Paricalcitol attenuates indoxyl sulfate-induced apoptosis through the inhibition of MAPK, Akt, and NF-kB activation in HK-2 cells
Open Access
- 1 January 2019
- journal article
- research article
- Published by Korean Association of Internal Medicine in The Korean Journal of Internal Medicine
- Vol. 34 (1), 146-155
- https://doi.org/10.3904/kjim.2016.298
Abstract
Background/Aims: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear factor-kappa B (NF- kappa B) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of NF-kappa B was measured by luciferase assays and apoptosis was determined by flow cytometry of cells stained with fluorescein isothiocyanate-conjugated Annexin V protein. Results: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, NF-kappa B p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, NF-kappa B p65, and Akt in HK-2 cells. NF-kappa B promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, NF-kappa B, and Akt signaling pathway in HK-2 cells.Keywords
Funding Information
- National Research Foundation of Korea
- Ministry of Education (2014R1A1A2008333)
- Ministry of Science, ICT and Future Planning (2016R1A2B4007870, 2014M3C1A3053036, 2017M3A9E8023001)
- Korea Health Industry Development Institute
- Ministry of Health and Welfare (HI14C2084)
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