Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells1

Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells.BackgroundClinical and experimental observations suggest that proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. Filtered proteins are taken up by tubular cells, and overwhelming this system may lead to tubular synthesis of various proinflammatory and profibrogenic cytokines, including transforming growth factor-beta (TGF-β). TGF-β acts by first binding to specific receptors. We studied in an in vitro system using a well-defined mouse proximal tubular cell line (MCT cells) whether fatty acid-free bovine albumin modulates expression of specific receptors for TGF-β.MethodsMCT (and LLC-PK1) cells were challenged in serum-free medium with different concentrations of albumin. Activation of a local renin-angiotensin system was tested by real-time polymerase chain reaction (PCR) for renin and angiotensinogen transcripts and determination of secreted angiotensin II (Ang II) by enzyme-linked immunosorbent assay (ELISA). Some cells were also treated with the AT1 receptor antagonist losartan. TGF-β receptor types I and II mRNA levels were determined by Northern analysis whereas protein abundance was measured by Western blots. To test for a functional consequence of up-regulated TGF-β receptors, MCT cells were preincubated with albumin and subsequently treated with low-dose TGF-β that normally does not induce collagen type IV expression by itself. Downstream signaling events were detected by Western blots for phosphorylated Smad2. Scatchard assays with [125I]TGF-β1 were performed to estimate affinity and number of specific binding sites. Different length TGF-β type II promoter constructs linked to CAT reporter were transiently transected into MCT cells to determine transcriptional activity.ResultsIncubation of MCT cells with 0.5 to 10 mg/mL albumin leads to an increase in type II TGF-β receptor mRNA and protein expression without influencing type I receptors. An increase in type II TGF-β receptor protein expression was detected after 12 hours of albumin incubation and was still detectable after 48 hours. The albumin-mediated increase in type II TGF-β receptor mRNA was attenuated in the presence of 1 μmol/L losartan, suggesting involvement of a local renin-angiotensin system. MCT cells treated with albumin significantly increased expression of angiotensinogen and renin transcripts and also secreted more Ang II into the culture supernatant. Analysis of transcriptional activity showed that promoter segments containing activating protein (AP-1)-binding sites are necessary for albumin-induced transcription of the TGF-β type II receptor. Binding assays revealed that albumin treatment significantly increased the overall binding sites as well as the affinity for TGF-β. This effect had functional consequences because MCT cells pretreated with albumin reacted with a stronger TGF-β–mediated phosphorylation of down-stream Smad2 and also increased collagen IV expression compared with control cells.ConclusionOur findings indicate that albumin up-regulates ligand-binding TGF-β receptors on cultured proximal tubular cells. Albumin-induced activation of local Ang II production appears to be responsible for this effect. This may amplify the matrix-stimulatory actions of TGF-β on tubular cells and could be a novel mechanism for how proteinuria exhibits pathophysiologic effects on tubular cells ultimately leading to tubulointerstitial fibrosis

Role of insulin and IGF1 receptors in proliferation of cultured renal proximal tubule cells

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia

Glomerular expression of p27Kip1 in diabetic db/db mouse: Role of hyperglycemia

Glomerular expression of p27Kip1 in diabetic db/db mouse: Role of hyperglycemia Early diabetic nephropathy is characterized by glomerular hypertrophy. Previous studies in vitro have demonstrated that mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and express increased levels of the cyclin-dependent kinase inhibitor p27Kip1. The present study was performed to investigate the renal expression of p27Kip1 in db/db mice, a model of diabetes mellitus type II. Glomerular p27Kip1 protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates. Immunohistochemical studies revealed that this stimulated expression was mainly restricted to the nuclei of mesangial cells and podocytes, but glomerular endothelial cells occasionally also stained positively. Quantification of p27Kip1 positive glomerular cells showed a significant increase of these cells in db/db mice compared with non-diabetic db/+ animals. Although tubular cells revealed a positive staining for p27Kip1 protein, there was no difference between db/+ and db/db mice. Immunoprecipitation experiments revealed that p27Kip1 protein associates with Cdk2 and Cdk4, but not with Cdk6. To test for the influence of hyperglycemia on cell cycle arrest and p27Kip1 expression, mesangial cells were isolated from db/+ and db/db mice. There was a similar basal proliferation when these cells were grown in normal glucose-containing medium (100 mg/dl). However, raising the glucose concentration to 275 to 450 mg/dl induced cell cycle arrest in db/+ as well as db/db mesangial cells. Increasing the medium osmolarity with D-mannitol failed to induce p27Kip1 expression in mesangial cells. Transfection of cells with p27Kip1 antisense, but not missense, phosphorothioate oligonucleotides facilitated cell cycle progression equally well in db/+ and db/db mesangial cells. Furthermore, p27Kip1 expression was comparable in both cell lines in normal glucose, but increased in high glucose medium. Our studies demonstrate that p27Kip1 expression is enhanced in diabetic db/db animals. This induction appears to be due to hyperglycemia. Expression of p27Kip1 may be important in cell cycle arrest and hypertrophy of mesangial cells during early diabetic nephropathy

Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-ß antibody in \u3ci\u3edb/db\u3c/i\u3e diabetic mice

Emerging evidence suggests that transforming growth factor-(TGF-β) is an important mediator of diabetic nephropathy. We showed previously that short-term treatment with a neutralizing monoclonal anti-TGF-antibody (αT) in streptozotocin-diabetic mice prevents early changes of renal hypertrophy and increased matrix mRNA. To establish that overactivity of the renal TGF-system mediates the functional and structural changes of the more advanced stages of nephropathy, we tested whether chronic administration of αT prevents renal insufficiency and glomerulosclerosis in the db/db mouse, model of type 2 diabetes that develops overt nephropathy. Diabetic db/db mice and nondiabetic db/m littermates were treated intraperitoneally with α or control IgG, 300 µg three times per week for 8 wk. Treatment with αT, but not with IgG, significantly decreased the plasma TGF-β1 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 α 1(IV) collagen and fibronectin. On the other hand, treatment with α 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 α treatment. We conclude that chronic inhibition of the biologic actions of TGF-with neutralizing monoclonal antibody in db/db mice prevents the glomerulosclerosis and renal insufficiency resulting from type diabetes

Effects of high glucose and TGF-β1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes

Effects of high glucose and TGF-β1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes.BackgroundThe 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-β (TGF-β).MethodsThe effects of high glucose and exogenous TGF-β1 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-β system were assayed. An inhibitor of TGF-β signaling was used to determine whether some of the high glucose effects might be mediated by the TGF-β system.ResultsCompared 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-β1 (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 α1(IV) collagen by 51% and α5(IV) by 90%, whereas treatment with TGF-β1 (2 ng/mL) for 24 hours decreased the mRNA level of α1(IV) by 36% and α5(IV) by 40%. Consistent with these effects on mRNA expression, Western blotting showed that HG increased α1(IV) protein by 44% and α5(IV) by 28%, while TGF-β1 decreased α1(IV) protein by 29% and α5(IV) by 7%. In contrast to their opposing actions on α1 and α5(IV), both HG and exogenous TGF-β1 increased α3(IV) collagen and VEGF, with TGF-β1 having the greater effect. An inhibitor of the TGF-β type I receptor (ALK5) was able to prevent the stimulation of α3(IV) and VEGF proteins by HG. Unlike in other renal cell types, HG did not increase TGF-β1 mRNA or protein in the podocyte, but HG did induce the expression of the ligand-binding TGF-β type II receptor (TβRII). Because HG had up-regulated TβRII after two weeks, the addition of physiological-dose TGF-β1 (0.010 ng/mL) for 24 hours stimulated the production of α3(IV) and VEGF proteins to a greater extent in high than in normal glucose. Up-regulation of TβRII in the podocyte was corroborated by immunohistochemistry of the kidney cortex in the db/db mouse, a model of type 2 diabetes.ConclusionsHigh glucose and exogenous TGF-β1 exert disparate effects on the expression of α1 and α5(IV) collagen. However, high glucose and TGF-β1 coordinately induce the production of α3(IV) collagen and VEGF in the podocyte. The HG-induced increases in α3(IV) collagen and VEGF proteins are mediated by the TGF-β system. By increasing the expression of TβRII, high glucose may augment the response of the podocyte to ambient levels of TGF-β1

Emerging Therapies for Diabetic Nephropathy Patients: Beyond Blockade of the Renin-Angiotensin System

Diabetic nephropathy is a leading cause of end-stage renal disease worldwide. The mainstay of treatment has been glycemic control and blood pressure lowering using agents blocking the renin-angiotensin system. Clinical trials are currently under way using novel agents for the treatment of patients with diabetic nephropathy. Promising agents emerging from some of the completed trials include pirfenidone and bardoxolone methyl, which have been shown in two recent randomized controlled trials in patients with diabetic nephropathy to result in an improved estimated glomerular filtration rate compared to placebo. Also, paricalcitol has been shown to decrease the urinary albumin-to-creatinine ratio, whereas sulodexide failed to do so in a large randomized double-blind placebo-controlled trial. Of note, pyridoxamine has also shown promise in the treatment of diabetic nephropathy if started early in the disease course. These preliminary trials have shown significant promise for managing patients with diabetic nephropathy, sparking active research in this field and providing the rationale for further clinical testing in long-term, hard-outcomes trials

Stimulation of Collagen Gene Expression and Protein Synthesis in Murine Mesangial Cells by High Glucose Is Mediated by Autocrine Activation of Transforming Growth Factor-/3

Abstract Previous investigations have demonstrated that growing mesangial cells in high glucose concentration stimulates extracellular matrix synthesis and also increases the expression of TGF-ft. We tested whether the stimulation of extracellular matrix production is mediated by autocrine activation of TGF-13, a known prosclerotic cytokine. Addition of neutralizing anti-TGF-ft antibody, but not normal rabbit IgG, significantly reduced the high glucose-stimulated incorporation of31HIproline. 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-fi antibody. Treatment of mesangial cells grown in normal (100 mg/dl) D-glucose with 2 ng/ml recombinant TGF-f,1 mimicked the effects of high glucose. Furthermore, the anti-TGF-#t antibody significantly reduced the increase in mRNA levels encoding a2(I) and al (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-f activation. We postulate that the interception of the glomerular activity of TGF-li may be an effective intervention in the management of diabetic nephropathy. (J