Transforming growth factor-beta1 reduces megalin- and cubilin-mediated endocytosis of albumin in proximal-tubule-derived opossum kidney cells.

Transforming growth factor (TGF)-beta1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial-mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF-beta1-induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin-cubilin-dependent receptor-mediated endocytosis. In this study we used a well established cell culture model (proximal-tubule-derived opossum kidney (OK) cells) in order to test the hypothesis that TGF-beta1 reduces megalin-cubilin-mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF-beta1 led to a time- and dose-dependent downregulation of megalin-cubilin-mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF-beta1 action does not involve mitogen-activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF-beta1-induced downregulation of megalin-cubilin-mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF-beta1. In conclusion our data indicate that enhanced levels of TGF-beta1 occurring in various nephropathies can lead to downregulation of megalin-cubilin-dependent endocytosis. Probably, TGF-beta1 leads to Smad2- and Smad3-dependent expression of negative regulators of receptor-mediated endocytosis.

Protein uptake disturbs collagen homeostasis in proximal tubule-derived cells.

BACKGROUND: Interstitial fibrosis is of major importance for the deterioration of renal function, leading to uremia. Interaction of filtered proteins with proximal tubular cells is important for the onset and development of tubulointerstitial damage. METHODS: We investigated the effects of protein endocytosis on collagen homeostasis and signaling pathways of proximal tubule-derived cells (OK cells, LLC-PK1 cells), which express the endocytic machinery typical for the proximal tubule (megalin and cubilin), and compared it to renal epithelial cells with low endocytic activity (MDCK, IHKE1, NHE3-deficient OK cells). Collagen homeostasis was assessed by proline incorporation, ELISA, and Western blot. Matrix metalloproteinase (MMP) activity was assessed by gelatinase assay. Signaling pathways were monitored by reporter gene assay. RESULTS: Albumin, glycated albumin, fatty acid-free albumin, or globulins led to an increase of secreted collagen (types I, III, and IV) in OK and LLC-PK1 cells. In cells with low protein uptake activity, albumin exposure inhibited collagen secretion. Western blot analysis showed an increase of cellular collagen. MMP activity was significantly decreased by albumin exposure. Furthermore, albumin exposure led to activation of the NF-kappa B-, AP1-, NFAT-, SRE-, and CRE-pathways. Inhibition of NF-kappa B, PKC, or PKA partially reversed the effects of albumin. In addition, inhibition of albumin endocytosis reduced collagen secretion and activation of the signaling pathways. Discussion. The data show that endocytic uptake of proteins disturbs collagen homeostasis in proximal tubular cells. This disturbed matrix homeostasis probably supports the progression of interstitial fibrosis, which is of importance for the development of renal insufficiency.