Rag1-null Dahl SS rats reveal that adaptive immune mechanisms exacerbate high protein-induced hypertension and renal injury

Abstract

The present study, performed in Dahl salt-sensitive (SS) and SS- Rag1^-/- rats lacking T and B lymphocytes, tested the hypothesis that immune cells amplify salt-sensitive hypertension and kidney damage in response to a high-protein diet. After being weaned, SS and SS- Rag1^-/- rats were placed on an isocaloric, 0.4% NaCl diet containing normal (18%) or high (30%) protein. At 9 wk of age, rats were switched to a 4.0% NaCl diet containing the same amount of dietary protein and maintained on the high-salt diet for 3 wk. After being fed the high-salt diet, SS rats fed high protein had amplified hypertension and albumin excretion (158.7 ± 2.6 mmHg and 140.8 ± 16.0 mg/day, respectively, means ± SE) compared with SS rats fed normal protein (139.4 ± 3.6 mmHg and 69.4 ± 11.3 mg/day). When compared with the SS rats, SS- Rag1^-/- rats fed high protein were protected from exacerbated hypertension and albuminuria (142.9 ± 5.8 mmHg and 66.2 ± 10.8 mg/day). After 3 wk of the high-salt diet, there was a corresponding increase in total leukocyte infiltration (CD45⁺) in the kidneys of both strains fed high-protein diet. The SS- Rag1^-/- rats fed high-protein diet had 74-86% fewer CD3⁺ T lymphocytes and CD45R⁺ B lymphocytes infiltrating the kidney versus SS rats, but there was no difference in the infiltration of CD11b/c⁺ monocytes and macrophages, suggesting that the protective effects observed in the SS- Rag1^-/- rats are specific to the reduction of lymphocytes. With the SS- Rag1^-/- rats utilized as a novel tool to explore the effects of lymphocyte deficiency, these results provide evidence that adaptive immune mechanisms contribute to the exacerbation of salt-induced hypertension and renal injury mediated by increased dietary protein intake.

Keywords: T lymphocytes; dietary protein; kidney disease; salt-sensitive hypertension.

Fig. 1.

A: body weight in Dahl salt-sensitive (SS) and SS-Rag1^−/− rats fed either the normal (18%) or high (30%)-protein chow. B: steady-state sodium excretion between SS and SS-Rag1^−/− rats fed either the normal (18%) or high (30%)-protein chow during low salt (0.4% NaCl) and throughout 3 wk of high salt (4.0% NaCl). Two-way repeated-measures ANOVA with Holm-Sidak post hoc test, **P < 0.001 vs. week 6. Here, 18% SS, n = 8; 30% SS, n = 7; 18% SS-Rag1^−/−, n = 8; 30% SS-Rag1^−/−, n = 5–7.

Fig. 2.

Mean arterial blood pressure (MAP, A), urinary protein excretion (B), and urinary albumin excretion (C) after 3 wk of 4.0% high-salt challenge in Dahl salt-sensitive (SS) and SS-Rag1^−/− rats fed either the normal (18%) or high (30%)-protein chow. Two-way ANOVA with Holm-Sidak post hoc test, *P < 0.01 and **P < 0.001 vs. 18% SS, #P < 0.01 and ##P < 0.001 vs. 30% SS. Here, 18% SS, n = 8; 30% SS, n = 7; 18% SS-Rag1^−/−, n = 8; 30% SS-Rag1^−/−, n = 5–7.

Fig. 3.

A: circulating immune cell profile after 3 wk of 4.0% high-salt challenge in Dahl salt-sensitive (SS) and SS-Rag1^−/− rats fed either the normal (18%) or high (30%)-protein chow. CD45, leukocytes; CD11b/c, monocytes and macrophages; CD3, T lymphocytes; CD45R, B lymphocytes. Two-way ANOVA with Holm-Sidak post hoc test, *P < 0.01 and **P < 0.001 vs. SS. Here, 18% SS, n = 8; 30% SS, n = 7; 18% SS-Rag1^−/−, n = 8; 30% SS-Rag1^−/−, n = 7. B: representative flow cytometric plots demonstrating T and B lymphocyte depletion in SS-Rag1^−/− rats.

Fig. 4.

A: renal infiltrating immune cell profile after 3 wk of 4.0% high-salt challenge in Dahl salt-sensitive (SS) and SS-Rag1^−/− rats fed either the normal (18%) or high (30%)-protein chow. CD45, leukocytes; CD11b/c, monocytes and macrophages; CD3, T lymphocytes; CD45R, B lymphocytes. Two-way ANOVA with Holm-Sidak post hoc test, *P < 0.01 and **P < 0.001 vs. SS, #P < 0.01 and ##P < 0.001 vs. 18% SS. Here, 18% SS, n = 8; 30% SS, n = 7; 18% SS-Rag1^−/−, n = 8; 30% SS-Rag1^−/−, n = 7. B: representative flow cytometric plots demonstrating T lymphocyte depletion in SS-Rag1^−/− rats.

Fig. 5.

A: representative light microscopy images of trichrome-stained kidneys after 3 wk of high-salt challenge in Dahl salt-sensitive (SS) and SS-Rag1^−/− rats fed either the normal (18%) or high (30%)-protein chow (×4), with the quantification of the percentage of outer medullary protein casts summarized in B. C: light microscopy images of representative glomeruli (×40) from kidneys obtained from SS and SS-Rag1^−/− rats after 4.0% NaCl normal protein or high-protein diet, with the percentage of damaged glomeruli summarized in D. Two-way ANOVA with Holm-Sidak post hoc test, **P < 0.001 vs. 18% SS, ##P < 0.001 vs. 30% SS. Here, 18% SS, n = 8; 30% SS, n = 7; 18% SS-Rag1^−/−, n = 8; 30% SS-Rag1^−/−, n = 6.

Fig. 6.

A: urinary albumin excretion after 3 wk of 4.0% high-salt challenge in Dahl salt-sensitive (SS) and SS-Cd247^−/− rats fed either the normal (18%) or high (30%)-protein chow. B: quantification of the percentage of outer medullary protein casts in SS and SS-Cd247^−/− rats after 3 wk of high salt. C: renal infiltrating immune cell profile after 3 wk of high-salt challenge in SS and SS-Cd247^−/− rats with representative flow cytometric plots demonstrating T lymphocyte depletion in SS-Cd247^−/− rats. CD45, leukocytes; CD11b/c, monocytes and macrophages; CD3, T lymphocytes; CD45R, B lymphocytes. Two-way ANOVA with Holm-Sidak post hoc test, *P < 0.01 and **P < 0.001 vs. 18% SS, ##P < 0.001 vs. 30% SS. Here, 18% SS, n = 5–6; 30% SS, n = 5; 18% SS-Cd247^−/−, n = 6; 30% SS-Cd247^−/−, n = 5.