Background. Gentamicin, a widely used antibiotic for the treatment of bacterial infection, can cause nephrotoxicity. Tetramethylpyrazine (TMP) is a compound purified from the rhizome of Ligusticum wallichi (called chuanxiong in Chinese). Besides its protection against ischaemia–reperfusion injury and nephritis in mice, we previously reported that TMP reverses gentamicin-induced apoptosis in rat kidneys. Haem oxygenase-1 (HO-1) induction by TMP has also been shown to attenuate myocardial ischaemia/reperfusion injury in rats. Methods. We used rat renal tubular (NRK-52E) cells, transformed cells with HO-1 overexpression or knockdown, and an adenovirus carrying the HO-1 gene (Adv-HO-1) as gene therapy targeting murine kidneys to explore the role of HO-1 in protection by TMP against gentamicin-induced toxicity both in vitro and in vivo . We evaluated the protective effects of HO-1 on several apoptotic parameters induced by gentamicin: cleaved caspases-3 and -9, cycloxygenase-2 (Cox-2) and subcellular localization of nuclear factor kappa B-p65 (NF-κB-p65), Bcl-xl and HS-1-associated protein (Hax-1) in NRK-52E cells. Results. NRK-52E cells treated with TMP exhibited transcriptional upregulation of the HO-1 protein by approximately twofold. Overexpression of HO-1 in NRK-52E cells significantly increased mitochondrial protein levels of the antiapoptotic molecules, Bcl-xL and Hax-1, and markedly decreased the NADPH oxidase activity and proinflammatory molecules, NF-κB-p65 and Cox-2, which might decrease gentamicin-induced activation of caspases-9 and -3. Conversely, NRK-52E cells with HO-1 knockdown significantly exacerbated gentamicin-induced tubular cell apoptosis. Additionally, the concomitant HO-1 induction by TMP was also evident in vivo , and HO-1 therapy markedly attenuated gentamicin-induced renal apoptosis to a similar extent as TMP pretreatment. Conclusions. Collectively, we suggest that HO-1 induced by TMP might, at least in part, protect against gentamicin-induced nephrotoxicity through antiapoptotic and anti-inflammatory mechanisms, and that it may have therapeutic potential for patients with renal disease. This is also the first demonstration that HO-1 increases Hax-1 mitochondrial localization.

We have previously reported that perfluorooctanesulfonate (PFOS) causes cell apoptosis in renal tubular epithelial cells (RTCs). Here, we extend our findings and provide evidence of epithelial-mesenchymal transition (EMT)-associated renal fibrosis caused by PFOS and the protection by l-carnitine. Our results demonstrate that PFOS increased the expression of EMT and renal injury biomarkers (eg, N-cadherin, vimentin, Snail, Kim1, and Lcn2). In addition, PFOS caused EMT induction through Sirt1-mediated PPARγ deacetylation and inactivation. l-carnitine reversed the EMT induction caused by PFOS and alleviated PFOS-mediated increases in cell migration by reactivating PPARγ through the inhibition of Sirt1 activity. The critical role of Sirt1 in this process was validated by using Sirt1 overexpression, resveratrol (a pharmacologic activator of Sirt1), nicotinamide (a Sirt1 inhibitor) and siSirt1. Nicotinamide and siSirt1, but not Sirt1 overexpression and resveratrol, alleviated PFOS-mediated EMT induction, suggesting that increased Sirt1 activity contributed to the alterations. Furthermore, through PPARγ overexpression and pharmacologic interventions, we validated the crucial role of increased PPARγ deacetylation caused by aberrant increased Sirt1 activity in RTC transformation. Similar to PPARγ overexpression, rosiglitazone (a PPARγ agonist) alleviated the effects of PFOS on the EMT-related features, whereas GW9662 (a PPARγ antagonist) mimicked the effects. The protective effect of l-carnitine was also verified in a mouse model of chronic PFOS exposure, in which decreased EMT biomarker levels and renal fibrosis by l-carnitine were observed in Western blot and histological analyses. Accordingly, l-carnitine alleviated EMT-associated renal fibrosis caused by PFOS through a Sirt1- and PPARγ-dependent mechanism.

Perfluorinated chemicals (PFCs) are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS) caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs). In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1) by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP) assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α) by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE) in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation.