Chronic treatment with an inhibitor of nitric oxide synthase reduces protein expression of tumor necrosis factor‐alpha receptor type 1 in renal cortical tissues in mice
Authors: Majid Dewan S. A. , Prieto Minolfa A. , Castillo Alexander A. 
Published: November 2023

Tumor necrosis factor‐alpha (TNF‐α) production, stimulated by chronic high salt (HS) intake, has been implicated in salt sensitive hypertension (SSH) though the mechanism is not yet clearly defined. TNF‐α exerts its biological responses via interaction with two cell surface receptors, TNF‐α receptor type 1 (TNFR1) and type 2 (TNFR2). While TNFR2 activation enhances renal injury, TNFR1 activation induces natriuretic response, suggesting a counter‐regulatory mechanism in SSH opposing salt retention. Earlier, we have demonstrated that the TNFR1 protein expression decreases but TNFR2 protein expression increases in renal cortical tissues during development of SSH induced by angiotensin II. We hypothesize that such differential protein expressions of TNFR1 and TNFR2 are also involved in the development of SSH induced by nitric oxide (NO) deficiency. To examine this, experiments were performed in C57BL6 strain of mice (n=7 in each group) chronically treated with or without NO synthase inhibitor, nitro‐L‐ arginine methyl ester (L‐NAME; 0.05 mg/min/gm of body weight by osmotic mini‐pump) which were fed either normal (NS; 0.4% NaCl) or HS (4% NaCl) containing diet for 6 weeks. Systemic blood pressure (SBP) was measured using tail‐cuff plethysmography and 24 hour urine collections were made using metabolic cages at the start of the experiment and at the end of every week during the experimental period. At the end of the treatment period, the mice were sacrificed and the kidneys were isolated and processed for tissue analysis. Immuno‐ histochemical analysis of TNFR1 and TNFR2 protein expressions using appropriate antibodies in renal cortical tissues was performed. The staining area as well as the intensity of TNFR1 and TNFR2 immunoreactivity in renal cortical sections was analyzed using NIS Elements Software AR (version 3.0 for Windows; Nikon), which allowed a computerized determination of the area of positive staining (μm) and the intensity of immunoreactivity (density of positive staining in an analyzed area). As usual, HS intake alone did not alter mean SBP (NS, 76 ±2.1 vs HS, 78 ±1.4 mmHg) but it caused an exaggeration of L‐NAME induced increases in mean SBP (NS, 95 ±3.9 vs HS, 104 ±2.8 mmHg; P=0.06) at the end of the 6 week treatment period. The urinary excretion rate of TNF‐α was undetected in mice at the start of the experimental period but it was increased (31±7 pg/day; P<0.05) in L‐NAME treated mice fed with HS diet for 6 weeks. As expressed in percent area of positive staining, it was observed that TNFR1 immunoreactivity is lower in L‐NAME treated mice compared to untreated mice both in NS (1.0±0.1 % vs 2.7±0.6%; P<0.05) and HS (1.4±0.2% vs 4.1±0.5%; P<0.01) intake conditions. The intensity of TNFR1 immunoreactivity also showed the similar results. TNFR2 immunoreactivity was minimal in untreated as well as in L‐NAME treated mice. Collectively, these data indicate that NO synthase inhibition downregulates TNFR1 activity in the kidney. Such decrease in TNFR1 activity minimizes TNF‐α induced natriuretic response leading to further increase in salt retention and thus, causes HS induced exaggerated hypertensive response in NO deficient condition.