Abstract 462: Gene-targeting of Npr1 Reveals the Retinoic Acid-mediated Enhanced Expression of Guanylyl Cyclase-A/Natriuretic Peptide Receptor-A and Attenuation of Renal Fibrosis in Mutant Mice

Abstract

Mice lacking functional Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor-A, GC-A/NPRA) exhibit hypertension and kidney disease, however, the mechanisms regulating Npr1 expression are not well understood. The objective of the present study was to determine the effect of all- trans retinoic acid (ATRA) on Npr1 gene expression and reduction of renal fibrosis in gene-targeted mutant mice. Adult (24-week old) male Npr1 gene-disrupted heterozygous (1-copy; +/-), wild-type (2-copy; +/+), and gene-duplicated (3-copy; ++/+) mice were treated by injecting ATRA (0.5 mg/Kg/day) intraperitoneally for 2-weeks. After treatment with ATRA, the renal Npr1 mRNA levels was increased in 1-copy mice by 4.6-fold, (1.4 ± 0.05 vs. control, 0.3 ± 0.02; p < 0.05), 2-copy mice by 3-fold, (3.7 ± 0.2 vs. control 1.2 ± 0.1; p < 0.05), and 3-copy mice by 2-fold (3.2 ± 0.3 vs. control 1.6 ± 0.2; p < 0.05). The retinoic acid increased renal cGMP levels (pmol/mg protein) in 1-copy (28.9 ± 1.1 vs. control, 7.1 ± 0.4; p < 0.05), 2-copy (55.8 ± 1.9 vs. control 22.6 ± 1.8; p < 0.05), and 3-copy (85.8 ± 4.3 vs. control 47.8 ± 3.2; p < 0.05) mice. A significant decrease in systolic blood pressure (SBP) was observed in ATRA-treated 1-copy mice (114.6 ± 2.2, p < 0.01) vs. 1-copy control mice (128.2 ± 2.1). Higher tubulo-interstitial fibrosis present in 1-copy mice was attenuated after treatment with ATRA (35%, p < 0.05). Immunohistochemistry and Western blot analyses exhibited the reduction in renal expression of α-SMA (25%, p < 0.05 and 35%, p < 0.05, respectively) in ATRA-treated 1-copy mice compared with 1-copy control mice. Interestingly, ATRA significantly enhanced the levels of immunosuppressive cytokine IL-10 (pg/mg protein) in the kidneys of 1-copy (43.3 ± 3.1 vs. control 23.4 ± 1.8; p < 0.05), 2-copy (67.1 ± 5.5 vs. control 56.5 ± 3.7; p < 0.05), and 3-copy (102.7 ± 6.7 vs. control 81.4 ± 3.2; p < 0.05) mice. The results show that ATRA induces Npr1 gene expression and function, lowers SBP, and attenuates renal fibrosis in 1-copy Npr1 mice. The findings of this study indicate that ATRA can serve as a possible therapeutic agent in attenuation of renal fibrosis under reduced Npr1 gene copy number, which will have important implications in prevention of hypertension and renal remodeling process.