MD-NOS1 KO
Description
Background: Acute kidney injury (AKI) causes rapid loss of renal function and leads to high morbidity and mortality. Our previous research has shown that neuronal nitric oxide synthase (NOS1) influences nitric oxide (NO)-mediated dilation of the afferent arteriole, thereby inhibiting tubuloglomerular feedback (TGF), which plays a critical role in glomerular filtration rate (GFR). Methods: We generated inducible macula densa–specific NOS1 knockout mice (NKCC2-Cre- NOS1 flox/flox) and introduced AKI by 18 min bilateral renal ischemia at 37 °C, followed by 48 h reperfusion. The kidney injury was assessed by measuring GFR, plasma creatinine, histology, cytokines, apoptosis, fibrotic factors, and proteomics. Results: Deletion of NOS1 was confirmed through immunofluorescence double staining of NKCC2 and NOS1. The results showed that crossing NKCC2 cre line with NOS1 flox line induces a complete deletion of NOS1 from the macula densa cells. In response to IR-AKI, compared with wild-type controls, NOS1 knockouts showed a dramatic GFR decline (236 ± 66 to 24 ± 22 µL/min) and elevated creatinine, alongside more severe tubular damages evidenced by H&E staining. Cytokine array analysis showed chemokines such as MCP-1, 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 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. Conclusions: These findings highlight a novel role of NOS1 in AKI pathophysiology and suggest targeting NOS1 could be a therapeutic strategy to mitigate AKI severity, identified Hp as a downstream plasma signal of NOS1-dependent AKI responses, suggesting potential translational value pending human validation.