Macula densa nitric oxide synthase 1β restoration by kidney alkalization enhances renal graft outcomes

Ischemia-reperfusion injury (IRI) remains a critical challenge to the survival of kidney transplantation (KTX) graft, with no effective prevention or treatment strategies currently available. Neuronal nitric oxide synthase 1β (NOS1β), the predominant splice variant of NOS1 and the main source of NO in the macula densa (MD), mediates tubuloglomerular feedback and regulates glomerular filtration rates. NOS1β activity in the MD is influenced by renal pH; however, the role of pH-dependent regulation of NOS1β in mitigating IRI and protecting transplanted kidney graft function remains unclear. To explore this, C57BL/6J mice were given oral sodium bicarbonate (NaHCO₃) or NaCl for 2 wk before KTX. Blood and urine pH, NOS1β expression, NO levels, and transplant outcomes were evaluated. MD-specific NOS1 knockout (MD-NOS1KO) mice were used to assess the direct role of NOS1β. NOS1β expression decreased by approximately 60% 3 days after KTX. MD-NOS1β deletion exacerbated graft injury. NOS1β activities showed a strong tubular pH dependence, with maximal activity near pH 8.0. Bicarbonate treatment increased NOS1β expression in the MD by 65% and significantly improved graft outcomes, lowering plasma creatinine by ∼30% relative to NaCl-treated group. These protective effects were absent in MD-NOS1KO mice. Proteomic analysis revealed 718 differentially expressed proteins, with several showing enrichment in NO signaling, tissue repair, and inflammatory response pathways. In summary, MD-NOS1β downregulation after transplantation contributes to graft injury. Raising renal pH with bicarbonate enhances NOS1β activity and protects graft function, suggesting a potential therapeutic strategy to reduce IRI in kidney transplants.NEW & NOTEWORTHY This study reveals that raising renal tubular pH with oral bicarbonate enhances macula densa-specific NOS1β activity, protecting against ischemia-reperfusion injury in kidney transplants. These benefits are lost in macula densa NOS1β knockout mice, confirming its key role in graft protection. The findings suggest that modulating renal pH is a promising, noninvasive strategy to improve transplant outcomes by targeting macula densa-NOS1β.