Indexed on: 22 Oct '04Published on: 22 Oct '04Published in: Kidney International
Clinical 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-beta). TGF-beta 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-beta.MCT (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-beta 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-beta receptors, MCT cells were preincubated with albumin and subsequently treated with low-dose TGF-beta 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-beta1 were performed to estimate affinity and number of specific binding sites. Different length TGF-beta type II promoter constructs linked to CAT reporter were transiently transected into MCT cells to determine transcriptional activity.Incubation of MCT cells with 0.5 to 10 mg/mL albumin leads to an increase in type II TGF-beta receptor mRNA and protein expression without influencing type I receptors. An increase in type II TGF-beta 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-beta receptor mRNA was attenuated in the presence of 1 micromol/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-beta type II receptor. Binding assays revealed that albumin treatment significantly increased the overall binding sites as well as the affinity for TGF-beta. This effect had functional consequences because MCT cells pretreated with albumin reacted with a stronger TGF-beta-mediated phosphorylation of down-stream Smad2 and also increased collagen IV expression compared with control cells.Our findings indicate that albumin up-regulates ligand-binding TGF-beta 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-beta on tubular cells and could be a novel mechanism for how proteinuria exhibits pathophysiologic effects on tubular cells ultimately leading to tubulointerstitial fibrosis.