Genetic disruption of Npr1 depletes regulatory T cells and provokes high levels of proinflammatory cytokines and fibrosis in the kidneys of female mutant mice

Abstract

The present study was designed to determine the effects of gene knockout of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) on immunogenic responses affecting kidney function and blood pressure (BP) in Npr1 (coding for GC-A/NPRA)-null mutant mice. We used female Npr1 gene-disrupted (Npr1^-/-, 0 copy), heterozygous (Npr1^+/-, 1 copy), wild-type (Npr1^+/+, 2 copy), and gene-duplicated (Npr1^++/++, 4 copy) mice. Expression levels of Toll-like receptor (TLR)2/TLR4 mRNA were increased 4- to 5-fold in 1-copy mice and 6- to 10-fold in 0-copy mice; protein levels were increased 2.5- to 3-fold in 1-copy mice and 4- to 5-fold in 0-copy mice. Expression of proinflammatory cytokines and BP was significantly elevated in 1-copy and 0-copy mice compared with 2-copy and 4-copy mice. In addition, 0-copy and 1-copy mice exhibited drastic reductions in regulatory T cells (Tregs). After rapamycin treatment, Tregs were increased by 17% (P < 0.001) in 0-copy mice and 8% (P < 0.001) in 1-copy mice. Renal mRNA and protein levels of TLR2 and TLR4 were decreased by 70% in 0-copy mice and 50% in 1-copy mice. There were significantly higher levels of Tregs and very low levels of TLR2/TLR4 expression in 4-copy mice (P < 0.001). These findings indicate that the disruption of Npr1 in female mice triggers renal immunogenic pathways, which transactivate the expression of proinflammatory cytokines and renal fibrosis with elevated BP in mutant animals. The data suggest that rapamycin treatment attenuates proinflammatory cytokine expression, dramatically increases anti-inflammatory cytokines, and substantially reduces BP and renal fibrosis in mutant animals.

Keywords: cytokines; fibrosis; gene targeting; guanylyl cyclase receptor; natriuretic peptides; rapamycin; regulatory T cells.

Fig. 1.

Assessment of regulatory T (Treg) cell, Foxp3 protein, and mRNA expressions in natriuretic peptide receptor-A (Npr1) gene-targeted mice with and without rapamycin (RA) treatment. A: bar graph showing a representative FACS analysis of Treg cell percentage in splenocytes, including Foxp3⁺, CD4⁺, and CD25⁺ expression in Npr1 gene-targeted null mutant (Npr1^−/−, 0-copy), heterozygous (Npr1^+/−, 1-copy), wild-type (Npr1^+/+, 2-copy), and gene-duplicated (Npr1^++/++, 4-copy) mice. B: renal levels of Foxp3 protein expression determined by Western blot analysis. β-Actin was used as an internal control for normalization. C and D: relative densitometry values from Western blot results in B (C) and Foxp3 mRNA expression (D) of a Foxp3-targeted gene normalized to β-actin mRNA in kidney tissues. Values are expressed as means ± SE; n = 6/group. *$#P < 0.05, control vs. rapamycin-treated Npr1^−/− mice, Npr1^+/− vs. Npr1^+/+ mice, and Npr1^+/+ vs. Npr1^++/++ mice. C, control; EP, enalapril.

Fig. 2.

Analysis of Toll-like receptors (TLRs) and mammalian target of rapamycin (mTOR) expression in natriuretic peptide receptor-A (Npr1) gene-targeted kidneys in untreated mice and mice treated with rapamycin. A–C: relative mRNA expression of TLR2-, TLR4-, and TLR6-targeted genes normalized to β-actin mRNA in kidney tissues. D: renal expression levels of TLR4 protein were determined by Western blot analysis. E: immunofluorescence of TLR4 in rapamycin-treated and vehicle-treated Npr1 gene-targeted mouse kidneys. A marked increase in TLR4 expression occurred in Npr1 gene knockout kidneys but not in the kidneys of wild-type controls. Rapamycin decreased expression of TLR4 in tubular epithelial cells of kidneys. Red fluorescence indicates TLR4 protein expression; blue fluorescence indicates a nucleus stained with DAPI. F: quantitative fluorescence intensity analysis of TLR4 protein expression was done by measuring intracellular fluorescence using MetaMorph software. G: mTOR protein expression was determined by Western blot analysis. β-Actin was used as an internal control for normalization. Densitometric analyses of the respective bands were done with an Alpha Innotech phosphoimager. Values are expressed as means ± SE; n = 6/group. *P < 0.05, control and Npr1^++/++ vs. rapamycin-treated Npr1^++/++ mice; **P < 0.01, control Npr1^+/+ and Npr1^++/++ vs. rapamycin-treated Npr1^+/+ and Npr1^++/++ mice; ***P < 0.001, control Npr1^+/− and Npr1^−/− vs. rapamycin-treated Npr1^+/−and Npr1^−/− mice.

Fig. 3.

Effect of rapamycin on proinflammatory and anti-inflammatory cytokine gene expression in the kidneys of natriuretic peptide receptor-A (Npr1) gene-targeted mice. A–E: relative mRNA expression of proinflammatory [T helper (Th)1] cytokine-targeted genes [IL-2, interferon (IFN)-γ, TNF-α, IL-17, and IL-17F]. F–I: relative mRNA expression of anti-inflammatory (Th2) cytokine-targeted genes (IL-4, IL-5, IL-10, and IL-13) normalized with β-actin mRNA in kidney tissues treated or not treated with rapamycin. Values are expressed as means ± SE; n = 6/group. *P < 0.05, control Npr1^++/++ vs. rapamycin-treated Npr1^++/++ mice; **P < 0.01, control Npr1^+/− and Npr1^+/+ mice vs. rapamycin-treated Npr1^+/− and Npr1^+/+ mice; ***P < 0.001, control Npr1^+/− and Npr1^−/− mice vs. rapamycin-treated Npr1^+/−and Npr1^−/− mice.

Fig. 4.

Renal protein levels of proinflammatory and anti-inflammatory cytokine and mammalian target of rapamycin (mTOR) proteins in natriuretic peptide receptor-A (Npr1) gene-targeted mice after treatment with rapamycin. A–E: Western blot analysis of proinflammatory cytokines (TNF-α, IL-1α, IL-1β, and IL-6) as well as anti-inflammatory cytokine IL-10 protein expression and densitometry analyses of these cytokines in the kidney tissues of untreated mice and mice treated with rapamycin (β-actin was used as loading control). Values are expressed as means ± SE; n = 6/group. *P < 0.05, control Npr1^+/+ vs. rapamycin-treated Npr1^+/+ mice; ***P < 0.001, control Npr1^+/− and Npr1^−/− mice vs. rapamycin-treated Npr1^+/−and Npr1^−/− mice.

Fig. 5.

Quantitative analysis of plasma proinflammatory and anti-inflammatory cytokines in natriuretic peptide receptor-A (Npr1) gene-targeted mice. A–F: plasma levels of T helper (Th)1 proinflammatory cytokines [IL-1β, IL-2, IL-6, interferon (IFN)-γ, TNF-α, and IL-17A]. G–I: plasma levels of Th2 anti-inflammatory cytokines (IL-5, IL-10, and IL-13). Values are expressed as means ± SE; n = 6/group. *P < 0.05, control Npr1^+/+ and Npr1^++/++ mice vs. rapamycin-treated Npr1^+/+ and Npr1^++/++ mice; **P < 0.01, control Npr1^+/− and Npr1^+/+ mice vs. rapamycin-treated Npr1^+/− and Npr1 ^−/− mice; ***P < 0.001, control Npr1^+/− and Npr1^−/− mice vs. rapamycin-treated Npr1^+/−and Npr1^−/− mice.

Fig. 6.

Quantitative analysis of renal proinflammatory and anti-inflammatory cytokines in the kidneys of natriuretic peptide receptor-A (Npr1) gene-targeted mice. A–F: concentrations of proinflammatory cytokines [IL-1β, IL-2, IL-6, interferon (IFN)-γ, TNF-α, and IL-17A]. G–I: concentrations of anti-inflammatory cytokines (IL-5, IL-10, and IL-13) in kidney tissues. Values are expressed as means ± SE; n = 6/group. *P < 0.05, control Npr1^+/− and Npr1^−/− mice vs. rapamycin-treated Npr1^+/− and Npr1^−/− mice; **P < 0.01, control Npr1^+/− and Npr1^+/+ mice vs. rapamycin-treated Npr1^+/− and Npr1^−/− mice; ***P < 0.001, control Npr1^+/− and Npr1^+/+ mice vs. rapamycin-treated Npr1^+/− and Npr1^−/− mice.

Fig. 7.

Comparative analysis of renal histology and mesangial matrix expansion in natriuretic peptide receptor-A (Npr1) gene-targeted mice. A: kidney sections stained with hematoxylin and eosin indicating matrix mesangial expansion (MME) of Npr1 gene-targeted kidneys of control and rapamycin-treated mice (shown by arrows). B: accumulation of collagen (renal fibrosis) in sections of Npr1 gene-targeted kidneys of control and experimental mice after staining with Masson’s trichrome (shown by arrows). C and D: quantitative analyses of experimental and control mice. n = 6/group. ***P < 0.01, control Npr1^+/− and Npr1^+/+ mice vs. rapamycin-treated Npr1^+/− and Npr1^−/− mice.

Fig. 8.

Proposed model depicting the effects of natriuretic peptide receptor-A (NPRA) ablation and signaling in renal remodeling, including the renoprotective role of rapamycin. Npr1 gene ablation leads to impaired renal function, which triggers structural and molecular changes in the kidneys of 0-copy and 1-copy mice. Activated innate or adaptive signals (e.g., NPRA disruption, gene toxicity, and Toll-like receptors) trigger a mammalian target of rapamycin (mTOR) signaling cascade. Activation of mTOR signaling can boost the propensity of cells to be skewed toward the T helper (Th)1 phenotype, which leads to the activation of transcription of proinflammatory (Th1) cytokines, increased blood presure, and infiltration of inflammatory cells. However, treatment with rapamycin inhibits mTOR activation and, hence, downregulates proinflammatory molecules and upregulates anti-inflammatory (Th2) molecules. The increased levels of regulatory T cells, downregulation of proinflammatory cytokines, and normalized blood pressure lead to homeostasis. ANP, atrial natriuretic peptide; Npr1, gene coding for natriuretic peptide receptor-A; GCD, guanylyl cyclase catalytic domain; DAMPs, damage-associated molecular patterns; MyD88, myeloid differentiation primary response 88; TRIF, TIR-domain-containing adapter-inducing interferon-β; mTORC1/C2, mTOR complex 1/complex 2.