by approximately 90%. Northern analysis demonstrated that the steady-state TGF-beta1 mRNA level was increased nearly twofold after 48 hours of growth in high glucose. This increase was not due to increased stability, as the half-life of the message was approximately five hours in both normal and high glucose conditions. Transcriptional activity of the TGF-beta1 gene (nuclear run-on assay) was increased by 73% in cells grown in high glucose for 24 hours. Transiently transfected MMCs with CAT constructs containing varying lengths of the murine TGF-beta1 promoter demonstrated that high glucose selectively increased the expression of only one of the constructs, pA835. Sequence inspection revealed the presence of a putative glucose responsive element, CACGTG, within this construct. High glucose in MMC culture for 24 hours increased nuclear protein binding to a probe containing this element when analyzed using EMSA., Conclusions: High glucose stimulates total TGF-beta1 protein production and bioactivity as well as the steady-state level of TGF-beta1 mRNA. The latter effect is due primarily to stimulation of gene transcription rate rather than message stability. Transcriptional activation by high glucose may involve a region in the TGF-beta1 promoter containing a putative glucose-response element.

We have previously shown that estradiol suppresses types I and IV collagen synthesis by mesangial cells grown in the presence of serum. In the present study, we examined the interaction between estradiol and transforming growth factor-beta (TGF-beta) on collagen IV synthesis. In a luciferase reporter gene construct containing the type IV collagen promoter and 1-chain regulatory sequences, we found that TGF-beta1 (2 ng/ml) stimulated alpha1-collagen IV gene transcription in serum-free media (140.5 +/- 6.2 relative luciferase units, expressed as a percent of control untreated cells, P < 0.001). Estradiol reversed the stimulatory effects of TGF-beta1 on reporter gene transcription in a dose-dependent manner [for 2.5 x 10(-9) M, 114.2 +/- 0.2, P < 0.002 vs. TGF-beta1; for 10(-7) M, 89.5 +/- 4.0, P < 0.001 vs. TGF-beta1 and P = not significant (NS) vs. control]. Using immunoprecipitation techniques, we found that estradiol (10(-7) M) reversed TGF-beta1-stimulated type IV collagen synthesis (175.3 +/- 14.7 vs. 111.6 +/- 7.1, expressed as a percent of control untreated cells, P < 0.001) but did not affect TGF-beta1-stimulated type I collagen synthesis (166.9 +/- 18.8 vs. 162.2 +/- 16.2, P = NS). These results were confirmed with Western blotting. Nuclear extracts from mesangial cells treated with TGF-beta1 showed increased binding to a Sp1 consensus binding sequence oligonucleotide and to an Sp1 binding site in the collagen IV promoter. Estradiol reversed this enhanced binding. These data suggest that estradiol antagonizes TGF-beta1-stimulated type IV collagen synthesis at a transcriptional level and that this effect may be mediated by interactions with the transcription factor Sp1.

The prosclerotic cytokine transforming growth factor beta 1 (TGF-beta1) has been causally implicated in renal pathobiology in diabetes. We sought evidence that the TGF-beta system participates in the nephropathic process in the db/db mouse, a hyperinsulinemic model of genetic diabetes that develops abnormalities in renal morphology and function that parallel those in human diabetic nephropathy. In support of this hypothesis, we found that steady state levels of mRNA encoding the TGF-beta type II receptor were significantly increased in renal cortex from db/db diabetic mice. Additionally, the translated TGF-beta type II receptor protein, assessed by immunoblot, also was increased in diabetic kidneys. However, in contrast to rodents with insulin-deficient diabetes, steady state levels of mRNA encoding TGF-beta1 in the renal cortex of diabetic db/db mice did not differ from those in cortex from nondiabetic (db/m) littermate controls. Further, concentrations of TGF-beta protein, measured by immunoassay and bioassay, were significantly lower in extracts prepared from renal cortex of diabetic animals compared with those from nondiabetic controls. Urine and serum concentrations of immunoreactive TGF-beta1 also were reduced in diabetic mice. The findings are consistent with upregulation of TGF-beta type II receptor activity as a consequence of hyperglycemia in the hyperinsulinemic db/db mouse and suggest that hyperinsulinemia inhibits TGF-beta1 production. The results further suggest that type II receptor upregulation is a contributing factor to the increased gene expression of renal cortical mRNAs encoding the extracellular matrix proteins fibronectin and alpha 1 (IV) collagen and to the renal abnormalities observed in this animal model.

Albumin modified by Amadori glucose adducts, formed in increased amounts in diabetes, stimulates collagen IV production and gene expression in renal glomerular mesangial cells, and induces mesangial matrix accumulation accompanied by increased mRNA encoding alpha 1 (IV) collagen and fibronectin in diabetic animals. These effects contribute to the pathogenesis of diabetic nephropathy, and resemble biologic activities of the cytokine TGF-beta 1, which also has been causally implicated in diabetic renal disease. We postulated that Amadori-modified glycated albumin modulates TGF-beta 1 expression in mesangial cells, and that TGF-beta 1 participates in mediating the glycated albumin-induced increases in mesangial cell matrix production. To test this hypothesis, we measured mRNA encoding TGF-beta 1, the TGF-beta Type II receptor and fibronectin, a key matrix component of the TGF-beta 1 tissue response, after incubation of mesangial cells with glycated albumin. Steady state levels of the mRNAs encoding for these proteins were stimulated when mesangial cells were cultured in the presence of albumin containing Amadori glucose adducts compared with levels in cells cultured with the nonglycated, glucose-free counterpart. The glycated protein-induced changes in mRNA expression were observed with concentrations of glycated albumin encompassing those found in clinical specimens and in media containing physiologic (5.5 mM) glucose concentrations, indicating that they were due to the glucose-modified protein and not to a hyperglycemic milieu. Further, they were accompanied by increased translated fibronectin protein, which was prevented with TGF-beta neutralizing antibody, as was the glycated albumin-induced increase in fibronectin mRNA. The findings indicate that Amadori-modified glycated albumin stimulates mesangial cell TGF-beta 1 gene expression by mechanisms that are operative under normoglycemic conditions. These data provide the first link between elevated glycated serum albumin concentrations and increased TGF-beta 1 bioactivity in the pathogenesis of mesangial matrix accumulation in diabetes.

We examined the hypothesis that fetal calf serum (FCS) stimulates murine mesangial cell alpha 1 type IV collagen (COL4A1) gene transcription by increasing autocrine production of transforming growth factor-beta (TGF-beta) through a platelet-derived growth factor (PDGF)-dependent mechanisms. PDGF-stimulated COL4A1 gene transcription was inhibited by neutralizing antibody to TGF-beta (119.3 +/- 3.6 vs. 106.0 +/- 6.2 relative luciferase units, expressed as a percentage of control untreated cells, P < 0.003). FCS-stimulated gene transcription was inhibited by neutralizing antibody to PDGF (148.3 +/- 4.1 vs. 136.7 +/- 0.3 relative luciferase units, P < 0.002) and by neutralizing antibody to TGF-beta (148.3 +/- 4.1 vs. 127.1 +/- 3.4 relative luciferase units, P < 0.036). The inhibitory effect of combined treatment with anti-PDGF and anti-TGF-beta antibody on gene transcription was no greater than that of anti-TGF-beta antibody alone [129.5 +/- 0.53 vs. 127.1 +/- 3.4 relative luciferase units, P = not significant (NS)]. FCS-stimulated gene transcription was also inhibited by estradiol (10(-7) M) (148.4 +/- 3.1 vs. 119.4 +/- 8.1 relative luciferase units, P < 0.019). In the presence of estradiol, anti-TGF-beta antibody failed to further reduce serum-stimulated gene transcription (119.4 +/- 8.1 vs. 115.6 +/- 9.8, P = NS), suggesting that estradiol reverses FCS-stimulated COL4A1 gene transcription by antagonizing the actions of TGF-beta. Measurement of type IV collagen synthesis by Western blotting confirmed that the intact gene responded in a manner analogous to the promoter construct.

Renal injury in diabetes mellitus is a major cause of morbidity and mortality. Several manifestations of diabetic nephropathy may be a consequence of altered production and/or response to cytokines or growth factors. Transforming growth factor-beta (TGF-beta) is one such factor because it promotes renal cell hypertrophy and regulates the production of extracellular matrix molecules. In addition, high ambient glucose increases TGF-beta1 mRNA and protein level in cultured proximal tubular cells and glomerular epithelial and mesangial cells. Neutralizing anti-TGF-beta antibodies or antisense TGF-beta1 oligodeoxynucleotides prevents the hypertrophic effects of high glucose and the stimulation of matrix synthesis in renal cells. Several reports have described overexpression of TGF-beta in the glomeruli and tubulointerstitium of experimental and human diabetes mellitus. We recently provided evidence that the kidney in diabetic patients displays net renal production of immunoreactive TGF-beta1, whereas there is net renal extraction in nondiabetic subjects. We also demonstrated that short-term treatment of streptozotocin-diabetic mice with neutralizing monoclonal antibody directed against TGF-beta significantly reduces kidney weight and glomerular hypertrophy, and attenuates the increase in extracellular matrix mRNA levels. The factors that mediate increased renal TGF-beta activity involve hyperglycemia per se and the intermediary action of other potent mediators such as angiotensin II, thromboxane, endothelins, and platelet-derived growth factor.

Diabetic nephropathy is a common complication in patients with either type I or type II diabetes. The pathogenesis of diabetic nephropathy is thought to involve both metabolic and vascular factors leading to chronic accumulation of glomerular mesangial matrix. In this context, both transforming growth factor-beta (TGF-beta) and endothelin may contribute to these processes. To determine if diabetic patients demonstrate increased renal production of TGF-beta and endothelin, aortic, renal vein, and urinary levels of these factors were measured in 14 type II diabetic patients and 11 nondiabetic patients who were undergoing elective cardiac catheterization. Renal blood flow was measured in all patients to calculate net mass balance across the kidney. Diabetic patients demonstrated net renal production of immunoreactive TGF-beta1 (830 +/- 429 ng/min [mean +/- SE]), whereas nondiabetic patients demonstrated net renal extraction of circulating TGF-beta1 (-3479 +/- 1010 ng/min, P < 0.001). Urinary levels of bioassayable TGF-beta were also significantly increased in diabetic patients compared with nondiabetic patients (2.435 +/- 0.385 vs. 0.569 +/- 0.190 ng/mg creatinine, respectively; P < 0.001). Renal production of immunoreactive endothelin was not significantly increased in diabetic patients. In summary, type II diabetes is associated with enhanced net renal production of TGF-beta1, whereas nondiabetic patients exhibit net renal extraction of circulating TGF-beta1. Increased renal TGF-beta production may be an important manifestation of diabetic kidney disease.

One pathological feature of chronic cyclosporin A (Cs-A) nephrotoxicity is striped interstitial fibrosis, the pathogenesis of which is not completely understood. We have previously reported that Cs-A directly stimulates the synthesis of various collagens in a cultured murine proximal tubular cell line (MCT cells). In addition, we have recently observed that Cs-A stimulates the synthesis of transforming growth factor beta (TGF-beta) in MCT cells. To this end, we investigated whether Cs-A directly stimulates transcription of TGF-beta in this cell line. A single dose of 2,000 ng/ml Cs-A stimulates the expression of TGF-beta1 transcripts in MCT cells. This stimulation is abrogated in the presence of 150 and 250 ng/ml actinomycin D without significantly influencing basal mRNA expression, suggesting that the increase in mRNA is due to stimulated transcriptional activity. Furthermore, nuclear runoff experiments demonstrate that Cs-A directly stimulates the transcriptional activity of the TGF-beta1 gene in MCT cells. The results of transient transfection of reporter gene constructs containing regulatory elements of the murine TGF-beta1 promoter into MCT cells and subsequent treatment with Cs-A further suggest direct effects of Cs-A on TGF-beta1 gene transcription. This is the first report demonstrating Cs-A-mediated TGF-beta1 gene transcription in renal cells. Considering the well-known profibrogenic effects of TGF-beta, our results suggest a novel mechanism of Cs-A nephrotoxicity.

Diabetic nephropathy is characterized by renal hypertrophy, thickening of basement membranes, and accumulation of extracellular matrix in the glomerular mesangium and the interstitium. Our previous investigations have shown that high glucose concentration increases transforming growth factor (TGF)-beta1 mRNA in mesangial and proximal tubule cells and that treatment with anti-TGF-beta antibody results in prevention of the effects of high glucose on cell growth (e.g., induction of cellular hypertrophy) and the stimulation of collagen biosynthesis. We evaluated in vivo the functional role of the renal TGF-beta system in diabetic kidney disease by treatment of streptozotocin-induced diabetic mice with either a neutralizing monoclonal antibody against TGF-beta1, -beta2, and -beta3 (alphaT) or nonimmune murine IgG for 9 days. Diabetic mice given IgG demonstrated total kidney and glomerular hypertrophy, significantly elevated urinary TGF-beta1 protein, and increased mRNAs encoding TGF-beta1, type II TGF-beta receptor, alpha1(IV) collagen, and fibronectin. Treatment of diabetic mice with alphaT prevented glomerular hypertrophy, reduced the increment in kidney weight by approximately 50%, and significantly attenuated the increase in mRNA levels without having any effect on blood glucose. The antibody was without significant effect on mRNA levels in nondiabetic mice. This is the first demonstration that the early characteristic features of diabetic renal involvement, which include hypertrophy and increased matrix mRNAs, are largely mediated by increased endogenous TGF-beta activity in the kidney and that they can be significantly attenuated by treatment with neutralizing anti-TGF-beta antibodies.

Angiotensin II (Ang II) stimulates cellular hypertrophy of cultured murine proximal tubular cells (MCT cells). This Ang II-mediated hypertrophy depends on the endogenous induction and autocrine action of transforming growth factor-beta (TGF-beta). We have previously demonstrated that permanent transfection of MCT cells with the c-mas oncogene, whose protein product encodes a serpentine receptor-like moiety coupled to G proteins without an hitherto identified ligand, changes the hypertrophic actions of Ang II into a proliferative response (Am J Physiol 263: F931-F938, 1992). The present study demonstrated that Ang II failed to stimulate induction of TGF-beta 1 protein in c-mas transfected MCT cells under the control of SV 40 promoter/enhancer (pSV2mas) as measured by mink cell bioassay and specific ELISA for TGF-beta 1. Moreover, in contrast to either wild-type MCT cells or to cells permanently transfected with the SV 40 based expression plasmid only (pSV2 cells), Ang II stimulated gene transcription and mRNA expression of TGF-beta 1 were decreased in c-mas transfected cells. Our findings demonstrate that the Ang II-induced proliferation of c-mas transfected MCT cells most likely depends on failure of TGF-beta 1 induction in these cells. c-mas transfected cells are a useful tool to further investigate the complex relationships between activation of second messengers subsequent to binding of Ang II to AT1-receptors and gene regulation like transcription of TGF-beta 1.

Renal cells are a rich source of transforming growth factor (TGF)-beta, and they serve as targets for its actions. Our hypothesis that activation of the TGF-beta system in the kidney is implicated in the development of diabetic renal disease stems from the close similarity of actions of TGF-beta and high ambient glucose on renal cell growth and extracellular matrix metabolism. Proximal tubule cells and glomerular mesangial cells cultured in high glucose concentration express increased TGF-beta 1 mRNA and protein levels, and treatment with anti-TGF-beta antibodies results in prevention of the effects of high glucose to induce cellular hypertrophy and stimulate collagen biosynthesis. Several in vivo studies by different groups of investigators have reported overexpression of TGF-beta in the glomeruli in human and experimental diabetes. We have also observed that the development of renal hypertrophy in the insulin-dependent diabetic BB rat and NOD mouse is associated with increased expression of TGF-beta 1 in the kidney and that short-term administration of antibodies capable of neutralizing the activity of TGF-beta in the streptozotocin mouse model of diabetes results in attenuation of whole kidney and glomerular hypertrophy and overexpression of mRNAs encoding matrix components. Together, these findings are consistent with the hypothesis that the diabetic state stimulates TGF-beta expression in the kidney and that in turn this growth factor may mediate, in an autocrine/paracrine manner, some of the principal early manifestations of diabetic renal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Atrial natriuretic peptide (ANP) is a growth suppressor for a variety of different cell types including cultured mesangial cells. This study examined the effects of ANP on the expression and synthesis of transforming growth factor-beta (TGF-beta) in a cultured murine mesangial cell line (MMC). Full-length rat ANP as well as 8-bromo-cGMP, but not ring-deleted ANP analogues, induced, in a dose-dependent manner, TGF-beta 1 mRNA expression in MMC. Moreover, full-length ANP and 8-bromo-cGMP stimulated the synthesis and release of TGF-beta 1 into cell culture supernatants as measured by a specific enzyme-linked immunosorbent assay and mink cell bioassay. The induction of TGF-beta 1 by 10(-6) M ANP and 10(-4) M 8-bromo-cGMP for 24 h was also documented by western blotting of MMC culture supernatants. However, transient transfection studies in MMC with three different murine TGF-beta 1 reporter gene constructs revealed only little stimulation of activity after ANP treatment, suggesting that posttranscriptional activation may mainly contribute to TGF-beta mRNA induction. Functional studies demonstrated that the ANP-mediated inhibition of mitosis, induced by 5% fetal calf serum, is partly abolished in the presence of a neutralizing anti-TGF-beta 1-3 antibody. In addition, the antiproliferative effects of exogenous 8-bromo-cGMP are also attenuated by anti-TGF-beta 1-3 antibody. These findings indicate that ANP stimulates TGF-beta expression in MMC and that the antiproliferative effects of ANP in this cell line may be mediated, at least to some extent, by the endogenous induction of TGF-beta. An increase in intracellular cGMP is most likely the mediator of this TGF-beta 1 induction. Considering the fibrogenic effects of TGF-beta, these findings may be important in the development of glomerulosclerosis in situations with an increase in local or systemic ANP.

Transforming growth factor-beta (TGF beta) is a multifunctional cytokine showing growth effects on many cell types. In the present study effects of TGF beta 1 on mitogen-induced Ca2+ responses were investigated in an immortalized murine mesangial cell line where TGF beta 1 effects on growth are inhibitory. TGF beta 1 was found to inhibit intracellular Ca2+ mobilization induced by platelet-derived growth factor (PDGF). This effect was completely reversed by previous addition of the non-specific serine/threonine kinase inhibitor H-7, but was unaffected by GF 109203X, a specific inhibitor of protein kinase C (PKC). These findings suggest that inhibition of mitogen-induced Ca2+ mobilization by TGF beta 1 appears not to involve prior activation of PKC, but may participate in the mechanisms whereby mesangial cell growth is inhibited by TGF beta 1.

Renal hypertrophy is an early feature of diabetes, and it may predispose the kidney to the eventual development of parenchymal dysfunction. Since we have previously demonstrated that short-term culture in high glucose concentration stimulates production of transforming growth factor-beta 1 (TGF-beta 1) in proximal tubular and glomerular mesangial cells, we postulated that this cytokine, which has potent regulatory effects on cellular growth and extracellular matrix production, is important in mediating diabetic renal disease. In this study we evaluated the gene and protein expression of TGF-beta 1 in the kidney of two rodent models of spontaneous insulin-dependent diabetes mellitus [the biobreeding (BB) rat and the nonobese diabetic (NOD) mouse]. In association with the appearance in both models of significant renal hypertrophy, TGF-beta 1 mRNA levels were increased threefold in the kidney of the diabetic BB rat after 3 days of diabetes and also threefold after 7-9 days in the NOD mouse. There was no increase in TGF-beta 1 transcripts in the livers of the diabetic animals, suggesting that this response is tissue specific. Immunohistochemical studies revealed that TGF-beta 1 protein is concordantly elevated in the cortical tubular cells of the diabetic kidney in both models. These results suggest that the stimulated expression of renal TGF-beta is an early manifestation of the involvement of the kidney by diabetes. Whether increased TGF-beta production in the diabetic kidney is a key promoter of diabetic renal manifestations (e.g., hypertrophy) deserves additional studies.

Previous investigations have demonstrated that growing mesangial cells in high glucose concentration stimulates extracellular matrix synthesis and also increases the expression of TGF-beta. We tested whether the stimulation of extracellular matrix production is mediated by autocrine activation of TGF-beta, a known prosclerotic cytokine. Addition of neutralizing anti-TGF-beta antibody, but not normal rabbit IgG, significantly reduced the high glucose-stimulated incorporation of 3[H]proline. Denaturing SDS-PAGE revealed that mainly collagen types I and IV were stimulated by high (450 mg/dl) D-glucose. This high glucose-mediated increase in collagen synthesis was reduced by the anti-TGF-beta antibody. Treatment of mesangial cells grown in normal (100 mg/dl) D-glucose with 2 ng/ml recombinant TGF-beta 1 mimicked the effects of high glucose. Furthermore, the anti-TGF-beta antibody significantly reduced the increase in mRNA levels encoding alpha 2(I) and alpha 1(IV) collagens induced by high glucose. Thus, the high glucose-stimulated increase of collagen production in mesangial cells is mediated, at least in part, by autocrine TGF-beta activation. We postulate that the interception of the glomerular activity of TGF-beta may be an effective intervention in the management of diabetic nephropathy.

Previous studies by our group have demonstrated that angiotensin II (ANG II), as a single factor in serum-free medium, induces cellular hypertrophy of a cultured murine proximal tubular cell line (MCT). The present study was performed to test the hypothesis that this growth effect was mediated by activation of endogenous transforming growth factor-beta (TGF-beta). Exogenous TGF-beta 1 (1 ng/ml) mimicked the growth effects observed with 10(-8) M ANG II (inhibition of DNA synthesis and induction of cellular hypertrophy). A neutralizing anti-TGF-beta antibody attenuated the ANG II-induced increase in de novo protein and total RNA synthesis as well as total protein content. This antibody also abolished the ANG II-mediated inhibition of [3H]thymidine incorporation into quiescent MCT cells. Control IgG or an unrelated antibody had no effect. A bioassay for TGF-beta using mink lung epithelial cells revealed that MCT cells treated with ANG II released active TGF-beta into the cell culture supernatant. Northern blot analysis and semi-quantitative cDNA amplification demonstrated increases in steady-state levels for TGF-beta 1 mRNA after ANG II stimulation of MCT cells, but not in a syngeneic murine mesangial cell line. Our data indicate that the ANG II-induced hypertrophy in MCT cells is mediated by synthesis and activation of endogenous TGF-beta. It is intriguing to speculate that TGF-beta may play a role in the early tubular cell hypertrophy and the subsequent interstitial scarring observed in several models of chronic renal injury that are characterized by increased activity of intrarenal ANG II.