Role of KCa3.1 channel on renal fibrosis in Doxorubicin-induced feline kidney cell line

Abstract

Feline renal fibrosis is the most common outcome of chronic kidney disease in cats. Nowadays, there is no specific treatment and the mechanism of fibrosis in cats is still unknown. Intermediate conductance Ca2+ activated K+ channel (KCa3.1) plays an important role in novel therapy for organ fibrosis in many species. Transforming growth factor- β (TGF-β)/Smad signaling pathway and KCa3.1 has been shown to relate to renal fibrosis. This study aimed to investigate the relationship between KCa3.1 and feline renal fibrosis via TGF-β/Smad signaling pathway. CRFK cultures were induced with doxorubicin (DOX) to generate feline renal fibrosis model. Triarymethane-34 (TRAM-34), selective inhibitor of KCa3.1 was used for blocking KCa3.1 function pretreatment before fibrosis induction. TGF-β receptor type 2 (TGF-βR2), Smad2/3, α-Smooth muscle actin (α-SMA) which are fibrotic markers in TGF-β/Smad signaling cascade were measured using western blot to compare the fibrosis production between the normal KCa3.1 expression and KCa 3.1 blockage CRFK cells. The cell viability assay was performed alongside with protein expression measurement. CRFK cells pretreatment with TRAM-34 before DOX induction has no significant difference of percentage of cell viability when compared to negative control group. CRFK with DOX incubation alone significantly declined the percentage of cell viability compared to negative control (p<0.05). Interestingly, the expression of TGF-βR2 and Smad2/3 were not significant difference when compared between all study groups but α-SMA level in DOX induction group with and without TRAM-34 pretreatment were significant decrease when compared to negative control group (p<0.05). These findings indicated that KCa3.1 may not be involved in fibrogenesis in feline kidney cells via TGF-β/Smad signaling pathway and DOX may affect to α-SMA production in CRFK cells. The further investigation of KCa3.1 in feline renal fibrosis on another fibrotic partway is needed to be evaluated in the future.