| The mechanisms leading to targeted necrosis of proximal straight tubular (PST) cells following (ischemic renal injury) IRI are not completely understood. Following IRI, the anaerobic glycolytic capacity is selectively inhibited in the medullary PST cells. Nonetheless, the exact mechanism of inhibition of anaerobic ATP synthesis following IRI is not elucidated. We demonstrated using in vitro and in vivo models that GAPDH is poly(ADP-ribosyl)ated by poly(ADP-ribose) polymerase-1 (PARP-1) and as a consequence, its activity and anaerobic glycolysis are inhibited under hypoxic conditions. Inhibition of PARP-1 activity restored GAPDH activity and ATP levels. Supplementation of NAD+ in simulated hypoxia had only a minimal effect on ATP synthesis indicating that glycolytic inhibition is not due to depletion of NAD+ as previously proposed. Inhibition of GAPDH using iodoacetate, however, exacerbated ATP depletion and necrotic cell death under hypoxic conditions when compared to controls, while inhibition of PARP-1 activity protected the cells. Following IRI, activation of several stress signaling pathways, resulting in the generation of reactive oxygen species, increased intracellular calcium levels, and decreased ATP levels, converge on the mitochondria to mediate necrosis and/or apoptosis and subsequent renal dysfunction. However, the mechanisms of mitochondrial-mediated necrosis of PST cells to cause renal dysfunction following IRI have not been investigated. Our results demonstrated that absence of the mitochondrial form of cyclophilin, cyclophilin D, (CypD) results in both functional and histopathological protection following IRI. Renal proximal tubular necrosis, erythrocyte congestion, tubular dilation and tubular cast formation were significantly lower in CypD deficient mice compared to wild type mice following IRI. Our in vitro data showed that CypD mediates necrotic cell death via opening of mitochondrial permeability transition (MPT) pore and subsequent MPT. Collectively, our studies demonstrated that PARP-1- and/or CypD-mediated downstream signaling cascades lead to targeted necrosis of PST following IRI. Overall, our data implicate that identifying the molecules involved in necrotic cell death and developing therapeutic agents to inhibit their activities may help to minimize/prevent PST cell injury and demise to improve renal functions post-IRI. |
