Proteolytic processing of the catalytic subunit of protein kinase A (PKA C) by meprin β in ischemia/reperfusion‐induced acute kidney injury

Ischemic/reperfusion (IR) is associated with acute kidney injury. Meprin metalloproteases are the most abundant proteins in the brush border membranes (BBMs) of proximal kidney tubules. Presence of meprins was previously shown to enhance kidney injury in IR. However, the mechanisms by which meprins modulate kidney injury in IR are not fully understood. We are gaining insights from identification of kidney meprin targets, which include extracellular matrix (ECM) proteins, cytoskeletal proteins, tight junction proteins, and members of the AGC kinase family of proteins (e.g protein kinase A and protein kinase C). Meprins are redistributed from the BBM to the cytosol and basolateral membranes in ischemia/reperfusion bringing them in close contact with proteins in these cell compartments. We recently showed that meprins cleave the catalytic subunit of protein kinase A (PKA C) in an isoform specific manner. While both meprin A and meprin B could cleave PKA Cβ1, only meprin B cleaved PKA Cα and PKA Cβ2. In mice subjected to IR‐induced acute kidney injury, fluorescent microscopy showed a decrease in total PKA C in the proximal tubules (which express meprins) but not in distal tubules (which lack endogenous meprins). In fact the levels of PKA C in distal tubules increased at 6h post‐IR, suggesting that the PKA pathway plays a role in the hypoxia response associated with IR. The objective of the current study was to determine how proteolytic processing of PKA C by meprin β affects the levels PKA C isoforms in mice with IR‐induced acute kidney injury. To this end, surgical procedures were used for unilateral nephrectomy coupled with clamping of renal arteries for 27 minutes to induce IR‐associated injury in the contralateral kidneys of 12‐week old male mice on a C57BL/6 background (n=6 meprin β knockout; n=5 wild‐type). Blood samples were collected pre and post IR injury to phenotype kidney. The mice were sacrificed at 6h post IR and kidney tissues processed for proteomic analysis. Serum creatinine levels were determined by ELISA to confirm kidney failure. Western blot analysis with anti‐PKA C and anti‐PKA Cα specific antibodies was used to evaluate the levels of total PKA C and the PKA Cα isoform in kidney proteins. The intensities of the PKA C protein bands were quantified using a calibrated densitometer. Statistical analysis utilized 2‐way ANOVA. Our data showed a decrease in the kidney levels of total PKA C and PKA Cα in WT kidneys subjected to IR. This decrease was not observed in meprin β knockout kidneys. The data suggests increased in vivo proteolytic processing of PKA C present in kidneys by meprin β in IR. Meprin β may thus enhance renal tubular damage via cleaving of PKA C. We will evaluate the impact on downstream targets of the PKA pathway and hypoxia response genes. Support or Funding Information NIH/NIGMS Grant # SC3GM102049