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.

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.

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.

GYY4137, a H2S donor, ameliorates kidney injuries in diabetic mice by modifying renal ROS-associated enzymes.

Diabetic nephropathy (DN) is a common microvascular complication of both type 1 and type 2 diabetes mellitus and often advances to end-stage renal disease. Oxidative stress plays an important role in the pathogenesis and progress of DN. Hydrogen sulfide (H2S) is considered as a promising candidate for the management of DN. But the antioxidant effects of H2S in DN have not been fully studied. In mouse model induced by high-fat diet and streptozotocin, GYY4137, a H2S donor, ameliorated albuminuria at weeks 6 & 8 and decreased serum creatinine at week 8, but not hyperglycemia. Renal nitrotyrosine and urinary 8-isoprostane were reduced along with the suppressed levels of renal laminin and kidney-injury-molecule 1. Renal NADPH oxidase (NOX) 2 was lower but heme oxygenase (HO) 2, paraoxonase (PON) 1, PON2 were higher in DN+GYY than DN group. NOX1, NOX4, HO1, superoxide dismutases 1-3 were similar between groups. Except for a rise at HO2, all the affected enzymes were unchanged in mRNA levels. The affected reactive-oxygen-species (ROS) enzymes were mainly located in the renal sodium-hydrogen-exchanger positive proximal tubules with similar distribution but changed immunofluorence in GYY4137 treated DN mice. Kidney morphological alterations in DN mice under light and electrical-microscopes were also improved by GYY4137. Thus, exogenous H2S administration may improve the renal oxidative damage in DN by reducing ROS production and enhancing ROS cleavage in kidney via the affected enzymes. This study may shed a light on therapeutic applications in diabetic nephropathy with H2S donors in the future.

Renal CD81 interacts with sodium potassium 2 chloride cotransporter and sodium chloride cotransporter in rats with lipopolysaccharide-induced preeclampsia.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

A low-salt diet with candesartan administration is associated with acute kidney injury in nephritis by increasing nitric oxide.

A low-salt diet may activate the renin-angiotensin-aldosterone system (RAAS) and is often applied simultaneously with RAAS inhibitors, especially for treatment of proteinuric nephritis. To explore the effect of a low-salt diet combined with angiotensin receptor blockers (ARB) on kidney function, the proteinuric nephritis model was induced by single intravenous injection of doxorubicin, and then the SD rats were administrated with candesartan intraperitoneal injection and fed with different salt diets. Rats with low-salt plus candesartan, not either alone, experienced acute kidney injury (AKI) at day 7 and could not self-restore when extending the experiment time from 7 days to 21 days, unless switching low-salt to normal-salt. Among three nitric oxide synthetases (NOS), endothelial NOS (eNOS) was obviously elevated and PI3K-Akt-eNOS signal pathway was activated. NG-Nitro-L-Arginine Methyl Ester (L-NAME), an eNOS inhibitor, reversed the decreased blood pressure and recovered the kidney dysfunction induced by low-salt with candesartan. The increased TUNEL-positive cells, Bax/Bcl-2 and cleaved-caspase3 protein abundance was ameliorated by L-NAME in vivo. In vitro, sodium nitroprusside, a nitric oxide donor, can also increase Bax/Bcl-2 and cleaved-caspase3 protein level in HK-2 cell. Thus, low-salt diet combined with candesartan in nephritis rats led to AKI, and the mechanism involved the increase of eNOS/NO, which linked to the decrease of blood pressure and the increase of apoptosis. This study provides practical guidance for salt intake in cases of RAS inhibitor usage clinically.

Long non-coding RNA PCDRlnc1 confers docetaxel resistance in prostate cancer by promoting autophagy.

Docetaxel resistance seriously affects its clinical application in prostate cancer (PCa). Long noncoding RNAs (lncRNAs) influence the chemosensitivity of various cancers. However, the potential involvement of lncRNAs in docetaxel sensitivity remains largely unknown in PCa. In the present study, we used RNA sequencing to compare the expression profiles of lncRNAs in docetaxel-resistant PCa cells and their parental cells and identified a novel lncRNA, ENSG00000234147, termed as PCa docetaxel resistance-associated lncRNA1 (PCDRlnc1). Our results indicated that PCDRlnc1 is closely associated with docetaxel resistance in PCa, and PCDRlnc1 knockout markedly sensitized the resistant cells to docetaxel in vitro and in vivo. In addition, PCDRlnc1 inhibition markedly suppressed docetaxel-induced autophagy. Conversely, PCDRlnc1 overexpression promoted autophagy. Mechanistically, PCDRlnc1 interacted with UHRF1 (ubiquitin-like with plant homeodomain and ring finger domains 1) and promoted its transcription level in PCa cells, leading to the activation of autophagic Beclin-1 signaling. Together, our data demonstrate that PCDRlnc1 is a novel key regulator of PCa docetaxel resistance, suggesting that it may be used as a potential biomarker of docetaxel resistance and therapeutic target in PCa.

Glucose-induced decrease of cystathionine ß-synthase mediates renal injuries.

Exogenous hydrogen sulfide (H2 S) protects kidneys from diabetic injuries in animal models. In order to explore the role of endogenous H2 S in diabetic nephropathy, we determined the renal H2S producing enzymes in vivo and in vitro. In diabetic mice, H2 S levels in blood and kidney were decreased while cystathionine ß-synthase (CBS), mainly located in mouse renal proximal convoluted tubules (PCT), was reduced selectively. In cultured mouse PCT cells treated with high glucose, CBS protein and activity was reduced while ubiquitinated CBS was increased, which was abolished by a proteasome inhibitor MG132 at 1 hour; high glucose drove CBS colocalized with proteasome 26S subunit ATPase6, indicating an involvement of ubiquitination proteasome degradation. At 48 hours, high glucose also selectively decreased CBS protein, concentration-dependently, but increased the ubiquitination of CBS; silence of CBS by siRNA increased nitrotyrosine, a marker for protein oxidative injury. Nitrotyrosine was also increased by high glucose treatments. The increases of nitrotyrosine either by cbs-siRNA or by glucose were restored by GYY4137, indicating that the H2 S donor may protect kidney from oxidative injury induced by CBS deficiency. In diabetic kidneys, ubiquitinated CBS and nitrotyrosine were increased but restored by GYY4137. The treatment also ameliorated albuminuria and renal morphologic changes in diabetic mice. Our findings suggest that high glucose induces reduction of renal CBS protein and activity in vivo and in vitro that is critical to the pathogenesis of diabetic kidney disease.

FOXM1 modulates docetaxel resistance in prostate cancer by regulating KIF20A.

BACKGROUND: Docetaxel resistance affects prognosis in advanced prostate cancer (PCa). The precise mechanisms remain unclear. Transcription factor Forkhead box M1 (FOXM1), which participates in cell proliferation and cell cycle progression, has been reported to affect the sensitivity of chemotherapy. This study explores the role of FOXM1 in PCa docetaxel resistance and its association with kinesin family member 20 A (KIF20A), which is known to promote therapeutic resistance in some cancers. METHODS: We monitored cell growth using MTT and colony formation assays, and cell apoptosis and cell cycle progression using flow cytometry. Wound-healing and transwell assays were used to detect cell invasion and migration. mRNA and protein expression were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. We monitored FOXM1 binding to the KIF20A promoter using a ChIP assay. Tumorigenicity in nude mice was used to assess in vivo tumorigenicity. RESULTS: FOXM1 knockdown induced cell apoptosis and G2/M cell cycle arrest, suppressing cell migration and invasion in docetaxel-resistant PCa cell lines (DU145-DR and VCaP-DR). Exogenous FOXM1 overexpression was found in their parental cells. Specific FOXM1 inhibitor thiostrepton significantly weakened docetaxel resistance in vitro and in vivo. We also found that FOXM1 and KIF20A exhibited consistent and highly correlated overexpression in PCa cells and tissues. FOXM1 also regulated KIF20A expression at the transcriptional level by acting directly on a Forkhead response element (FHRE) in its promoter. KIF20A overexpression could partially reverse the effect on cell proliferation, cell cycle proteins (cyclinA2, cyclinD1 and cyclinE1) and apoptosis protein (bcl-2 and PARP) of FOXM1 depletion. CONCLUSIONS: Our findings indicate that highly expressed FOXM1 may help promote docetaxel resistance by inducing KIF20A expression, providing insight into novel chemotherapeutic strategies for combatting PCa docetaxel resistance.

FOXM1 contributes to docetaxel resistance in castration-resistant prostate cancer by inducing AMPK/mTOR-mediated autophagy.

Docetaxel-mediated chemotherapy is the first line therapy for metastatic castration-resistant prostate cancer (CRPC) patients, but its therapeutic benefit is limited by the development of resistance. Although Forkhead box protein M1 (FOXM1) has been implicated in prostate tumorigenesis and metastasis, its role in docetaxel resistance has not been studied. Here, we showed that FOXM1 expression was upregulated in the docetaxel resistant CRPC cell lines (PC3-DR and VCaP-DR) and knockdown of FOXM1 sensitized the cells to docetaxel both in vitro and in vivo. In addition, autophagy was found to be significantly enhanced in resistant cells. Moreover, FOXM1 overexpression cells showed increased autophagic flux and higher numbers of autophagosomes. Knockdown of ATG7, beclin-1 or cotreatment with chloroquine, partly restored sensitivity to docetaxel in the FOXM1-overexpressing cells. Mechanistically, FOXM1 targeted AMPK/mTOR to activate the autophagy pathway and altered docetaxel response in CRPC. These findings identify the role of FOXM1 as well as the mechanism underlying FOXM1 action in docetaxel sensitivity and may, therefore, aid in design of CRPC therapies.

Effect of miR-132 on bupivacaine-induced neurotoxicity in human neuroblastoma cell line.

BACKGROUND: Local anesthetics (LAs) may generate neurotoxicity in neurons. In the current study, we explored the mechanisms by which microRNA-132 (miR-132) regulated the neurotoxicity of human neuroblastoma cells (SH-SY5Y) induced by bupivacaine (BUP). METHODS: CCK-8, flow cytometry, EdU detection, qRT-PCR and western blotting were used to explore the cell viability, apoptosis and gene expression, respectively. RESULTS: In this study, we found that 600 µM BUP dramatically inhibited SH-SY5Y cells viability. In addition, BUP induced cell apoptosis and neurotoxicity via increasing active caspase-3 and cleaved PARP1 levels. More importantly, the level of miR-132 was significantly up-regulated in BUP-treated cells, which was significantly reversed by miR-132 inhibitor. In addition, dual-luciferase assay indicated IGF1R was the directly binding target of miR-132 in cells. Our study further indicated that the level of IGF1R was markedly decreased by BUP interference, while miR-132 inhibitor exerted the opposite effect. Furthermore, BUP induced apoptosis and neurotoxicity in SH-SY5Y cells were attenuated by IGF1, which further confirmed IGF1R was the downstream target of BUP in SH-SY5Y cells. CONCLUSION: In the present study, miR-132 played important roles in regulating BUP-induced neurotoxicity through IGF1R and may act as a promising molecular target for the treatment of human neurotoxicity induced by BUP.