Elsevier

Translational Research

Volume 148, Issue 3, September 2006, Pages 134-141
Translational Research

Original article
TGF-β1 and integrin synergistically facilitate the differentiation of rat podocytes by increasing α-smooth muscle actin expression

https://doi.org/10.1016/j.trsl.2006.03.008Get rights and content

Phenotypic changes can be found in certain glomerular diseases, and the cell origin is not defined. This study was designed to identify whether podocytes can differentiate by the expression of α-smooth muscle actin (α-SMA), under the effects of TGF-β1 (transforming growth factor-β1) and integrin. Western and Northern blot analyses were performed to identify the protein and mRNA (messenger ribonucleic acid) expression of α-SMA. The number of podocytes, which express α-SMA, was measured by immunocytochemical staining. The results showed that TGF-β1 dose-dependently increased α-SMA protein and mRNA expression at 4 and 2 days, respectively. TGF-β1 also dose-dependently increased the α-SMA staining of podocytes. The α-SMA-positive podocytes showed front-end and back-end polarity. The integrinα3β1 antagonists, anti-integrinβ1 monoclonal antibody and Gly-Arg-Gly-Asp (GRGD), decreased the expression of α-SMA protein and the percentage of α-SMA positive cells stimulated by TGF-β1 (both P < 0.01). The addition of calphostin [inhibitor of protein kinase C (PKC)] and genistein [inhibitor of focal adhesion kinase (FAK)] also decreased the expression of α-SMA protein and the percentage of α-SMA positive cells stimulated by TGF-β1 (both P < 0.01). In conclusion, this study indicated that TGF-β1 may act synergistically with integrins, through activation of PKC and FAK, to induce the phenotypic changes of rat podocytes with increasing α-SMA expression.

Section snippets

Podocyte cell culture

To generate primary cultures of rat podocytes, glomeruli were isolated from Sprague–Dawley rats (50-g body weight) by the sieving method.31, 32 Renal cortical tissue homogenates were carefully passed through sequential stainless-steel sieves of 212 μm and then 106-μm pore size. The isolated glomeruli were collected from a sieve of 75-μm pore size and inspected with phase contrast microscopy for confirmation of successful extraction. Isolated glomeruli were plated in plastic culture flasks that

α-SMA protein expression of podocytes after TGF-β1 stimulation

The expression of α-SMA protein induced by TGF-β1 stimulation for 4 days was analyzed by Western blotting. Western blotting of cell lysates showed that TGF-β1 induced a dose-dependent increase in the expression of α-SMA protein in cultured rat podocytes. A faint band existed for α-SMA protein in podocytes grown on collagen-coated plates with free-serum medium. A significant increase of α-SMA protein occurred after TGF-β1 stimulation with dosage of 2.5, 7.5, 10, 25, and 50 ng/mL (P < 0.05, Fig. 2

Discussion

In this study, it has been demonstrated that rat podocytes can undergo phenotypic change with increased expression of α-SMA under the influence of TGF-β1. The cell adhesion/integrin activation is needed for the induction of phenotypic change by TGF-β1.

Myofibroblasts play an important role in wound healing and scarring. Myofibroblast-like cells have been detected within glomeruli, crescents, and interstitium in experimental glomerulonephritis and humans with progressive renal diseases.3, 8, 9, 37

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