Inhibition of Na‐H exchanger 3 ameliorates lower limb ischemia/reperfusion‐induced acute kidney injury through preservation of mitochondrial biogenesis in mice
Authors: Packialakshmi Balamurugan , Stewart Ian J. , Burmeister David M. , Zhou Xiaoming , Chung Kevin K. , Li Xiao C. , Soleimani Manoocher , Zhuo Jia L. , MacMillan‐Crow Lee Ann 
Published: May 2022

Despite advances in our understanding of acute kidney injury (AKI), its mortality rate remains high. Novel therapeutic targets are therefore needed. Mitochondrial dysfunction is a pivotal factor in the pathogenesis of AKI. The Na+‐H+ exchanger 3 (NHE3) plays a critical role in ATP‐dependent Na+ absorption in the proximal tubules. As such, inhibition of NHE3 may reduce metabolic demand for mitochondria thus reducing mitochondrial stress and dysfunction. In the present study, we sought to determine whether inhibition of NHE3 alleviated lower limb ischemia/reperfusion‐induced AKI. We induced lower limb ischemia/reperfusion by applying tourniquets bilaterally on the inguinal areas of male B6 or transgenic mice for 76 minutes followed by monitoring in metabolic cages for 22 hours. Control mice were treated in the same way except for no tourniquets. We used acetazolamide and kidney epithelial cell‐specific knockout to inhibit NHE3 in mice and monensin to mimic NHE3 in LLC‐PK1 cells. We assessed the renal function with urine output, transcutaneous GFR (tGFR) and serum urea nitrogen (SUN) and histology with H&E staining. We measured mitochondrial biogenesis by mRNA levels of PGC‐1α, a master regulator of mitochondrial biogenesis, and its targeted genes NDUFS1 and ATP5o. Compared with the control, tourniquets reduced urinary output by 62%, tGFR by 95% and increased SUN by 774% in B6 mice. Acetazolamide, given by oral gavage (40 mg/kg), reduced NHE3 protein abundance by 37% in the kidney cortex of tourniquet‐injured mice, attenuated tourniquet‐induced reductions in urinary output by 100% and tGFR by 77%, and increase in SUN by 76%. It also attenuated tourniquet‐induced peritubular focal hemorrhage, tubular degeneration and nuclear condensation in the renal cortex. Tourniquets reduced mRNA levels of PGC‐1α, NDUFS1 and ATP5o by 45%, 44%, and 52%, respectively. Acetazolamide significantly blunted all these effects. Compared with no tourniquet‐injured mice, tourniquets reduced tGFR by 98% and increased SUN by 825% in the NHE3Flx/Flx mice, which served as a control for NHE3 knockouts. However, tourniquets only reduced tGFR by 25% and increased SUN by 105% in the kidney epithelial cell‐specific NHE3 knockouts. The difference in the effects of tourniquets between these two strains was significant (p<0.05). Inhibition of NHE3 would reduce intracellular Na+concentrations. To determine whether intracellular Na+ regulates mitochondrial biogenesis, we treated LLC‐PK1 cells with monensin to raise intracellular Na+ levels. Monensin (10 μM) for 24 hours significantly reduced mRNA levels of PGC‐1α by 42%, NDUFS1 by 37% and ATP5o by 38% and activities of mitochondrial complex I by 100% and V by 58% in the cells. In conclusion, inhibition of NHE3 ameliorates tourniquet‐induced AKI through preservation of mitochondrial biogenesis, and this effect is most likely mediated by reducing intracellular Na+ concentration.