Nos3 Gene Knockout in Mice Enhances Kidney Sensitivity to Furosemide

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Abstract

Nitric oxide (NO) regulates renal hemodynamics and inhibits tubular sodium reabsorption. NO formation is catalyzed by NO synthases (NOS), and it is important to study the role of individual NO synthases for renal functions. The purpose of the study was to obtain a line of mice with a knockout of the nos3 gene (NOS KO) and to evaluate their ion-regulatory renal function. A homozygous line of NOS KO mice was developed at the background of the C57Bl/6 line using the CRISPR-Cas9 editing method. The physiological study included 39 animals (10 female and 10 male C57Bl/6 mice; 10 female and 9 male NOS KO mice); genotyping was carried out using PCR and sequencing methods at the age of 4 weeks. To identify differences in the transport of sodium and potassium in the kidneys of NOS KO mice, experiments were conducted to assess ion excretion in animals when given water (control), with a NaCl load (7.7 μmol/g) and with the administration of a loop diuretic. In the control, no differences were detected in the excretion of sodium and potassium ions in NOS KO and C57Bl/6 mice. Under conditions of excess NaCl intake, no significant interstrain differences were also revealed: sodium and potassium excretion increased by 8.8 and 1.3 times in NOS KO mice and by 8.4 and 1.7 times in wild-type mice, respectively. The natriuretic effect of furosemide (5 μg/g) in NOS KO mice was greater than in C57Bl/6 mice. Urinary sodium excretion was 4.1 ± 0.3 µmol/g during 2 hours of observation in NOS KO and 2.7 ± 0.2 µmol/g in wild-type mice (p < 0.001). Thus, the work revealed for the first time an increase in sensitivity to furosemide in mice with a knockout of the nos3 gene, which may indicate the importance of the activity of endothelial NO synthase for the regulation of sodium transport in the thick ascending limb of the loop of Henle.

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About the authors

A. V. Kutina

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences; Saint Petersburg State University

Author for correspondence.
Email: kutina_anna@mail.ru
Russian Federation, Saint Petersburg; Saint Petersburg

G. V. Belyakov

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences; Saint Petersburg State University

Email: kutina_anna@mail.ru
Russian Federation, Saint Petersburg; Saint Petersburg

E. V. Balbotkina

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: kutina_anna@mail.ru
Russian Federation, Saint Petersburg

A. V. Chirinskaite

Saint Petersburg State University

Email: kutina_anna@mail.ru
Russian Federation, Saint Petersburg

J. V. Sopova

Saint Petersburg State University

Email: kutina_anna@mail.ru
Russian Federation, Saint Petersburg

E. I. Leonova

Saint Petersburg State University

Email: kutina_anna@mail.ru
Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. Schematic of the deletion in the nos3 gene. The intron sequence is underlined, the sequence deleted in NOS KO mice is highlighted in larger and bold font.

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3. Fig. 2. Somatometric indices of male (♂) and female (♀) NOS KO and wild-type (C57Bl/6) mice. (a) - Body weight of mice between 3 and 9 months of age. (b) - Body length of mice at 9 months of age. Significance of differences (p < 0.05): * - when comparing mice of two lines, # - when comparing females and males.

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4. Fig. 3. Excretion of sodium (a) and potassium (b) ions by kidneys over 2 h in male (♂) and female (♀) NOS KO and wild-type (C57Bl/6) mice after administration of 10 μL/g water. Significance of differences (p < 0.05): # - when comparing females and males.

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5. Fig. 4. Renal excretion of sodium (a) and potassium (b) ions over 2 h in male (♂) and female (♀) NOS KO and wild-type (C57Bl/6) mice after NaCl loading assay (7.7 μmol/g). Significance of differences (p < 0.05): # - when comparing females and males.

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6. Fig. 5. Renal excretion of sodium (a) and potassium (b) ions over 2 h in male (♂) and female (♀) NOS KO and wild-type (C57Bl/6) mice after injection of 5 μg/g furosemide.

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7. Fig. 6. Dynamics of renal sodium ion excretion in male (♂) and female (♀) NOS KO and wild-type (C57Bl/6) mice after injection of 5 μg/g furosemide. Significance of differences (p < 0.05): & - for intergroup comparisons at individual time points. Basal natriuresis (0 min) was estimated from urine samples collected in the morning in unexposed animals.

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