Rosuvastatin protects against podocyte apoptosis in vitro

Abstract

Background: Clinical studies suggest that statins reduce proteinuria and slow the decline in kidney function in chronic kidney disease. Given a rich literature identifying podocyte apoptosis as an early step in the pathophysiological progression to proteinuria and glomerulosclerosis, we hypothesized that rosuvastatin protects podocytes from undergoing apoptosis. Regarding a potential mechanism, our lab has shown that the cell cycle protein, p21, has a prosurvial role in podocytes and there is literature showing statins upregulate p21 in other renal cells. Therefore, we queried whether rosuvastatin is prosurvival in podocytes through a p21-dependent pathway.

Methods: Two independent apoptotic triggers, puromycin aminonucleoside (PA) and adriamycin (ADR), were used to induce apoptosis in p21 +/+ and p21 -/- conditionally immortalized mouse podocytes with or without pre-exposure to rosuvastatin. Apoptosis was measured by two methods: Hoechst 33342 staining and fluorescence-activated cell sorting (FACS). To establish a role for p21, p21 levels were measured by western blotting following rosuvastatin exposure and p21 was stably transduced into p21 -/- mouse podocytes.

Results: Rosuvastatin protects against ADR- and PA-induced apoptosis in podocytes. Further, exposure to rosuvastatin increases p21 levels in podocytes in vitro. ADR induces apoptosis in p21 -/- mouse podocytes, but rosuvastatin's protective effect is not seen in the absence of p21. Reconstituting p21 in p21 -/- podocytes restores rosuvastatin's prosurvival effect.

Conclusion: Rosuvastatin is prosurvival in injured podocytes. Rosuvastatin exerts its protective effect through a p21-dependent antiapoptotic pathway. These findings suggest that statins decrease proteinuria by protecting against podocyte apoptosis and subsequent podocyte depopulation.

Fig. 1

Low-dose rosuvastatin does not affect podocyte viability in vitro. Growth-restricted conditionally immortalized mouse podocytes were incubated with escalating doses of rosuvastatin or DMSO as a vehicle control. Cell viability was determined by MTT assay at 24, 48 and 72 h time points (^*P < 0.05).

Fig. 2

Rosuvastatin protects podocytes from puromycin aminonucleoside-induced apoptosis. Growth-restricted mouse podocytes pre-incubated with rosuvastatin (20 μM) or DMSO (vehicle) for 24 h were exposed to puromycin aminonucleoside (PA, 30 μg/ml) for 48 h. Hoechst staining was performed (A) and the percentage of apoptosis was measured (B).

Fig. 3

Rosuvastatin protects podocytes from adriamycin-induced apoptosis. Podocytes pre-incubated for 24 h with rosuvastatin or DMSO were exposed to adriamycin (ADR, 0.25 μg/ml) for 48 h. Hoechst staining was performed (A) and the percentage of apoptosis was measured (B). Additionally, cells were dual-labeled with FITC-Annexin V and propidium iodide and fluorescent-activated cell sorting (FACS) performed. Representative contour plots (C) show virtually all of the cells in control and rosuvastatin-treated groups were viable, as detected in the lower-left quadrant (FITC⁻, PI⁻). ADR exposure significantly increased the percentage of apoptotic (FITC⁺, PI⁻) cells, as detected in the lower-right quadrant. Pre-incubation with rosuvastatin largely abrogated the rightward shift induced by ADR, consistent with a prosurvival effect. The results of three separate FACS experiments were pooled and averaged results are shown graphically.

Fig. 4

Rosuvastatin increases protein levels of p21 in podocytes in vitro. Growth-restricted mouse podocytes were pre-incubated for 24 h with rosuvastatin or DMSO and protein levels of p21^WAF1/CIP1 +/+ measured by Western blot analysis. A representative blot is shown (A). β-actin was used as a housekeeping protein to ensure equal protein loading and densitometric analysis performed (B). Values are averages of three separate experiments and normalized for differences in protein loading.

Fig. 5

The prosurvival effect of rosuvastatin is negated in the absence of p21. p21 −/− growth-restricted conditionally immortalized mouse podocytes were pre-incubated with rosuvastatin or DMSO. Cells were exposed to ADR and apoptosis measured 48 h later by Hoechst staining (A) and FACS analysis (B).

Fig. 6

Reconstituting p21 expression in p21 −/− podocytes restores the prosurvival effect of rosuvastatin. Growth-restricted p21 −/− mouse podocytes were stably transfected with a human p21 vector or GFP vector as a control. Western blotting for p21^WAF1/CIP1 +/+ showed successful re-expression of p21 in the p21 −/− podocytes (A). Following stable transfection, cells were pre-incubated with rosuvastatin or DMSO and ADR-induced apoptosis was measured by FACS analysis (B). The prosurvival effects of rosuvastatin in podocytes as a function of p21 status are summarized in (C).