Genetic Abrogation of Adenosine A3 Receptor Prevents Uninephrectomy and High Salt-Induced Hypertension

Abstract

Background: Early-life reduction in nephron number (uninephrectomy [UNX]) and chronic high salt (HS) intake increase the risk of hypertension and chronic kidney disease. Adenosine signaling via its different receptors has been implicated in modulating renal, cardiovascular, and metabolic functions as well as inflammatory processes; however, the specific role of the A3 receptor in cardiovascular diseases is not clear. In this study, gene-modified mice were used to investigate the hypothesis that lack of A3 signaling prevents the development of hypertension and attenuates renal and cardiovascular injuries following UNX in combination with HS (UNX-HS) in mice.

Methods and results: Wild-type (A3 (+/+)) mice subjected to UNX-HS developed hypertension compared with controls (mean arterial pressure 106±3 versus 82±3 mm Hg; P<0.05) and displayed an impaired metabolic phenotype (eg, increased adiposity, reduced glucose tolerance, hyperinsulinemia). These changes were associated with both cardiac hypertrophy and fibrosis together with renal injuries and proteinuria. All of these pathological hallmarks were significantly attenuated in the A3 (-/-) mice. Mechanistically, absence of A3 receptors protected from UNX-HS-associated increase in renal NADPH oxidase activity and Nox2 expression. In addition, circulating cytokines including interleukins 1β, 6, 12, and 10 were increased in A3 (+/+) following UNX-HS, but these cytokines were already elevated in naïve A3 (-/-) mice and did not change following UNX-HS.

Conclusions: Reduction in nephron number combined with chronic HS intake is associated with oxidative stress, chronic inflammation, and development of hypertension in mice. Absence of adenosine A3 receptor signaling was strongly protective in this novel mouse model of renal and cardiovascular disease.

Keywords: NADPH oxidase; adenosine receptor; cardiovascular disease; high salt diet; inflammation; kidney; nephron number; uninephrectomy.

Figure 1

Blood pressure and heart histology. A, The protocol and timeline of experiments. UNX in combination with HS (UNX‐HS) significantly increased the systolic and diastolic arterial pressure in A₃ ^+/+ but not in A₃ ^−/− mice (B and C). 2×2 ANOVA revealed significant (P<0.0001) interaction (ie, effects of genotype on the response to UNX‐HS). UNX‐HS also increased the heart/body weight ratio in A₃ ^+/+ mice; the A₃ ^−/− mice had a higher baseline heart/body weight ratio compared with A₃ ^+/+ mice but were not affected by UNX‐HS (D). Representative pictures of hematoxylin and eosin staining in the heart are shown in (E). The A₃ ^+/+ mice showed significantly worse pathological changes in the heart following UNX‐HS, which was evaluated as fibrosis and myocyte histological scores (F). The thickness of the intraventricular septum was significantly increased in the A₃ ^+/+ mice after UNX‐HS, but there were no changes in the A₃ ^−/− group (G). Data are shown as mean±SEM, except in (F), in which results are presented as median and interquartile range. *P<0.05, n=8 to 12 per group. DEXA indicates dual‐emission x‐ray absorptiometry; GFR, glomerular filtration rate; HS, high salt; IPGTT, intraperitoneal glucose tolerance test; PND, postnatal day; RPF, renal plasma flow; UNX, uninephrectomy.

Figure 2

Renal function and histology. UNX in combination with HS (UNX‐HS) caused significant reduction of GFR in both genotypes (A), but RPF was reduced only in A₃ ^+/+ mice (B). 2×2 ANOVA did not reveal any interaction (ie, effects of genotype on the response to UNX‐HS) for GFR, but significant (P<0.0001) interaction was found for RPF. Plasma creatinine and proteinuria were significantly increased after UNX‐HS in A₃ ^+/+ mice but were not changed in A₃ ^−/− mice (C and D). Urinary protein excretion after UNX‐HS was also significantly lower in A₃ ^−/− mice compared with A₃ ^+/+ mice (D). UNX‐HS induced multiple renal histopathological changes in both genotypes, but these were more pronounced in the A₃ ^+/+ mice after UNX‐HS compared with A₃ ^−/− mice (G). Representative pictures of hematoxylin and eosin staining in the kidney are shown in (E). Kidney weight are similar between genotypes both at baseline and after UNX‐HS (F). Data are shown as mean±SEM, except in (G), in which results are presented as median and interquartile range. *P<0.05, n=8 to 12 per group. GFR indicates glomerular filtration rate; HS, high salt; RPF, renal plasma flow; UNX, uninephrectomy.

Figure 3

Nox activity and expression in kidney. Nox‐derived ${\mathrm{O}}_2^{-}$ generation and Nox2 protein level were significantly increased in the renal cortex in A₃ ^+/+ mice after UNX in combination with HS (UNX‐HS), but none of these were changed following UNX‐HS in the A₃ ^−/− mice (A–C). UNX‐HS associated with increased mRNA expressions of Nox2, whereas Nox1 and Nox4 were not significantly changed in A₃ ^+/+ mice (D–F). p22phox and p47phox expression were increased in A₃ ^+/+ mice but not in the A₃ ^−/− mice following UNX‐HS (G and H). No significant changes in p67phox were observed, although expression trended higher in A₃ ^+/+ compared with A₃ ^−/− mice after UNX‐HS (I). Data are shown as mean±SEM, *P<0.05, n=8 to 12 per group. CLU indicates chemiluminescence unit; HS, high salt; Nox, NADPH oxidase; UNX, uninephrectomy.

Figure 4

Plasma markers of oxidative stress and nitric oxide synthase activity. Urinary excretion of the oxidative stress markers 8‐OH‐dG (A), 8,12‐iso‐iPF2α‐VI (B), and its metabolite 2,3‐dinor‐8‐iso iPF2α (C) were increased in A₃ ^+/+ mice after UNX in combination with HS (UNX‐HS) but were not significantly changed in the A₃ ^−/− mice. UNX‐HS caused significant increase of plasma citrulline/arginine ratio in A₃ ^−/− mice (D) and significant reduction of plasma ornithine/arginine ratio in A₃ ^+/+ mice only (E). Baseline levels of both citrulline/arginine and ornithine/arginine ratios were lower in the A₃ ^−/− mice compared with A₃ ^+/+ mice. A₃ ^−/− mice had significantly lower plasma ADMA and SDMA levels (F and G). Although UNX‐HS did not influence circulating ADMA and SDMA levels, the SDMA level after UNX‐HS was significantly lower in A₃ ^−/− compared with A₃ ^+/+ mice. Data are shown as mean±SEM, *P<0.05, n=8 to 12 per group. ADMA indicates asymmetric dimethylarginine; HS, high salt; SDMA, symmetric dimethylarginine; UNX, uninephrectomy.

Figure 5

Glucose handling, fat content, and metabolic markers in plasma. UNX in combination with HS (UNX‐HS) significantly impaired glucose clearance ability in the A₃ ^+/+ mice but not in the A₃ ^−/− mice (A and B). 2×2 ANOVA revealed significant (P<0.05) interaction (ie, effects of genotype on the response to UNX‐HS) for the glucose tolerance responses. Although fat content increased in both genotypes after UNX‐HS, the A₃ ^−/− mice showed significantly less total body fat content and abdominal fat content at both baseline and after UNX‐HS compared with A₃ ^+/+ mice (C and D). GLP‐1, insulin, and leptin levels in the A₃ ^+/+ mice were significantly increased after UNX‐HS, but the levels of these metabolic hormones were not changed with UNX‐HS in the A₃ ^−/− mice (E–G). Insulin and leptin were also significantly lower in the A₃ ^−/− UNX‐HS mice compared with A₃ ^+/+ UNX‐HS mice (F and G). No significant differences in circulating glucagon were observed (H). Adiponectin levels trended toward being reduced in A₃ ^+/+ mice but increased in A₃ ^−/− mice after UNX‐HS (I). Data are shown as mean±SEM, *P<0.05, n=8 to 12 per group (A–D), n=8 to 10 per group (E–I). AUC indicates area under the curve; GLP‐1, glucagon‐like peptide 1; HS indicates high salt; IPGTT, intraperitoneal glucose tolerance test; UNX, uninephrectomy.

Figure 6

Systemic cytokines. Plasma IL‐1β, IL‐6, IL‐12, and IL‐10 levels were all significantly increased after UNX in combination with HS (UNX‐HS) in the A₃ ^+/+ mice (A–D). UNX‐HS did not affect plasma cytokines levels in A₃ ^−/− mice; however, A₃ ^−/− mice had significantly higher baseline IL‐1β, IL‐6, and IL‐10 compared with A₃ ^+/+ mice. Data are shown as mean±SEM, *P<0.05, n=8 to 12 per group. HS indicates high salt; IL, interleukin; UNX, uninephrectomy.

Figure 7

Antigen‐presenting cells in the kidney. A, The gating of the cell population. In kidneys from A₃ ^−/− mice, there was significant less proportion of major histocompatibility complex class II (H2iab⁺) cells in both CD11b⁺ macrophages and CD11c⁺ dendritic cells compared with those from A₃ ^+/+ mice (B). Data are shown as mean±SEM, *P<0.05, n=9 per group. HS indicates high salt; UNX, uninephrectomy.

Figure 8

Bone marrow‐derived macrophages. NADPH oxidase‐derived ${\mathrm{O}}_2^{-}$ production was significantly increased in the BMDMs following 24 hours of LPS stimulation (10 ng/mL), with this increase being greater in A₃ ^−/− BMDMs (A and B). As indicated by MFI, both M1 markers (PD‐L1 and CD86) and M2 marker (PD‐L2) were significantly increased after LPS stimulation in both genotypes (C). Moreover, cells from A₃ ^−/− expressed higher surface markers after LPS compared with A₃ ^+/+. No differences of CD206 level were observed between groups. LPS significantly increased Nox2, p22phox, and p67phox protein levels in A₃ ^−/− BMDMs but not in the A₃ ^+/+ cells (D and E). The p47phox protein was increased in both genotypes following LPS stimulation; however, after LPS stimulation, the Nox2, p22phox, and p47phox levels were significantly higher in cells from A₃ ^−/− mice than from A₃ ^+/+ mice. The IL‐1β, IL‐12, KC/GRO, and IL‐10 levels in the cell culture medium were significantly increased after LPS stimulation (24 hours) in both genotypes, but the cytokine levels of IL‐1β, IL‐12, and KC/GRO were significantly higher in BMDMs from A₃ ^−/− compared with A₃ ^+/+ mice (F–I). Nitrite levels in the culture medium were significantly increased in both genotypes following LPS stimulation (J); however, nitrite was significantly lower in the A₃ ^−/− group compared with the A₃ ^+/+ group. In agreement with the nitrite levels, both mRNA (K) and protein levels (L and M) of iNOS showed similar patterns. Data are shown as mean±SEM, *P<0.05, n=6 per group (A and B), n=4 to 5 per group (C–J), n=3 to 6 per group (K–M). BMDM indicates bone marrow‐derived macrophage; CLU, chemiluminescence unit; IL, interleukin; iNOS, inducible nitric oxide synthase; KC/GRO, keratinocyte chemoattractant/human growth‐regulated oncogene; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; Nox2, NADPH oxidase 2; PD‐L, programmed death ligand.

Figure 9

Proposed mechanisms contributing to a protective renal and cardiovascular phenotype in adenosine A₃ knockout mice following reduction in nephron number at early age and chronic feeding with high salt diet. Early age reduction of nephron number (UNX) combined with an HS diet (UNX‐HS) induces hypertension, which is associated with increased renal oxidative stress and inflammation in wild‐type mice. Our study demonstrates that gene‐modified mice lacking the adenosine A₃ receptor are protected from the development of hypertension and organ injuries in this chronic model of renal and cardiovascular disease. Mechanistically, we proposed a novel role of the A₃ receptors in modulating immune system development and homeostasis, which affects the ability to fight chronic inflammation and oxidative stress during conditions of renal and cardiovascular disease. Red lines denote inhibition or negative feedback, whereas the green arrows represent stimulation of a particular response. The dashed green arrows indicate a vicious cycle once hypertension is established, contributing to oxidative stress and progressive inflammation as well as accelerated renal and cardiac injuries following UNX‐HS. HS indicates high salt; UNX, uninephrectomy.