Acetazolamide Protects Against Limb Ischemia/Reperfusion-Induced Acute Kidney Injury via NHE3 and Cathepsin D Inhibition

Prolonged tourniquet (TQ) application can cause ischemia/reperfusion injury in limbs, leading to systemic inflammation and acute kidney injury (AKI) with no specific treatment. Reduced urinary Na excretion is observed during AKI, while increased excretion correlates with better outcomes in patients. The Na-H exchanger 3 (NHE3) on proximal tubules is pivotal for Na absorption in the kidney. An increase in NHE3 protein level has been implicated in the reduced urinary Na excretion in AKI in both patients and animal models. Excessive activation of cathepsin D, a lysosomal protease, promotes apoptosis and necrosis in AKI. The FDA-approved acetazolamide (AZ) reduces NHE3 activity by decreasing proton generation through inhibiting carbonic anhydrase. We examined AZ's effectiveness in mitigating TQ-induced AKI in mice. C57BL/6 mice underwent bilateral thigh TQ application for 76 minutes followed by 22 hours of urine collection in metabolic cages. AZ (40 mg/kg) was given orally immediately after TQ application. Kidney function was assessed by urine output (UO) and glomerular filtration rate measured with a transcutaneous method (tGFR), and blood urea nitrogen (BUN) levels. Kidney structural damage was evaluated by histopathology in a blinded fashion and apoptosis, measured by caspase-3 activation and DNA fragmentation (TUNEL assay) across four groups: Sham, TQ with vehicle, AZ without TQ, and AZ with TQ. TQ with vehicle reduced UO by 62%, tGFR by 95%, and increased BUN by 774% compared with Sham. However, TQ with AZ had no significant impact on UO, only a 22% reduction in tGFR and 187% increase in BUN compared with AZ without TQ. The improvements with AZ were significant (Two-way ANOVA, p<0.05). TQ induced tubular degeneration from histology analysis and apoptosis, both were alleviated by AZ. To understand AZ's mitigation of TQ effects, we screened 48 molecules targeting different pathways, using Western blot analysis. TQ increased cathepsin C and D mRNA and protein levels, but AZ only reduced cathepsin D protein and mRNA levels. AZ also decreased NHE3 protein abundance, correlating with a drop in SGK1 protein, known for increasing NHE3 activity and stability. Treating pig proximal tubule LLC-PK1 cell line with monensin, an NHE3 mimetic, raised protein levels of cathepsin D by 60% and kidney injury molecule (KIM)-1 by 139%, further linking NHE3 to cathepsin D. Thus, AZ protects against TQ-induced AKI likely by inhibiting NHE3-dependent cathepsin D expression. Disclaimer The content and views expressed in this article are the sole responsibility of the authors and do not necessarily reflect the views or policies of the Uniformed Services University, Henry Jackson Foundation or Department of Defense. Mention of trade names, commercial products, or organizations does not imply endorsement by the Uniformed Services University, Henry Jackson Foundation or Department of Defense. This work was supported by the DoD grants W81XWH-22-2-0068. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.