Candesartan upregulates angiotensin-converting enzyme 2 in kidneys of male animals by decreased ubiquitination.

Candesartan is a common angiotensin-II receptor-1 blocker used for patients with cardiovascular and renal diseases. Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of blood pressure (BP), and also a major receptor for coronaviruses. To determine whether and how candesartan upregulates ACE2, we examined BP and ACE2 in multi-organs from male and female C57BL/6J mice treated with candesartan (1 mg/kg, i.p.) for 7 days. Relative to the vehicle, candesartan lowered BP more in males than females; ACE2 protein abundances were increased in kidneys, not lungs, hearts, aorta, liver, spleen, brain, or serum, only from males. Ace2-mRNA was similar in kidneys. Candesartan also decreased BP in normal, hypertensive, and nephrotic male rats. The renal ACE2 was increased by the drug in normal and nephrotic male rats but not spontaneously hypertensive ones. In male mouse kidneys, ACE2 was distributed at sodium-hydrogen-exchanger-3 positive proximal-convoluted-tubules; ACE2-ubiquitination was decreased by candesartan, accompanied with increased ubiquitin-specific-protease-48 (USP48). In candesartan-treated mouse renal proximal-convoluted-tubule cells, ACE2 abundances and activities were increased while ACE2-ubiquitination and colocalization with lysosomal and proteosomal markers were decreased. The silence of USP48 by siRNA caused a reduction of ACE2 in the cells. Thus, the sex-differential ACE2 upregulation by candesartan in kidney from males may be due to the decreased ACE2-ubiquitination, associated with USP48, and consequent degradation in lysosomes and proteosomes. This is a novel mechanism and may shed light on candesartan-like-drug choice in men and women prone to coronavirus infections.

Up-Regulation of Glioma-Associated Oncogene Homolog 1 Expression by Serum Starvation Promotes Cell Survival in ER-Positive Breast Cancer Cells.

BACKGROUND/AIMS: Cancer cells are resistant to ischemia and starvation. Glioma-associated oncogene homolog 1 (Gli1) is a positive transcriptional activator of Hedgehog (Hh) pathway and plays an essential role in the development of cancers, including breast cancer. However, how Gli1 promotes cell survival remains elusive. The main purpose of this study is to investigate the pro-survival effect of Gli1 under serum starvation and its molecular mechanism in ER-positive breast cancer cells. METHODS: Gene expression was determined by quantitative real-time PCR (QRT-PCR) and Western blot. The survival of Gli1 stably transfected ER-positive breast cancer cell lines (Gli1-MCF-7 and Gli1-T47D cells) and their untransfected control cells was estimated by WST-8 assay. Microarray analysis was performed to screen downstream Hh/Gli1 target genes in Gli1-overexpressed MCF-7 cells. Transcriptional activities of NF-kappaB were measured by luciferase assays. ChIP analysis was performed to explore whether cIAP2 was a direct target gene of Gli1. RESULTS: Serum starvation significantly up-regulated the expression of Gli1 gene through activating PI3K/AKT pathway. Over-expression of Gli1 markedly promoted cell survival under serum starvation. Microarray analysis revealed that 338 genes were differentially expressed in Gli1-MCF-7 cells compared with those in the control cells. Among these genes, cellular inhibitor of apoptosis 2 (cIAP2), coding an anti-apoptosis and pro-survival protein, was significantly up-regulated not only by Hh/Gli1 pathway, but also by serum starvation. However, ChIP assay revealed no binding of Gli1 to cIAP2 promoter at the region of -1792 to -1568bp. Moreover, over-expression of Gli1 resulted in enhanced trans-activation of transcriptional factor NF-κB. Suppression of NF-κB signaling with NF-κB inhibitor Bay11-7082, significantly reduced the expression of cIAP2 and the cell survival under serum starvation. CONCLUSION: Serum starvation significantly up-regulated the expression of Gli1, which in turn increased its key target cIAP2 expression and enhanced NF-κB/cIAP2 pathway, resulting in promoting cell survival under serum starvation. These findings may provide new insights into the pro-survival mechanisms of Gli1 in breast cancer.

The salt sensitivity of Drd4-null mice is associated with the upregulations of sodium transporters in kidneys.

To explore the mechanism of the hypertension in dopamine receptor-4 (Drd4) null mice, we determined the salt sensitivity and renal sodium transport proteins in Drd4-/- and Drd4+/+ mice with varied salt diets. On normal NaCl diet (NS), mean arterial pressures (MAP, telemetry) were higher in Drd4-/- than Drd4+/+; Low NaCl diet (LS) tended to decrease MAP in both strains; high NaCl diet (HS) elevated MAP with sodium excretion decreased and pressure-natriuresis curve shifted to right in Drd4-/- relative to Drd4+/+ mice. Drd4-/- mice exhibited increased renal sodium-hydrogen exchanger 3 (NHE3), sodium-potassium-2-chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC), and outer medullary α-epithelial sodium channel (αENaC) on NS, decreased NKCC2, NCC, αENaC, and αNa+-K+-ATPase on LS, and increased αENaC on HS. NKCC2, NCC, αENaC, and αNa+-K+-ATPase in plasma membrane were greater in Drd4-/- than in Drd4+/+ mice with HS. D4R was expressed in proximal and distal convoluted tubules, thick ascending limbs, and outer medullary collecting ducts and colocalized with NKCC2 and NCC. The phosphorylation of NKCC2 was enhanced but ubiquitination was reduced in the KO mice. There were no differences between the mouse strains in serum aldosterone concentrations and urinary dopamine excretions despite their changes with diets. The mRNA expressions of renal NHE3, NKCC2, NCC, and αENaC on NS were not altered in Drd4-/- mice. Thus, increased protein expressions of NHE3, NKCC2, NCC and αENaC are associated with hypertension in Drd4-/- mice; increased plasma membrane protein expression of NKCC2, NCC, αENaC, and αNa+-K+-ATPase may mediate the salt sensitivity of Drd4-/- mice.

DOPAMINE D4 RECEPTOR DOWN-REGULATES RENAL SODIUM CHLORIDE COTRANSPORTER VIA UBIQUITINATION-ASSOCIATED LYSOSOME DEGRADATION.

BACKGROUND: The thiazide-sensitive sodium chloride cotransporter (NCC) is the major apical sodium transporter located in the mammalian renal distal convoluted tubule (DCT). The amount of sodium reabsorbed in the DCT through NCC plays an important role in the regulation of extracellular fluid volume and blood pressure. Dopamine and its receptors constitute a renal antihypertensive system in mammals. The disruption of Drd4 in mice causes kidney-related hypertension. However, the pathogenesis of D4R-deficiency associated hypertension is not well documented. METHOD: We assessed the effects of D4R on NCC protein abundances and activities of DCT in mice with renal or global Drd4-deficiencies and expressing human D4.7 variant and in cultured mouse DCT cells, and explored the molecular mechanism. RESULTS: NCC inhibitor hydrochlorothiazide enhanced the natriuresis in Drd4-/- mice. Renal NCC protein was greater while ubiquitination of NCC was less in Drd4-/- than Drd4+/+ mice. Silencing of D4R in cultured mouse DCT cells increased NCC protein but decreased NCC ubiquitination. D4R agonist had opposite effects that were blocked by the antagonist. In mouse kidneys and DCT cells D4R and NCC colocalized and co-immunoprecipitated. Moreover, D4R-agonist promoted the binding between the two proteins demonstrated by fluorescence resonance energy transfer. D4R agonism internalized NCC, decreased NCC in the plasma membrane, increased NCC in lysosomes and reduced NCC-dependent-intracellular-sodium transport. The lysosomal inhibitor chloroquine prevented the D4R-induced NCC-reduction. A shortened NCC half-life was suggested by its decay under cycloheximide-chase. Ubiquitin-specific-protease 48 (USP48, a deubiquitinating enzyme) was increased in the kidneys and cells with Drd4-deficiency while D4R stimulation decreased it in vitro and reduction of USP48 with siRNA decreased NCC expression. The mice carrying human D4.7 variant or with renal supcapsular-Drd4-siRNA-delivery developed hypertension with increased NCC. CONCLUSION: Our data demonstrates that D4R downregulates NCC by promoting USP48-associated deubiquitination and subsequent internalization, lysosome relocation and degradation.

Regulatory T Cells Accelerate the Repair Process of Renal Fibrosis by Regulating Mononuclear Macrophages.

BACKGROUND: We aimed to investigate the mechanisms of renal fibrosis and explore the effect of CD4+CD25+Foxp3+ regulatory T cells (Treg) on renal fibrosis after the obstruction was removed. METHODS: Fifty-five C57BL/6 mice were randomly divided into three groups: the unilateral ureteral obstruction (UUO) group, the relief for unilateral ureteral obstruction (RUUO) group, and the RUUO+Treg group. Renal fibrosis indexes of RUUO mice were evaluated using hematoxylin and eosin (HE) and, Masson staining and immunohistochemistry after CD4+CD25+Treg cells were injected into the tail vein at the moment of recanalization. We detected the levels of Treg, M1, and M2 markers by flow cytometry, and the levels of transforming growth factor (TGF)-ß1, interleukin (IL)-1ß, IL-6 and IL-10 using ELISA. RESULTS: The tubular necrosis score, AO value of α-SMA (smooth muscle actin), and collagen area on the 3rd and 14th days post RUUO were up-regulated compared with the 7th day post RUUO (P<0.05). After injection of Treg via tail vein, the tubular necrosis score, AO value of α-SMA, TGF-ß1 level, and collagen area in the RUUO+Treg group on the 14th day were down-regulated compared with the RUUO group (P<0.05). Moreover, Treg could transform M1 macrophages into M2 macrophages, manifesting as up-regulated expression of CD206 compared with the RUUO group (P<0.05). Treg could also down-regulate the secretion of IL-6 and IL-1ß while up-regulating the secretion of IL-10 in vitro compared with the M1 group (P<0.05). CONCLUSIONS: The kidney could deteriorate into a state of injury and fibrosis after the obstruction was removed, and Treg could effectively protect the kidney function.

RhoB regulates the function of macrophages in the hypoxia-induced inflammatory response.

Immune cells, particularly macrophages, play critical roles in the hypoxia-induced inflammatory response. The small GTPase RhoB is usually rapidly induced by a variety of stimuli and has been described as an important regulator of cytoskeletal organization and vesicle and membrane receptor trafficking. However, it is unknown whether RhoB is involved in the hypoxia-induced inflammatory response. Here, we investigated the effect of hypoxia on the expression of RhoB and the mechanism and significance of RhoB expression in macrophages. We found that hypoxia significantly upregulated the expression of RhoB in RAW264.7 cells, mouse peritoneal macrophages, and the spleen of rats. Hypoxia-induced expression of RhoB was significantly blocked by a specific inhibitor of hypoxia-inducible factor-1α (HIF-1α), c-Jun N-terminal kinase (JNK), or extracellular-signal regulated protein kinase (ERK), indicating that hypoxia-activated HIF-1α, JNK, and ERK are involved in the upregulation of RhoB by hypoxia. Knockdown of RhoB expression not only significantly suppressed basal production of interleukin-1 beta (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in normoxia but also more markedly decreased the hypoxia-stimulated production of these cytokines. Furthermore, we showed that RhoB increased nuclear factor-kappa B (NF-κB) activity, and the inhibition of NF-κB transcriptional activity significantly decreased the RhoB-increased mRNA levels of IL-1ß, IL-6, and TNF-α. Finally, we demonstrated that RhoB enhanced cell adhesion and inhibited cell migration in normoxia and hypoxia. Taken together, these results suggest that RhoB plays an important role in the hypoxia-induced activation of macrophages and the inflammatory response.Cellular & Molecular Immunology advance online publication, 21 September 2015; doi:10.1038/cmi.2015.78.