Regulation und pathophysiologische Rolle der Calcium-permeablen Kationenkanäle TRPM7 und TRPC6 in Abhängigkeit des KCa3.1-Kanals im Transdifferenzierungsprozess renaler Myofibroblasten

With a prevalence of up to 16%, chronic kidney disease is one of the most common diseases worldwide. Regardless of the aetiology, usually the pathologic process culminates in irreversible fibrosis. This process is hallmarked by an excessive production of extracellular matrix by so called myofibroblasts, which then promotes renal failure. One important regulator of the transdifferentiation from residential fibroblasts to collagen-producing myofibroblasts is the cytokin TGF-β. Previously it has been shown that the potassium channel KCa3.1 is involved in the process of renal fibrosis. Furthermore, intracellular concentration of calcium controls activation of fibroblasts. Conclusively, the calcium channels TRPM7 and TRPC6 were identified as regulators of fibrosis in multiple organ systems. In this thesis, immortalized rattus norvegicus renal fibroblasts (NRK-49F) were used to investigate the role of above-mentioned ion channels KCa3.1-, TRPM7- und TRPC6 during the process of transdifferentation in vitro. The expression-levels of these channels were analyzed using molecular biology and proteo-biochemical methods. Furthermore, their function was characterized using real-time calcium-imaging and electrophysiological measurements using the patch-clamp technique. Expression of KCa3.1-, TRPM7- und TRPC6 was experimentally verified in NRK-49F cells. It was shown that activation of KCa3.1 is followed by an influx of calcium ions. Upon stimulation with TGF-β, KCa3.1- and TRPC6-expression is increased. In addition, the application of the TRPC6-activator OAG resulted in an increased calcium influx in stimulated cells. It could be demonstrated that KCa3.1 and TRPM7 produced relevant currents. Whereas TRPM7 had an impact on the intracelluar calcium concentration, TGF-β-treatment did not significantly change expression or activation of TRPM7 in NRK-49F cells. The upregulation of KCa3.1 was previously described in different renal cell-culture models. These results approve a major role of this channel in the process of renal fibrosis. Regarding TRPC6, an essential function in renal fibroblasts is very likely besides its already described function in dermal and pulmonal fibroblasts. In this project, relevant currents generated by TRPM7 in NRK-49F cells could be shown for the first time. In contrast to the upregulation of this channel during the transdifferentiation in cardial fibroblasts, there were no significant changes in TRPM7-activity during NRK-49F transdifferentiation. Taken together, an interaction between the investigated channels seems to influence the intracelluar calcium concentration significantly. Hyperpolarisation caused by KCa3.1 enforces an increased calcium influx through TRPC6 and TRPM7. This again leads to enhanced activation of KCa3.1 channels and therefore increased production of extracelluar matrix. The therapeutical options for the treatment of renal fibrosis are limited. Due to multiple non-benefitial side-effects, upstream-inhibition of TGF-β signaling seems to be risky. Consequently, it is necessary to affect the subprocesses of renal fibrosis. The data given in this thesis contributes to enhanced understanding of the fundamental processes during renal fibrosis and reveals TRPC6 and TRPM7 as possible therapeutic targets.