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Methods: RT-PCR, Immunohistochemistry, Western blot analysis, NADPH-Oxidase activity. Results: Chemokines and their receptors are expressed on a variety of intrinsic and infiltrating glomerular cells and play a crucial role during glomerular injury. Recently a new CC Chemokine receptor, CCR10 and its ligand CCL28 have been cloned. So far little is known about the expression and signaling of the new chemokine/ chemokine receptor pair CCL28/CCR10 in the glomerulus. Since podocyte injury is a major player in the development of glomerular injury, it was intriguing to study the expression of CCR10 and its ligand CCL28 in glomerular podocytes. We here show by RT-PCR, immunohistochemistry and Western blot analysis, that both CCR10 and CCL28 are expressed in glomerular podocytes in vivo and in vitro. We demonstrate functional expression of CCR10 in cultured human differentiated podocytes: Podocytes showed a dose dependent increase in the free cytosolic Ca++ activity after stimulation with CCL28. Moreover, CCR10 signaling results in an increased phosphorylation of ERK. This effect could be blocked by the specific MEK inhibitor PD98059. By using immunohistchemistry we show that the expression of CCR10 is upregulated in membranous nephropathy and membranoproliferative GN. A positive correlation between proteinuria and CCL28 mRNA expression was found by real time PCR studies. CCL28 stimulated podocytes showed an increased NADPH dependent generation of ROS. It has been shown, that ROS play a major role in the pathogenesis of membranous nephropathy. Conclusions: We speculate, that the chemokine/chemokine receptor pair CCL28/CCR10 may contribute to podocyte injury via an activation of the MEK/ERK pathway and an increase in ROS generation. [ABSTRACT FROM AUTHOR]

Methods: Cell culture, RT-PCR, Western blot, NADPH-oxidase acitivity, Calcium activity measurement, cloning, cDNA expression array Results: Bone morphogenetic proteins (BMPs) are secreted signaling molecules, which have been implicated to play a major role in kidney development and disease. Here we show that differentiated mouse podocytes express mRNA for BMP-2 and for the BMP receptors BMPR1A, BMPR1B, BMPRII, ACVR1A, ACVR2, and ACVR2B. BMP-2 dose-dependently increases the free cytosolic Ca2+ concentration in podocytes. cDNA expression analysis and Western blot analysis show that BMP-2 increases the expression of Id-1, a negative regulator of basic helix-loop-helix transcription factors. To investigate the role of Id-1 for podocyte function, overexpression of Id-1 was induced in mouse podocytes. Id-1 overexpressing podocytes show an increased NADPH dependent activation of ROS. This effect can be mimicked by BMP-2 in untransfected podocytes. Preincubation of podocytes with anti-Id-1-antisense oligonucleotides abolished the effect of BMP-2 on Id-1 expression and NADPH-oxidase acitivity in untransfected podocytes. Conclusions: The data indicate that BMP-2 may -- via an increased expression of Id-1 and an increased generation of ROS -- contribute to injury in podocytes. Reactive oxygen species supposedly play a major role in the pathogenesis of membranous nephropathy. [ABSTRACT FROM AUTHOR]