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We have previously shown that podocyte injury increases the glomerular filtration of liver-derived angiotensinogen (Agt) and the generation of intrarenal angiotensin (Ang) II and that the filtered Agt is reabsorbed by proximal tubules in a manner dependent on megalin. In the present study, we aimed to study the role of megalin in the generation of renal Ang II and sodium handling during nephrotic syndrome. We generated proximal tubule-specific megalin knockout (KO) mice and crossed these animals with NEP25 mice, in which podocyte-specific injury can be induced by injection of the immunotoxin LMB2. Without podocyte injury, renal Agt staining was markedly diminished and urinary Agt increased in KO mice. However, renal Ang II was similar between KO and control mice on average: 117 (95% CI 101–134) vs. 101 (68–133) fmol/g tissue. We next tested the effect of megalin KO on intrarenal Ang II generation with podocyte injury. Control NEP25 mice showed markedly increased renal Agt staining and renal Ang II levels: 450 (336–565) fmol/g tissue. Megalin KO/NEP25 mice showed markedly diminished Agt reabsorption and attenuated renal Ang II: 199 (156–242) fmol/g tissue (p < 0.001). Compared with control NEP25 mice, megalin KO/NEP25 mice excreted 5-fold more sodium in the urine. Western blot analysis showed that megalin KO decreased NHE3 and the cleaved α and γ forms of ENaC. These data indicate that Agt reabsorbed by proximal tubules via megalin in nephrotic syndrome is converted to Ang II, which may contribute to sodium retention and edema formation by activating NHE3 and ENaC. SUMMARY: This study shows that in nephrotic syndrome, plasma angiotensinogen leaked into the renal tubular lumen is reabsorbed by proximal tubules via megalin and converted to angiotensin II, which may contribute to sodium retention and edema formation by activating NHE3 and ENaC.

Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-α and T53-phosphorylated Na + -Cl − cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na + -Cl − cotransporter phosphorylation. Accordingly, a Na + -Cl − cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-α and phosphorylated Na + -Cl − cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11β-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor–epithelial sodium channel–Na + -Cl − cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the phenotype of apparent mineralocorticoid excess.

Background: Renal mechanism of 11β-HSD2 deficiency for developing hypertension is to be evaluated because vascular mechanism associated with sympathetic nervous activity was prevailing in global Hsd11b2 knockout (KO) mice although brain-specific KO mice needed high salt intake to develop hypertension. We have demonstrated the importance of renal 11β-HSD2 deficiency on developing hypertension by using kidney-specific Hsd11b2 knockout ( Hsd11b2 Ksp-/- ; KS-KO) mice ( Hypertension , in press) and have continued the analysis of the systemic effect of renal 11β-HSD2 deficiency. Method: Blood pressure (BP) and heart rate (HR) was measured by using 24h telemetry. Amiloride (25 mg/L) and hydrochlorothiazide (HCTZ, 300 mg/L) were administered through drinking water. Pellet containing MR antagonist spironolactone (MRA; 50 mg/KgBW/day) was administered subcutaneously. Corticosterone concentration was determined by ELISA. Data are presented as mean±SE. Result: Systolic and diastolic BPs of KS-KO mice were significantly higher, although the HR was lower, than those of WT mice: SBP, 142.4±1.0 vs 122.4±0.8 mmHg; DBP, 103.9±0.8 vs 94.4±0.8 mmHg; HR, 492.5±2.7 vs 555.4±2.4 (n=7). Mean BP was decreased to the level of WT mice by reducing dietary sodium content from 0.3 % to 0.01 %. Plasma [K + ] was significantly lower in KS-KO mice: 2.9±0.2 vs 4.2±0.2 mEq/L (n=5). Renal membrane expressions of NCC, T53-phosphorylated NCC (pNCC), cleaved ENaCα and full-length ENaCα were upregulated in KS-KO mice. Correction of plasma [K + ] of KS-KO mice by using high KCl diet or amiloride downregulated the renal membrane expression of pNCC and decreased the MBP to the level of WT mice as well as chronic HCTZ-treated KS-KO mice. Subcutaneous administration of MRA decreased MBP of KS-KO mice and the renal membrane expressions of pNCC and cleaved ENaCα. Diurnal variation of plasma corticosterone concentration was diminished in KS-KO mice and the urinary excretion of corticosterone was higher compared to that in WT mice. Conclusion: Renal 11β-HSD2 deficiency is sufficient in developing hypertension via MR activation induced by excessive corticosterone, the systemic effect of which are also suggested.

Overactivation of the mineralocorticoid receptor signaling is implicated in cardiovascular disease, including hypertensive heart disease. Oxidative stress is suggested to augment mineralocorticoid receptor signal transduction, but the precise mechanisms remain unclear. Mineralocorticoid receptor activity is regulated by multiple factors, in addition to plasma ligand levels. We previously identified Rac1 GTPase as a modulator of mineralocorticoid receptor activity. Here we show that oxidative stress induces mineralocorticoid receptor activation in a ligand-independent, Rac1-depenent manner in cardiomyocytes. Oxidant stress was induced in rat cultured cardiomyocytes (H9c2) by l -buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO depleted intracellular glutathione and concomitantly increased reactive oxygen species (199%; P P N -acetylcysteine. The ligand independency of BSO action was indicated using a mutant mineralocorticoid receptor that does not bind ligands. With this mutant mineralocorticoid receptor, BSO-evoked mineralocorticoid receptor activation remained intact, whereas ligand-induced mineralocorticoid receptor activation was abolished. We next examined the involvement of Rac1. BSO increased active Rac1 in a redox-dependent fashion, and Rac inhibition suppressed the enhancing effect of BSO. Constitutively active Rac1, indeed, potentiated mineralocorticoid receptor transactivation. Furthermore, mineralocorticoid receptor transactivation by BSO was accompanied by enhanced nuclear accumulation of mineralocorticoid receptor. We conclude that alteration of redox state modulates mineralocorticoid receptor–dependent transcriptional activity via Rac1 in the heart. This redox-sensitive, ligand-independent mineralocorticoid receptor activation may contribute to the processes by which oxidant stress promotes cardiac injury.

A Rho family GTPase, Rac1, has emerged as an important molecule involved in cardiac remodeling. Some studies demonstrated the requirement of Rac1 in angiotensin II-induced cardiac hypertrophy and diabetic cardiomyopathy in association with generation of reactive oxygen species (ROS). However its role in pressure overload-induced cardiac remodeling is still unclear. On the other hand, we previously reported that Rac1 can activate mineralocorticoid receptor (MR) in cultured cardiomyocytes. Here we demonstrate the requirement of Rac1 in pressure-overload cardiac remodeling, and putative role of MR as a downstream pathway of Rac1. First, we performed sham or transverse aortic constriction (TAC) surgery in C57BL/6 mice, and examined the effect of Rac1 inhibitor ( NSC23766 ) and MR blocker (eplerenone). After 7 weeks, TAC caused severe hypertrophy and dysfunction of left ventricle with significant increase in active form of Rac1. In addition, the amount of MR protein in nuclear fraction, and the expression of some target genes of MR (including serpina3n and serpine-1) in left ventricle were also increased by TAC. NSC23766 significantly reduced the TAC-induced activation of Rac1, and both NSC23766 and eplerenone attenuated cardiac hypertrophy and dysfunction, along with inhibition of MR signaling. Furthermore, TAC significantly increased ROS production in left ventricle, which was also attenuated by both of the pharmacological interventions. We next generated cardiomyocyte-specific heterozygous Rac1-deficient mice (Rac1 CM +/− ) and littermate wild-type mice (WT), and performed Sham or TAC surgery. The TAC-induced hypertrophy and dysfunction of left ventricle were significantly suppressed in Rac1 CM +/− compared with WT (heart/body weight ratio: 6.4 ± 0.86 vs 10.8 ± 0.81 mg/g, ejection fraction 54.1 ± 6.5 vs 33.3 ± 7.3 %, p < 0.05), with inhibition of Rac1 activation. Nuclear translocation of MR protein and increases in expression of the target genes of MR were also significantly attenuated in Rac1 CM +/− compared with WT. These results indicate that Rac1 plays an essential role in the maladaptive cardiac hypertrophy induced by pressure overload, and that MR is an important downstream pathway of Rac1 in heart.