Previous studies have already demonstrated that mitochondria play a key role in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells. To further clarify the underlying mechanism of Pb-induced mitochondrial apoptosis, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and its regulatory components in Pb-induced apoptosis in rPT cells. Mitochondrial permeability transition pore (MPTP) opening together with disruption of mitochondrial ultrastructure, translocation of cytochrome c from mitochondria to cytoplasm and subsequent caspase-3 activation were observed in this study, suggesting that MPT is involved in Pb-induced apoptosis in rPT cells. Simultaneously, Pb-induced caspase-3 activation and apoptosis can be significantly inhibited by three MPTP inhibitors (CsA, DIDS, BA), which target different regulatory components of MPTP (Cyp-D, VDAC, ANT), respectively, demonstrating that Cyp-D, VDAC and ANT participate in MPTP regulation during lead exposure. Moreover, decreased ATP levels and increased ADP/ATP ratio induced by lead treatment can be significantly reversed by BA, indicating that Pb-mediated ANT dysfunction resulted in ATP depletion. In addition, up-regulation of VDAC-1, ANT-1 together with down-regulation of Cyp-D, VDAC-2 and ANT-2 at both the levels of transcription and translation were revealed in rPT cells under lead exposure conditions. In conclusion, Pb-mediated mitochondrial apoptosis in rPT cells is dependent on MPTP opening. Different expression levels in each isoform of three regulatory components contribute to alteration in their functions, which may promote the MPTP opening.

No abstract

Autophagy has an important renoprotective function and we recently found that autophagy inhibition is involved in cadmium (Cd)-induced nephrotoxicity. Here, we aimed to investigate the protective effect of trehalose (Tre), a novel autophagy activator, against Cd-induced cytotoxicity in primary rat proximal tubular (rPT) cells. First, data showed that Tre treatment significantly decreased Cd-induced apoptotic cell death of rPT cells via inhibiting caspase-dependent apoptotic pathway, evidenced by morphological analysis, flow cytometric and immunoblot assays. Also, administration with Tre protected rPT cells against Cd-induced lipid peroxidation. Inhibition of autophagic flux in Cd-exposed rPT cells was markedly restored by Tre administration, demonstrated by immunoblot analysis of autophagy marker proteins and GFP and RFP tandemly tagged LC3 method. Resultantly, Cd-induced autophagosome accumulation was obviously alleviated by Tre treatment. Meanwhile, blockage of autophagosome–lysosome fusion by Cd exposure was noticeably restored by Tre, which promoted the autophagic degradation in Cd-exposed rPT cells. Moreover, Tre treatment markedly recovered Cd-induced lysosomal alkalinization and impairment of lysosomal degradation capacity in rPT cells, demonstrating that Tre has the ability to restore Cd-impaired lysosomal function. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced cytotoxicity in rPT cells by inhibiting apoptosis and restoring autophagic flux.

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Puerarin (PU), a natural flavonoid, has been reported to possess anti-oxidative and anti-inflammatory activities. In the present study, female Sprague-Dawley rats received lead (Pb) nitrate (300 mg/L, via drinking water) and/or PU (400 mg/kg/day, orally) to investigate the protective effects of PU on Pb-induced renal damage. Renal toxicity was evaluated by detecting urinary proteins excretion as well as levels of serum urea nitrogen and serum creatinine. Ultrastructural observations and real-time quantitative polymerase chain reaction analyses were performed on kidney cortex tissues to identify the mitochondrial damage and quantify gene expression levels of cytochrome oxidase submits (COX-I/II/III), respectively. Renal cell damage was assessed by light microscopic examination. Lipid peroxidation (LPO) levels and antioxidant status in kidney were also evaluated. Animals that received both Pb and PU showed a better renal function than those that received Pb alone, with minor pathological damage. Moreover, PU significantly reduced LPO and markedly restored the enzymatic and non-enzymatic antioxidants levels in kidney of Pb-treated rats, which may be related to its restoring mitochondrial function. Furthermore, PU administration significantly increased urinary Pb excretion and decreased its level in the serum and kidney. In conclusion, these results suggested that PU reduces renal damage induced by chronic Pb administration through its antioxidant properties and chelating ability.