Pigment epithelium ‐ derived factor protects human glomerular mesangial cells from diabetes via NOXO 1 ‐ iNOS suppression

The authors previously reported that pigment epithelium-derived factor (PEDF) protects the diabetic kidney from fibrosis via its anti‐oxidative effects. However, the underlying molecular mechanism has never been revealed. The present study aimed to investigate how PEDF protects mesangial cells from diabetes induced damage. Human mesangial cells were exposed to a diabetic environment [30 mmol/l glucose and 200 mg advanced glycation end products (AGEs)] in the absence or presence of PEDF (200 mg/l). The superoxide and peroxynitrite productions were measured by fluorescent assay. The nicotinamide adenine dinucleotide phosphate (NAPDH) oxidases (NOXs; isoforms NOX1, NOX2, and NOX4), NADPH oxidase organizer 1 (NOXO1), DHFR, endothelial nitric oxide synthase (iNOS) and phospho‐p38MAPK (p‐p38) protein levels were also examined to explore the possible mechanism of the PEDF anti‐oxidative properties. The fibrogenesis of mesangial cells in diabetes was associated with increased superoxide generation and peroxynitrite production via iNOS induction and uncoupling. However, elevated transforming growth factor‐β level, reactive oxygen species (ROS) overproduction, iNOS induction and uncoupling were all reversed by NOXO1 suppression following PEDF treatment or NOXO1 silencing. Furthermore, the p38MAPK inhibition only attenuated the ROS/peroxynitrite production partially via abolishment of iNOS induction, however had no effect on iNOS uncoupling and its regulating enzyme: DHFR suppression. PEDF prevented oxidative stress and protected mesangial cells from fibrogenesis in a diabetic environment via dual effects mediated by NOXO1 inhibitory prevention of iNOS induction through p38MAPK inactivation and effects on iNOS coupling through DHFR restoration. Introduction Diabetic nephropathy (DN) is a leading cause of end-stage renal failure worldwide. The pathogenetic mechanisms for DN may start with reactive oxygen species (ROS) as the common link connecting to various signaling pathways that lead to kidney impairment (1‐4). The ROS are a family of molecules including superoxide anion (O2-), hydroxyl racial (HO-), hydrogen peroxide (H2O2), peroxynitrite (ONOO-), nitric oxide (NO) and lipid radicals. In mammalian cells, potential origins of ROS generation include mitochondrial respiratory chain, xanthine oxidase, NOXs, and nitric oxide synthase (NOS), with increasing evidence that NOXs appear to be the major sources or more specifically initial trigger of ROS generation in the diabetic complications including kidney (5‐7). Various subtypes of NOXs (e.g., NOX1, NOX2, NOX4) are discovered abundantly in the mesangial cells (8). Lee et al (9)showed that mitochondrial derived ROS was elevated in DN subsequent to NOX4 activation. Although NOX4 is likely implicated in the mediation of kidney fibrosis in diabetes including mesangial impairment, other subtypes such as NOX1, NOX2 and their cytosolic component NADPH oxidase organizer 1 (NOXO1) are also possible candidates involved in this pathogenesis (10). Additionally, other researches reported that NOS uncoupling instead of mitochondria dysfunction in the mesangial cells is responsible for the early glomerular pathology in diabetes and is associated with inflammation and oxidative stress of DN (11). Thus, the downstream of NOXs activation or the main source of oxidative stress is still in debate and should be under extensive investigation. Recently, both in vivo and in vitro studies have demonstrated that nitroxidative/nitrosative stress (ONOO-) has been actively involved in the development of DN or the hyperglycemia induced damage in mesangial cells (12). Therefore, NOS uncoupling as the most possible cause of ONOOproduction should be examined. It is well known that NOS coupling status is governed by its cofactor tetrahydrobiopterin (H4B) abundance, for instance: when H4B is deficient, NOS becomes dysfunctional to produce superoxide instead of NO which changes into ONOOon site instantly and vastly. Moreover, H4B synethesis is controlled by dihydrofolate reductase (DHFR), the key enzyme responsible for H4B salvage pathway, whose expression level would determine the abundance of H4B. Notably, DHFR suppression Pigment epithelium‐derived factor protects human glomerular mesangial cells from diabetes via NOXO1‐iNOS suppression LING GAO, YINGJIE HU and JING LI Department of Endocrinology, Renmin Hospital of Wuhan University, Wuhan 430060, P.R. China Received January 29, 2017; Accepted September 5, 2017 DOI: 10.3892/mmr.2017. 7563 Correspondence to: Professor Jing Li, Department of Endocrinology, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan, Hubei 430060, P.R. China E‐mail: lijing7823@medmail.com. cn