Modulation of APOL1-miR193a Axis Prevents Podocyte Dediffrentiation in High Glucose Milieu

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms’ tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.</jats:p

Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification