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Mild intracellular acidification by dexamethasone attenuates mitochondrial dysfunction in a human inflammatory proximal tubule epithelial cell model.

ABSTRACT: Septic acute kidney injury (AKI) associates with poor survival rates and often requires renal replacement therapy. Glucocorticoids may pose renal protective effects in sepsis via stimulation of mitochondrial function. Therefore, we studied the mitochondrial effects of dexamethasone in an experimental inflammatory proximal tubule epithelial cell model. Treatment of human proximal tubule epithelial cells with lipopolysaccharide (LPS) closely resembles pathophysiological processes during endotoxaemia, and led to increased cytokine excretion rates and cellular reactive oxygen species levels, combined with a reduced mitochondrial membrane potential and respiratory capacity. These effects were attenuated by dexamethasone. Dexamethasone specifically increased the expression and activity of mitochondrial complex V (CV), which could not be explained by an increase in mitochondrial mass. Finally, we demonstrated that dexamethasone acidified the intracellular milieu and consequently reversed LPS-induced alkalisation, leading to restoration of the mitochondrial function. This acidification also provides an explanation for the increase in CV expression, which is expected to compensate for the inhibitory effect of the acidified environment on this complex. Besides the mechanistic insights into the beneficial effects of dexamethasone during renal cellular inflammation, our work also supports a key role for mitochondria in this process and, hence, provides novel therapeutic avenues for the treatment of AKI.

SUBMITTER: Schirris TJJ

PROVIDER: S-EPMC5587643 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Mild intracellular acidification by dexamethasone attenuates mitochondrial dysfunction in a human inflammatory proximal tubule epithelial cell model.

Schirris T J J TJJ Jansen J J Mihajlovic M M van den Heuvel L P LP Masereeuw R R Russel F G M FGM

Scientific reports 20170906 1

Septic acute kidney injury (AKI) associates with poor survival rates and often requires renal replacement therapy. Glucocorticoids may pose renal protective effects in sepsis via stimulation of mitochondrial function. Therefore, we studied the mitochondrial effects of dexamethasone in an experimental inflammatory proximal tubule epithelial cell model. Treatment of human proximal tubule epithelial cells with lipopolysaccharide (LPS) closely resembles pathophysiological processes during endotoxaem ...[more]

PMID: 28878224

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Project description:Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.

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Mild intracellular acidification by dexamethasone attenuates mitochondrial dysfunction in a human inflammatory proximal tubule epithelial cell model.

Publications

Mild intracellular acidification by dexamethasone attenuates mitochondrial dysfunction in a human inflammatory proximal tubule epithelial cell model.

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