Khellin a promising natural compound for alleviation of diabetic nephropathy: Evidence from in-silico, in-vitro and in-vivo studies

This study aimed to evaluate the potential impacts of Khellin (Khe) therapeutic potential in streptozotocin (STZ)-induced diabetic nephropathy (DN) using integrated in silico, in vitro, and in vivo approaches. In silico studies were conducted to assess whether Khe could target DN-associated proteins. In vitro, the MTT assay assessed Khe's impact on normal kidney HK-2 cells. In vivo, male Sprague Dawley rats (n = 30) were randomly divided into six groups: normal control (Cnt), low (Khe-L, 50 mg/kg), and high (Khe-H, 100 mg/kg) dose Khe, DN, DN + Khe-L, and DN + Khe-H. Renal function markers, oxidative stress parameters, inflammatory cytokines, and apoptotic genes were evaluated. Histopathological examination of kidney tissues was also performed. Khe showed variable binding affinities and interaction patterns in a molecular docking study against seven proteins (TNFα, TGFβ1, ETS1, TIMP2, MAPK14, PI3K, and KIM1) involved in DN pathogenesis. In vitro, Khe improved the survival of high glucose (HG) treated HK-2 cells and inhibited the activation of MAPK14, PI3K, caspase 3, and TNFα induced by HG. Khe-treated rats (DN + Khe-L, and DN + Khe-H) exhibited significantly 1) decreased serum levels of creatinine, urea, and retinol-binding protein (RBP), 2) declined urinary proteins levels, 3) reduced renal levels of MDA, 4) increased activities of the antioxidant enzymes (SOD, CAT, GPx), 5) downregulated renal expression of TNFα, TGFβ1, ETS1, TIMP2, KIM1, MAPK14, PI3K, and Bax , and 6) upregulated expression of Bcl2 compared to the DN group. These biochemical and molecular results were corroborated by histopathological examination, which showed that the groups treated with Khe had much better kidney tissue integrity. The effectiveness of the treatment depended on the dosage. Khe exhibited therapeutic effects in DN, potentially mediated by its antioxidant, anti-apoptotic, and anti-inflammatory mechanisms. These findings underscore Khe's promise as an adjunctive therapy for DN.