Genetic mutation of recombination activating gene 1 in Dahl salt-sensitive rats attenuates hypertension and renal damage

Abstract

Hypertension and renal damage in Dahl SS rats are associated with increased infiltrating immune cells in the kidney. To examine the role of infiltrating immune cells in this disease process, a zinc finger nuclease targeting bases 672-706 of recombination-activating gene 1 (Rag1) was injected into the pronucleus of Dahl SS (SS/JrHsdMcwi) strain embryos and implanted in pseudopregnant females. This strategy yielded a rat strain with a 13-base frame-shift mutation in the target region of Rag1 and a deletion of immunoreactive Rag1 protein in the thymus. Flow cytometry demonstrated that the Rag1-null mutant rats have a significant reduction in T and B lymphocytes in the circulation and spleen. Studies were performed on SS and Rag1-null rats fed a 4.0% NaCl diet for 3 wk. The infiltration of T cells into the kidney following high-salt intake was significantly blunted in the Rag1-null rats (1.7 ± 0.6 × 10(5) cells/kidney) compared with the Dahl SS (5.6 ± 0.9 × 10(5) cells/kidney). Accompanying the reduction in infiltration of immune cells in the kidney, mean arterial blood pressure and urinary albumin excretion rate were significantly lower in Rag1-null mutants (158 ± 3 mmHg and 60 ± 16 mg/day, respectively) than in SS rats (180 ± 11 mmHg and 251 ± 37 mg/day). Finally, a histological analysis revealed that the glomerular and tubular damage in the kidneys of the SS rats fed a high-salt diet was also attenuated in the Rag1 mutants. These studies demonstrate the importance of renal infiltration of immune cells in the pathogenesis of hypertension and renal damage in Dahl SS rats.

Fig. 1.

Identification and genotyping of the Rag1 mutant strains. A: surveyor nuclease assay revealed three mutant founders (*), of which pups #1 and #2 were analyzed and bred. WT, wild-type Dahl salt-sensitive rat. B: Sanger sequencing revealed a 13-bp deletion mutation (SS-Rag1^em1Mcwi) in pup #1 and a 17-bp deletion mutation (SSRag1^em2Mcwi) in pup #2. C: in subsequent generations, fluorescent genotyping could easily distinguish wild-type from heterozygous and homozygous pups for breeding and phenotyping. Shown are representative example GeneMapper 4.0 plots for SSRag1^em1Mcwi animal genotypes.

Fig. 2.

A: Western blot of Rag1 and β-actin protein in homogenates of thymus obtained from Dahl SS and Rag1 mutant rats. Thymus (B) and body weight (C) of 12-wk-old Dahl SS and Rag1 mutant rats. *P < 0.05.

Fig. 3.

Flow cytometric identification of T lymphocytes (CD3+) and B lymphocytes (CD45R+) in circulating mononuclear cells from a representative Dahl SS (A) and Rag1 rat (B) and quantification of CD3+ and CD45R+ cells in the blood of SS and Rag1 mutant rats (C). *P < 0.05 vs. Dahl SS.

Fig. 4.

Flow cytometric and immunohistochemical identification of T cells and B cells in the spleen of Dahl SS (A–C) and Rag1 mutant rats (D–F). Flow cytometry indicates a deficit in CD3+ (T cells) and CD45R+ (B cells) in the Rag1 spleen. Immunohistochemical staining with anti-CD43, a T-cell marker, illustrates a deficit of T cells surrounding the central arterioles (indicated with arrowheads) of the spleen of the Rag1 mutant rat (E) compared with the Dahl SS (B). Similarly, staining with anti-CD79, a B-cell marker, shows a reduction in B cells in the spleen of the Rag1 (F) compared with the Dahl SS (C).

Fig. 5.

Changes in mean arterial pressure and albumin excretion rate in Dahl SS and Rag1 mutant rats fed AIN-76A diet containing 0.4% NaCl followed by 21 days of 4.0% NaCl chow. *P < 0.05 vs. values obtained on the final day of 0.4% NaCl chow. †P < 0.05 vs. Dahl SS on the same day.

Fig. 6.

Light microscopy of trichome-stained sections of the kidney (A and D, 1× original magnification) and renal cortex (B and E; 40× magnification) of Dahl SS rats (A and B) or Rag1 mutant rats (D and E) fed 4.0% NaCl chow for 3 wk. The percentage of the renal outer medulla consisting of protein casts (C) and the calculated glomerular injury score (F) in the SS and Rag1 mutant rats are also plotted. *P < 0.05 vs. SS.

Fig. 7.

T-cell counts in the kidney (A) and immunohistochemical images of CD43+ T cells in the renal cortex (B and D) and the outer medulla (C and E) of Dahl SS (B and C) and Rag1 mutant rats (D and E). *P < 0.05 vs. SS.