Nephroprotective effect of the cardiotonic steroid ouabain

Sammanfattning: It has been shown that Na,K-ATPase (NKA) is a signal transducer. Our group described the anti-apoptotic effect of ouabain to the signaling function of NKA. Exposure of renal cells to low concentrations of the highly specific NKA ligand, ouabain, will trigger a signaling cascade that involves interaction between the catalytic subunit of NKA and the inositol 1,4,5 triphosphate receptor (IP3R), intracellular calcium oscillations and activation of the NF-κB p65 subunit. It has been demonstrated that in nM ouabain can provide protection against apoptosis in fetal rat kidneys exposed to malnutrition. In this thesis we studied whether ouabain can prevent apoptosis in kidney tissue induced by Shiga toxin 2 (Stx2). We found that Stx2 at concentrations in the range of 3-4 ng/mL gave a reproducible level of apoptosis of rat proximal tubular cells (RPTC). In cells co-incubated with Stx and ouabain 5 nM the apoptotic effect was almost completely abolished. Stx2-induced apoptosis was due to stimulation Bax and the intrinsic apoptotic pathway activation. We show that the Stx2-induced down-regulation of the survival factor Bcl-xL in Stx2-treated renal epithelial cells was almost completely abolished by ouabain treatment. Treatment with ouabain protects kidneys from apoptosis by reversing an imbalance between Bcl-xL and Bax in mice inoculated with Stx2. In this thesis we tested whether other cardiotonic steroids (CTS) beside ouabain can play role in NKA-triggered Ca2+ oscillations. We found that cardenolide digoxin and the bufadienolide marinobufagenin may also trigger Ca2+ oscillations of similar frequency as ouabain. Binding of cardiotonic steroids to NKA will also stimulate tyrosine phosphorylation (Kometiani et al., 1998; Haas et al., 2000). We show that Src phosphorylation but not extracellular-signal-regulated (Erk) kinase is required for the initiation of the CTS evoked Ca2+ signaling pathway. Chronic kidney disease (CKD) is the 12th most common cause of death and the incidence is increasing worldwide. The albuminuria is a predictor of the progressive loss of kidney function, and is also considered a major cause of the progression of CKD (Anderson et al., 2009). In this thesis we tested whether ouabain can protect against albumin-induced apoptosis in vitro and on in vivo model of CKD. We found a time- and dose-dependent increase in apoptotic index in RPTC incubated with albumin. Observed apoptosis is an early sign of RPTC damage and precedes fibrosis. Co-incubation with ouabain resulted in substantial reduction of the apoptotic index and a fibroric marker TGF-beta 1 expression in cells incubated with albumin. We found that albumin enters the RPTC. We documented almost immediate translocation of Bax to the mitochondria accompanied by a depolarization of the mitochondrial membrane. Here we present evidence that ouabain counteracts the translocation of apoptotic factor Bax to mitochondria and the change in mitochondrial membrane potential in albumin-exposed RPTC. Treatment with ouabain restored albumin-induced down-regulated expression of the antiapoptotic factor Bcl-xL. To examine whether ouabain will also protect from loss of renal cells in proteinuric CKD, we have used a well known model of a proteinuric disease, Passive Heymann Nephritis (PHN). We found that chronic treatment with ouabain decreases the value of albuminuria, the apoptotic index (AI) in glomerular-tubular junction, glomerular-tubular disconnection, loss of podocytes as an indicators of permanent renal damage, expression of fibrotic markers. Serum creatinine, which was used as an end-point parameter in this study, was significantly lower in PHN rats treated with ouabain that in PHN rats treated with vehicle. In conclusion we have described a new downstream effect of the ouabain-induced NKA signaling mechanism preventing apoptosis throught regulation of mitochondrial intrinsic apoptotic pathway in acute and chronic kidney diseases.

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