Macula Densa Intracellular Alkalinization Activates NOS1β but Suppresses NOS1α during Tubuloglomerular Feedback
Authors: Zhang Jie , Wei Jin , Chan Jenna , Yadav Nikita , Patel Kshama , Thalakola Anish , Wang Lei , Patel Trushar , Buggs Jacentha , Liu Ruisheng 
Published: May 2022

Increased NaCl at the macula densa, the same stimulus of the tubuloglomerular feedback (TGF), also induces intracellular alkalinization (pH: from 7.4 to 7.8) and enhances nitric oxide (NO) generation. Neuronal NO synthase (NOS1) is the primary isoform of NOS expressed in the macula densa and its activity is strongly pH‐dependent. Nevertheless, the impact of pH on the catalytic capacity of NOS1 remains controversial with inconsistent observations. A potential explanation for this discrepancy may be due to the presence of different splice variants of NOS1 (NOS1α and NOS1β). Therefore, we hypothesized that the macula densa intracellular alkalinization during TGF activates NOS1β but suppresses NOS1α, which modulates the NO production and thus the TGF responsiveness. To test this hypothesis, we measured the NOS activity of purified NOS1α and NOS1β proteins in various pH environments using the Griess method. The pH‐dependent NO production curves of NOS1α and NOS1β were different. The optimal pH was ~6.5 for NOS1α, but ~8.5 for NOS1β. The maximum activities of NOS1α and NOS1β were similar at their optimal pH of 6.5 and 8.5, respectively. Then, we measured and compared the renal cortical NOS1 expression, macula densa NO generation, TGF response in vitro and in vivo, renal vascular resistance (RVR), and mean arterial pressure (MAP) in the Sprague Dawley (SD) rats and the spontaneously hypertensive rats (SHRs). While the total NOS1 expression was not significantly different between SD rats and SHRs, the alternative splicing patterns were contradictory. The ratio of NOS1α/NOS1β was greater in SHRs than SD rats (2.94±0.48 vs 0.36±0.07; n=6, p<0.01 vs SD), which was associated with the reduced NO production (21.6±4.5 vs 38.3±5.6 units/min; n=8, p<0.01 vs SD), enhanced TGF in vitro (4.9±0.4 vs 3.5±0.5 µm; n=10, p<0.05 vs SD) and in vivo (12.8±2.1 vs 9.1±1.7 mmHg; n=9, p<0.01 vs SD), increased RVR (23.9±1.3 vs 17.8±1.5 mmHg.ml‐1.min‐1.g kidney wt‐1; n=6, p<0.05 vs SD), and elevated MAP (127.3±6.6 vs 101.7±4.2 mmHg; n=6, p<0.05 vs SD). We also measured and compared the NOS1 expression in the renal biopsy (cortex) tissues of Caucasians (CAs) and African Americans (AAs). There was no significant difference in the total NOS1 expression between CAs and AAs, whereas the ratio of NOS1α/NOS1β was greater in AAs than CAs (3.53±1.64 vs 0.43±0.31; n=10‐11, p<0.01 vs CAs), which was associated with the higher serum creatinine level (1.25±0.08 vs 1.03±0.12 mg/dl; n=10‐11, p<0.05 vs CAs). Lastly, we determined the effects of pH on the macula densa NO generation as well as the TGF responsiveness in SD rats and SHRs. When the pH of the tubular perfusate was switched from 7.4 to 8.0, the macula densa NO production increased by 29.5±6.9% (from 39.7±4.5 to 51.4±5.7 units/min) in SD rats, whereas it decreased by 26.2±4.3% (from 22.5±3.4 to 16.6±4.2 units/min) in SHRs (n=7, p<0.05 vs 7.4); the TGF response in vitro decreased by 24.3±5.4% (from 3.7±0.4 to 2.8±0.6 µm) in SD rats, while it increased by 19.1±3.9% (from 4.7±0.4 to 5.6±0.5 µm) in SHRs (n=7, p<0.05 vs 7.4). In conclusion, the present study demonstrates that the macula densa intracellular alkalinization during TGF activates NOS1β but suppresses NOS1α, which regulates the NO generation, the TGF response and potentially the renal hemodynamics.