Abstract
Diabetic nephropathy (DN) is characterized by perturbations in metabolic/cellular signaling pathways with generation of reactive oxygen species (ROS). The ROS are regarded as a common denominator of various pathways, and they inflict injury on renal glomerular cells. Recent studies indicate that tubular pathobiology also plays a role in the progression of DN. However, the mechanism(s) for how high (25 mm) glucose (HG) ambience induces tubular damage remains enigmatic. myo-Inositol oxygenase (MIOX) is a tubular enzyme that catabolizes myo-inositol to d-glucuronate via the glucuronate-xylulose (G-X) pathway. In this study, we demonstrated that G-X pathway enzymes are expressed in the kidney, and MIOX expression/bioactivity was up-regulated under HG ambience in LLC-PK1 cells, a tubular cell line. We further investigated whether MIOX overexpression leads to accentuation of tubulo-interstitial injury, as gauged by some of the parameters relevant to the progression of DN. Under HG ambience, MIOX overexpression accentuated redox imbalance, perturbed NAD(+)/NADH ratios, increased ROS generation, depleted reduced glutathione, reduced GSH/GSSG ratio, and enhanced adaptive changes in the profile of the antioxidant defense system. These changes were also accompanied by mitochondrial dysfunctions, DNA damage and induction of apoptosis, accentuated activity of profibrogenic cytokine, and expression of fibronectin, the latter two being the major hallmarks of DN. These perturbations were largely blocked by various ROS inhibitors (Mito Q, diphenyleneiodonium chloride, and N-acetylcysteine) and MIOX/NOX4 siRNA. In conclusion, this study highlights a novel mechanism where MIOX under HG ambience exacerbates renal injury during the progression of diabetic nephropathy following the generation of excessive ROS via an unexplored G-X pathway.
Highlights
W bioactivity was up-regulated under high (25 mM) glucose (HG) ambience in LLC-PK1 s cells, a tubular cell line
The findings described here include events that indicate that following increased expression of myo-Inor sitol oxygenase (MIOX) in tubular cells under high glucose ambience, there is an accentuated perturbation in cellular redox and mitochondrial homeostasis, leading to cellular apoptosis
By RT-PCR analysis, expression of five enzymes (i.e. MIOX, ALR1, gulonate dehydrogenase, xylulose reductase, and xylulose kinase) could be demonstrated in kidney tissues (Fig. 2D), which confirmed the existence of a G-X pathway in the kidney cortical tubules, as it has been described in the eye lens previously and is akin to the polyol pathway [25,26,27,28]
Summary
Reagents—The reagents were purchased from the following vendors. From Sigma-Aldrich, we purchased polyclonal antifibronectin Assessment of Mitochondrial Membrane Potential (⌬⌿m)— To measure the loss of ⌬⌿m, confocal microscopy of cells stained with TMRE was employed [44] For these analyses, LLC-PK1 cells subjected to various treatments, high glucose versus low glucose, and those overexpressing MIOX and subjected to siRNA treatments were plated onto 35-mm glass bottom culture dishes (MatTak Corp.) and maintained at 37 °C in a defined medium. After hblocking, fixed cells were immersed in a solution containing itrabbit anti-COX IV or mouse anti-cytochrome c antibody age to high and low molecular weight mtDNA was evaluated by PCR amplification procedures, as described previously [46]. Data were analyzed by one-way analysis of variance, and a p value of Ͻ0.05 was considered statistically significant
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