Loss of α(E)-catenin potentiates cisplatin-induced nephrotoxicity via increasing apoptosis in renal tubular epithelial cells

Abstract

Cisplatin is one of the most potent and widely used antitumor drugs. However, the use of cisplatin is limited by its side effect, nephrotoxicity. Evidence has shown an increased incidence and severity of acute kidney injury (AKI) in the elderly. Previous studies from our laboratory demonstrate a decrease in α(E)-catenin expression in aged kidney. In this study, we investigated whether the loss of α(E)-catenin may increase cisplatin nephrotoxicity. To study the effects of reduced α(E)-catenin, a cell line with stable knockdown of α(E)-catenin (C2 cells) was used; NT3 is nontargeted control. C2 cells exhibited a significant loss of viability as determined by MTT assay compared with NT3 cells after cisplatin challenge, but showed no difference in lactate dehydrogenase (LDH) leakage. Increased caspase 3/7 activation and PARP cleavage was observed in C2 cells after cisplatin treatment. Z-VAD, a pan-caspase inhibitor, abolished the difference in susceptibility between NT3 and C2 cells. Interestingly, the expression of α(E)-catenin was further decreased after cisplatin treatment. Furthermore, in vivo data demonstrated a significant increase in serum creatinine at 72 h after a single dose of cisplatin in 24-month-old rats, but not in 4-month-old rats. Increased expression of KIM-1 and in situ apoptosis were also detected in aged kidney after cisplatin challenge. Taken together, these data suggest that loss of α(E)-catenin increases apoptosis of tubular epithelial cells which may contribute to the increased nephrotoxicity induced by cisplatin in aged kidney.

Keywords: AKI; aging; apoptosis; cisplatin; α-catenin.

FIG. 1.

Effect of decreased α(E)-catenin on susceptibility to cisplatin injury. (A) Cell viability was determined by MTT assay in cultures of NRK-52E cells seeded with the indicated densities and challenged with 150μM cisplatin in SF media for 24 h. The densities correspond with 100, 75, and 50% confluence. The results are presented as percentage viability of untreated control with corresponding cell density in SF media. The asterisks indicate significant differences from the 15 × 10³ group (n = 16). Cell viability determined by MTT (B) and neutral red (C) assays in confluent cultures of NT3 and C2 cells treated with the indicated concentrations of cisplatin in SF media for 24 h. (D) Cell viability determined by MTT assay upon treatment for the indicated time periods with 150μM cisplatin. The asterisks in (B), (C), and (D) indicate significant differences between NT3 and C2 cells for each treatment (n = 24). (E) Cell viability determined by MTT assay in NRK-52E cells treated with the indicated concentrations of cadherin antagonist (IC₅₀ = 0.09μM) alone or in the presence of 150μM cisplatin. Control peptide was used at a concentration of 1mM (n = 16). The results in (B), (C), (D), and (E) are presented as percentage viability of untreated control in SF media.

FIG. 2.

Susceptibility of NT3 and C2 cells to injuries induced by different chemicals. (A) Cytotoxicity was determined by LDH assay in confluent cultures of NT3 and C2 cells treated with the indicated concentrations of cisplatin in SF media for 24 h. The results are presented as the percentage cytotoxicity of the maximum LDH release control (n = 6). Cell viability was determined by MTT assay in cells treated with the indicated concentrations of mercury (B), cisplatin, and staurosporine (C). The results are presented as percentage viability of untreated control in SF media (B and C). The asterisks indicate significant differences between NT3 and C2 cells of each treatment (n = 24).

FIG. 3.

Apoptosis induced by cisplatin in NT3 and C2 cells. (A) Caspase 3/7 activity in confluent cultures of cells treated with the indicated concentrations of cisplatin in SF media for 24 h, was determined by luminescent assay. The asterisks indicate the significant differences between NT3 and C2 cells of each treatment (n = 8). Immunoblot analysis (B) and quantification of cleaved caspase 3/caspase 3 (C), cleaved caspase 7/caspase 7 (D) and PARP (cleaved-Asp214)/ β-actin (E) in whole cell lysates (70 μg protein) obtained from cells treated with 150μM cisplatin for the indicated time periods. The β-actin blot serves as the loading control. The significant difference between C2 and NT3 at each time point is indicated by asterisk (n = 2). (F) Cell viability determined by MTT assay in cells treated with 150μM cisplatin alone, in the presence of 50μM Z-VAD or 50μM Z-VAD alone in SF media for 24 h. The results are presented as percentage viability of untreated control in SF media. The asterisks indicate significant differences between NT3 and C2 cells of each treatment (n = 24).

FIG. 4.

The effect of cisplatin on α-catenin and N-cadherin expression. (A) qPCR for α(E)-catenin in NT3 and C2 cells with or without 100μM cisplatin treatment for 24 h. The result is normalized to untreated NT3 cells. The significant differences are indicated by asterisks (n = 3). Immunoblot analysis (B) and quantification of N-cadherin/β-actin (C) and α-catenin/β-actin (D) in whole cell lysates (40 μg protein) obtained from cells treated with the indicated concentrations of cisplatin in SF media for 24 h. The β-actin blot serves as loading control. The significant differences between C2 and NT3 are indicated by asterisks (n = 2). The hash symbol indicates significant difference from the untreated control group (n = 2).

FIG. 5.

Cisplatin or mercury(II) chloride-induced changes in young and aged kidney. Serum creatinine levels were measured after 72 h of cisplatin treatment (A) or 48 h of mercury(II) chloride treatment (B). Urine KIM-1 levels were measured after 72 h of cisplatin treatment (C). Equal volume of saline is used as control. The asterisk indicates significant differences from the control group (n = 5). Immunoblot (D) and quantification of N-cadherin/ β-actin (E) and α-catenin/β-actin (F) in kidney tissue lysates (100 μg protein) obtained from young and aged rats after 72 h of cisplatin treatment. The asterisk indicates significant differences between young and aged group of each treatment (n = 5). The pound indicates significant difference from the untreated group (n = 5).

FIG. 6.

In situ apoptosis induced by cisplatin in aged kidney. In situ apoptosis was detected (A) and quantified (B) via TUNEL assay. The white arrow heads point the apoptotic cells. The asterisks indicate significant difference from the untreated group (n = 5).