Macula Densa–Specific NOS1 Knockout Determines Susceptibility to Ischemic Acute Kidney Injury

Acute kidney injury (AKI) causes rapid loss of renal function with high morbidity and mortality. Our previous research showed that neuronal nitric oxide synthase (NOS1) mediates nitric oxide-dependent dilation of the afferent arteriole, inhibiting tubuloglomerular feedback (TGF), which critically regulates single nephron glomerular filtration (GFR). This mechanism could affect how vulnerable the organ is to ischemic injury, but this has not been directly tested. In this study, we generated inducible macula densa–specific NOS1 knockout mice (NKCC2-Cre-NOS1 flox/flox) using a new inducible NKCC2-Cre line crossed with NOS1 flox mice. AKI was induced by 18-minute bilateral renal pedicle clamping at 37°C, followed by 48-hour reperfusion. Kidney injury was assessed by measuring GFR, plasma creatinine, histology, cytokines, apoptosis, fibrotic factors, and proteomics. Complete NOS1 deletion in macula densa cells was confirmed through NKCC2/NOS1 immunofluorescence. Macula densa NOS1 deletion enhanced TGF response, which was further amplified following AKI compared to wild-type controls. Following AKI, compared with wild-type controls, NOS1 knockouts showed a significant lower GFR (236 66 to 24 22 µL/min) and higher plasma creatinine levels, alongside more severe tubular damage evidenced by H Western blot analysis showed cleaved caspase-3 levels were significantly increased, indicating enhanced apoptosis. Additionally, fibrosis markers TIMP1, collagen-3, and α-SMA were significantly upregulated at both mRNA and protein levels. We further observed increased hypoxia marker HIF-1α in MD-NOS1 KO mice. Global label-free proteomic profiling with targeted validation identified genotype-dependent responses involving haptoglobin, Tacstd2, and Cyp20a1, linking NOS1 deficiency to exaggerated inflammatory, fibrotic, and metabolic pathways. These findings highlight a novel role of macula densa NOS1 and TGF in AKI pathophysiology, suggesting NOS1 targeting as a therapeutic strategy. Haptoglobin was identified as a downstream plasma signal of NOS1-dependent AKI responses, indicating potential translational value.