Reactive oxygen species, endothelial dysfunction, inflammation, and mitogen-activated protein kinases have important roles in the pathogenesis of ischemia/reperfusion kidney injury. Stanniocalcin-1 (STC1) suppresses superoxide generation in many systems through induction of mitochondrial uncoupling proteins and blocks the cytokine-induced rise in endothelial permeability. Here we tested whether transgenic overexpression of STC1 protects from bilateral ischemia/reperfusion kidney injury. This injury in wild type mice caused a halving of the creatinine clearance; severe tubular vacuolization and cast formation; increased infiltration of macrophages and T cells; higher vascular permeability; greater production of superoxide and hydrogen peroxide; and higher ratio of activated ERK/activated JNK and p38, all compared to sham-treated controls. Mice transgenic for human STC1 expression, however, had resistance to equivalent ischemia/reperfusion injury indicated as no significant change from controls in any of these parameters. Tubular epithelial cells in transgenic mice expressed higher mitochondrial uncoupling protein 2 and lower superoxide generation. Pre-treatment of transgenic mice with paraquat, a generator of reactive oxygen species, before injury restored the susceptibility to ischemia/reperfusion kidney injury, suggesting that STC1 protects by an anti-oxidant mechanism. Thus, STC1 may be a therapeutic target for ischemia/reperfusion kidney injury.

AKI is associated with increased morbidity, mortality, and cost of care, and therapeutic options remain limited. Reactive oxygen species are critical for the genesis of ischemic AKI. Stanniocalcin-1 (STC1) suppresses superoxide generation through induction of uncoupling proteins (UCPs), and transgenic overexpression of STC1 inhibits reactive oxygen species and protects from ischemia/reperfusion (I/R) kidney injury. Our observations revealed high AMP-activated protein kinase (AMPK) activity in STC1 transgenic kidneys relative to wild-type (WT) kidneys; thus, we hypothesized that STC1 protects from I/R kidney injury through activation of AMPK. Baseline activity of AMPK in the kidney correlated with the expression of STCs, such that the highest activity was observed in STC1 transgenic mice followed (in decreasing order) by WT, STC1 knockout, and STC1/STC2 double-knockout mice. I/R in WT kidneys increased AMPK activity and the expression of STC1, UCP2, and sirtuin 3. Inhibition of AMPK by administration of compound C before I/R abolished the activation of AMPK, diminished the expression of UCP2 and sirtuin 3, and aggravated kidney injury but did not affect STC1 expression. Treatment of cultured HEK cells with recombinant STC1 activated AMPK and increased the expression of UCP2 and sirtuin 3, and concomitant treatment with compound C abolished these responses. STC1 knockout mice displayed high susceptibility to I/R, whereas pretreatment of STC1 transgenic mice with compound C restored the susceptibility to I/R kidney injury. These data suggest that STC1 is important for activation of AMPK in the kidney, which mediates STC1-induced expression of UCP2 and sirtuin 3 and protection from I/R.

RationaleWe have previously shown increased cardiac stanniocalcin-1 (STC1) in patients with idiopathic dilated cardiomyopathy. STC1 localizes to the inner mitochondrial membrane and transgenic over-expression of STC1 is associated with increased energy utilization.ObjectiveWe examined the hypothesis that STC1 uncouples mitochondrial oxidative phosphorylation - to suppress superoxide generation and modulate neurohormonal effects on cardiomyocytes.Methods and ResultsCompared to WT mouse heart, STC1 Tg heart displays: 2-fold higher uncoupling protein 3 (UCP3) levels, but no effect on UCP2 protein; 40% lower ATP levels; but similar activities of respiratory chain complexes I-IV. In cultured adult rat and freshly-isolated mouse cardiomyocytes, rSTC1 induces UCP3, but not UCP2. Treatment of cardiomyocytes with STC1 decreases mitochondrial membrane potential and suppresses baseline and angiotensin II (Ang II)-induced superoxide generation. Furthermore, baseline superoxide generation is higher in freshly-isolated adult UCP3−/− mouse cardiomyocytes compared to WT, suggesting an important role for UCP3 in regulating cardiomyocyte ROS under physiologic conditions. Treatment of UCP3−/− cardiomyocytes with rSTC1 failed to suppress superoxide generation, suggesting that the effects of STC1 on superoxide generation in cardiomyocytes are UCP3-dependent.ConclusionSTC1 activates a novel anti-oxidant pathway in cardiac myocytes through induction of UCP3, and may play an important role in suppressing ROS in the heart under normal physiologic conditions and ameliorate the deleterious effects of Ang II-mediated cardiac injury. Importantly, our data point to a critical role for the mitochondria in regulating ROS generation in response to Ang II.

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overexpression of stanniocalcin-1 in mice protects against ischemia-reperfusion kidney injury. We sought to determine the kidney phenotype after kidney endothelium-specific expression of stanniocalcin-1 small hairpin RNA (shRNA). We generated transgenic mice that express stanniocalcin-1 shRNA or scrambled shRNA upon removal of a floxed reporter (phosphoglycerate kinase-driven enhanced green fluorescent protein) and used ultrasound microbubbles to deliver tyrosine kinase receptor-2 promoter-driven Cre to the kidney to permit kidney endothelium-specific shRNA expression. Stanniocalcin-1 mRNA and protein were expressed throughout the kidney in wild-type mice. Delivery of tyrosine kinase receptor-2 promoter-driven Cre to stanniocalcin-1 shRNA transgenic kidneys diminished the expression of stanniocalcin-1 mRNA and protein throughout the kidneys. Stanniocalcin-1 mRNA and protein expression did not change in similarly treated scrambled shRNA transgenic kidneys, and we observed no Cre protein expression in cultured and tyrosine kinase receptor-2 promoter-driven Cre-transfected proximal tubule cells, suggesting that knockdown of stanniocalcin-1 in epithelial cells in vivo may result from stanniocalcin-1 shRNA transfer from endothelial cells to epithelial cells. Kidney-specific knockdown of stanniocalcin-1 led to severe proximal tubule injury characterized by vacuolization, decreased uncoupling of protein-2 expression, greater generation of superoxide, activation of the unfolded protein response, initiation of autophagy, cell apoptosis, and kidney failure. Our observations suggest that stanniocalcin-1 is critical for tubular epithelial survival under physiologic conditions.

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