4-(5-benzo(1-3)dioxol-5-yl-4-pyridin-2-yl-1h-imidazol-2-yl)benzamide and Kidney-Diseases

Zoledronate is a bisphosphonate that is widely used in the treatment of metabolic bone diseases. However, zoledronate induces significant nephrotoxicity associated with acute tubular necrosis and renal fibrosis when administered intravenously. There is speculation that zoledronate-induced nephrotoxicity may result from its pharmacological activity as an inhibitor of the mevalonate pathway but the molecular mechanisms are not fully understood. In this report, human proximal tubular HK-2 cells and mouse models were combined to dissect the molecular pathways underlying nephropathy caused by zoledronate treatments. Metabolomic and proteomic assays revealed that multiple cellular processes were significantly disrupted, including the TGFβ pathway, fatty acid metabolism and small GTPase signaling in zoledronate-treated HK-2 cells (50 μM) as compared with those in controls. Zoledronate treatments in cells (50 μM) and mice (3 mg/kg) increased TGFβ/Smad3 pathway activation to induce fibrosis and kidney injury, and specifically elevated lipid accumulation and expression of fibrotic proteins. Conversely, fatty acid transport protein Slc27a2 deficiency or co-administration of PPARA agonist fenofibrate (20 mg/kg) prevented zoledronate-induced lipid accumulation and kidney fibrosis in mice, indicating that over-expression of fatty acid transporter SLC27A2 and defective fatty acid β-oxidation following zoledronate treatments were significant factors contributing to its nephrotoxicity. These pharmacological and genetic studies provide an important mechanistic insight into zoledronate-associated kidney toxicity that will aid in development of therapeutic prevention and treatment options for this nephropathy.

Topics: Animals; Benzamides; Cell Line; Coenzyme A Ligases; Dioxoles; Epithelial Cells; Fatty Acids; Fenofibrate; Fibrosis; Humans; Kidney Diseases; Kidney Tubules; Lipid Metabolism; Male; Mice, Inbred C57BL; Mice, Mutant Strains; Oxidation-Reduction; Transforming Growth Factor beta; Zoledronic Acid

To investigate the mechanism of action of Fuzheng Huayu recipe (FZHY) and vitamin E (Vit E) against renal interstitial fibrosis related to transforming growth factor-beta1 (TGF-beta1) mediated tubular epithelial-to-mesenchymal transition.. Renal interstitial fibrosis was induced by administration of HgCl(2) at a dose of 8 mg/kg body weight once a day for 9 weeks. Rats were randomly divided into four groups: normal, model, FZHY, and Vit E group. Rats in the latter two groups were treated with the FZHY recipe and Vit E respectively. HK-2 cells were treated with TGF-beta1 for 24h, followed by incubation with either SB-431542 (a potent and specific inhibitor of TbetaR-I kinase) or FZHY drug-containing serum for another 24h. Hyp content in rat kidney tissue was assayed with Jamall's method and collagen deposition in kidney was visualized using Masson stain. Protein expression of TGF-beta1, TbetaR-I, Smad2, p-Smad2, Smad3, and p-Smad3 was analyzed by Western blotting. Protein expression and the location of Smad3 in kidney was assayed by immunohistochemistry, E-cadherin, cytokeratin 18 (CK-18), alpha-SMA and TGF-beta1 by immunofluorescent stain.. FZHY and Vit E inhibited renal collagen deposition and reduced Hyp content significantly. They upregulated E-cadherin protein expression and down-regulated the protein expression of alpha-SMA, TGF-beta1, p-Smad2, p-Smad3, and TbetaR-I. Lastly, they inhibited the nuclear translocation of Smad3 in fibrotic kidney tissue. FZHY drug-containing serum significantly upregulated the expression of CK-18 and down-regulated the expression of alpha-SMA, TbetaR-I, p-Smad2/3 in TGF-beta1 stimulated HK-2 cells.. The mechanism of action of FZHY and Vit E against renal interstitial fibrosis is related to the reversal of tubular EMT induced by TGF-beta1.

Topics: Adaptor Proteins, Signal Transducing; Animals; Benzamides; Biological Transport; Cadherins; Collagen; Dioxoles; Drugs, Chinese Herbal; Epithelial Cells; Fibrosis; Fungi; Humans; Hydroxyproline; Keratin-18; Kidney; Kidney Diseases; Male; Mesoderm; Phytotherapy; Plants, Medicinal; Random Allocation; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1; Vitamin E

A progressive tubulointerstitial nephropathy is mainly induced by aristolochic acid I (AAI), but a comprehensive understanding of this process is still missing. By using mouse primary renal tubular epithelial cells (RTECs) cultured in vitro and combining with two AAI treatment types (dose-response studies and time-response studies), we sought to investigate the nephrotoxicity of AAI further. Following our molecular and pharmacological studies, we found that high doses of AAI could lead to the death of RTECs within a short time, but low doses in a long duration only induce the epithelial cells to transform into myofibroblasts (MFs). This was also immediately identified by the increased expression of vimentin and de novo expression of alpha-smooth muscle actin (alpha-SMA) with the loss of cytokeratin 18 (CK18) by semiquantitative reverse transcriptase-PCR (RT-PCR) and immunofluorescence staining. The transcriptional level of transforming growth factor-beta1 (TGF-beta1) in the group treated with AAI significantly increased twice as much as the control. Smad2 mRNA level in the group with 50 ng/mL AAI declined by 23.4% at 24 hr, then increased by 180.0% at 36 hr; it was also evidently increased (217.4%) after being treated with 30 ng/mL AAI for 24 hr. Meanwhile, Smad7 mRNA level was down-regulated by AAI in dose- and time-dependence. Furthermore, by cotransfecting in mouse primary RTECs, the transcriptional level of Smad7 promoter-luciferase reporter gene was significantly down-regulated by AAI (300 ng/mL), and the expression of myofibroblast-specific markers induced by AAI was also suppressed by the specific antagonist of TGF-beta1 receptors (SB-431542). Collectively, the present results suggest that AAI may induce cytotoxicity through its conductive epithelial to mesenchymal transition, and TGF-beta1/Smad7 signaling can stimulate renal tubulointerstitial fibrosis induced by AAI.

Topics: Actins; Animals; Aristolochic Acids; Benzamides; Cells, Cultured; Dioxoles; Down-Regulation; Epithelial Cells; Fibrosis; Keratin-18; Kidney Diseases; Kidney Tubules; Male; Mice; RNA, Messenger; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta1; Vimentin