Macula densa-specific NOS1 knockout determines susceptibility to ischemic acute kidney injury.

Abstract

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 rate (GFR). This mechanism could affect how vulnerable the organ is to ischemic injury, but this has not been directly tested. In the present study, we generated inducible macula densa (MD)-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-min bilateral renal pedicle clamping at 37°C, followed by 48-h reperfusion. Kidney injury was assessed by measuring GFR, plasma creatinine, histology, cytokines, apoptosis, fibrotic factors, and proteomics. Complete NOS1 deletion in MD cells was confirmed through NKCC2/NOS1 immunofluorescence. MD NOS1 deletion enhanced the TGF response, which was further amplified following AKI compared with control (Cre -/-). Following AKI, compared with controls (Cre -/-), NOS1 knockouts showed a significantly lower GFR (236 ± 66 to 24 ± 22 μl/min) and higher plasma creatinine levels, alongside more severe tubular damage evidenced by H&E staining. Cytokine array analysis showed chemokines such as MCP-1 and CXCL1 and macrophage marker CD68 were significantly increased; Western blot analysis showed cleaved caspase-3 levels were significantly increased, indicating enhanced apoptosis. Additionally, fibrosis markers TIMP-1, collagen-3, and α-SMA were significantly up-regulated at both mRNA and protein levels. We further observed increased hypoxia marker hypoxia-inducible factor-1α in MD-specific NOS1 knockout mice (Cre +/-). 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 MD 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.