Search

AT2R (AngII [angiotensin II] type 2 receptor) is expressed in the distal nephrons. The aim of the present study is to examine whether AT2R regulates NCC (Na-Cl cotransporter) and Kir4.1 of the distal convoluted tubule. AngII inhibited the basolateral 40 pS K channel (a Kir4.1/5.1 heterotetramer) in the distal convoluted tubule treated with losartan but not with PD123319. AT2R agonist also inhibits the K channel, indicating that AT2R was involved in tonic regulation of Kir4.1. The infusion of PD123319 stimulated the expression of tNCC (total NCC) and pNCC (phosphorylated NCC; Thr53) by a time-dependent way with the peak at 4 days. PD123319 treatment (4 days) stimulated the basolateral 40 pS K channel activity, augmented the basolateral K conductance, and increased the negativity of distal convoluted tubule membrane. The stimulation of Kir4.1 was essential for PD123319-induced increase in NCC because inhibiting AT2R increased the expression of tNCC and pNCC only in wild-type but not in the kidney-specific Kir4.1 knockout mice. Renal clearance study showed that thiazide-induced natriuretic effect was larger in PD123319-treated mice for 4 days than untreated mice. However, this effect was absent in kidney-specific Kir4.1 knockout mice which were also Na wasting under basal conditions. Finally, application of AT2R antagonist decreased the renal ability of K excretion and caused hyperkalemia in wild-type but not in kidney-specific Kir4.1 knockout mice. We conclude that AT2R-dependent regulation of NCC requires Kir4.1 in the distal convoluted tubule and that AT2R plays a role in stimulating K excretion by inhibiting Kir4.1 and NCC. [ABSTRACT FROM AUTHOR]

Basolateral potassium channels in the distal convoluted tubule (DCT) are composed of inwardly-rectifying potassium channel 4.1 (Kir4.1) and Kir5.1. Kir4.1 interacts with Kir5.1 to form a 40 pS K + channel which is the only type K + channel expressed in the basolateral membrane of the DCT. Moreover, Kir4.1/Kir5.1 heterotetramer plays a key role in determining the expression and activity of thiazide-sensitive Na-Cl cotransport (NCC). In addition to Kir4.1/Kir5.1, Kir1.1 (ROMK) is expressed in the apical membrane of the late DCT (DCT2) and plays a key role in mediating epithelial Na + channel (ENaC)-dependent K + excretion. High dietary-K + -intake (HK) stimulates ROMK and inhibits Kir4.1/Kir5.1 in the DCT. Inhibition of Kir4.1/Kir5.1 is essential for HK-induced suppression of NCC whereas the stimulation of ROMK is important for increasing ENaC-dependent K + excretion during HK. We have now used the patch-clamp-technique to examine whether gender and Cl - content of K + -diet affect HK-induced inhibition of basolateral Kir4.1/Kir5.1 and HK-induced stimulation of ROMK. Single-channel-recording shows that basolateral 40 pS K + channel (Kir4.1/Kir5.1) activity of the DCT defined by NP o was 1.34 (1% KCl, normal K, NK), 0.95 (5% KCl) and 1.03 (5% K + -citrate) in male mice while it was 1.47, 1.02 and 1.05 in female mice. The whole-cell recording shows that Kir4.1/Kir5.1-mediated-K + current of the early-DCT (DCT1) was 1,170 pA (NK), 725 pA (5% KCl) and 700 pA (5% K + -citrate) in male mice whereas it was 1,125 pA, 674 pA and 700 pA in female mice. Moreover, K + -currents (I K ) reversal potential of DCT (an index of membrane potential) was -63 mV (NK), -49 mV (5% KCl) and -49 mV (5% K-citrate) in the male mice whereas it was -63 mV, -50 mV and -50 mV in female mice. Finally, TPNQ-sensitive whole-cell ROMK-currents in the DCT2 /initial-connecting tubule (CNT) were 910 pA (NK), 1,520 pA (5% KCl) and 1,540 pA (5% K + -citrate) in male mice whereas the ROMK-mediated K + currents were 1,005 pA, 1,590 pA and 1,570 pA in female mice. We conclude that the effect of HK intake on Kir4.1/Kir5.1 of the DCT and ROMK of DCT2/CNT is similar between male and female mice. Also, Cl - content in HK diets has no effect on HK-induced inhibition of Kir4.1/Kir5.1 of the DCT and HK-induced stimulation of ROMK in DCT2/CNT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Meng, Zhang, Meng, Jiang, Duan, Wang and Wang.)

Hypertension is one of the strongest risk factors for cardiovascular diseases, cerebral stroke, and kidney failure. Lifestyle and nutrition are important factors that modulate blood pressure. Hypertension can be controlled by increasing physical activity, decreasing alcohol and sodium intake, and stopping tobacco smoking. Chronic kidney disease patients often have increased blood pressure, which indicates that kidney is one of the major organs responsible for blood pressure homeostasis. The decrease of renal sodium reabsorption and increase of diuresis induced by high potassium intake is critical for the blood pressure reduction. The beneficial effect of a high potassium diet on hypertension could be explained by decreased salt reabsorption by sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT). In DCT cells, NCC activity is controlled by with-no-lysine kinases (WNKs) and its down-stream target kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1). The kinase activity of WNKs is inhibited by intracellular chloride ([Cl - ] i ) and WNK4 is known to be the major WNK positively regulating NCC. Based on our previous studies, high potassium intake reduces the basolateral potassium conductance, decreases the negativity of DCT basolateral membrane (depolarization), and increases [Cl - ] i . High [Cl - ] i inhibits WNK4-SPAK/OSR1 pathway, and thereby decreases NCC phosphorylation. In this review, we discuss the role of DCT in the blood pressure regulation by dietary potassium intake, which is the mechanism that has been best dissected so far.

Cytochrome P -450 (Cyp) epoxygenase-dependent metabolites of arachidonic acid (AA) have been shown to inhibit renal Na + transport, and inhibition of Cyp-epoxygenase is associated with salt-sensitive hypertension. We used the patch-clamp technique to examine whether Cyp-epoxygenase-dependent AA metabolites inhibited the basolateral 40-pS K + channel (Kir4.1/Kir5.1) in the distal convoluted tubule (DCT). Application of AA inhibited the basolateral 40-pS K + channel in the DCT. The inhibitory effect of AA on the 40-pS K + channel was specific because neither linoleic nor oleic acid was able to mimic the effect of AA on the K + channel. Inhibition of Cyp-monooxygenase with N -methylsulfonyl-12,12-dibromododec-11-enamide or inhibition of cyclooxygenase with indomethacin failed to abolish the inhibitory effect of AA on the 40-pS K + channel. However, the inhibition of Cyp-epoxygenase with N -methylsulfonyl-6-(propargyloxyphenyl)hexanamide abolished the effect of AA on the 40-pS K + channel in the DCT. Moreover, addition of either 11,12-epoxyeicosatrienoic acid (EET) or 14,15-EET also inhibited the 40-pS K + channel in the DCT. Whole cell recording demonstrated that application of AA decreased, whereas N -methylsulfonyl-6-(propargyloxyphenyl)hexanamide treatment increased, Ba 2+ -sensitive K + currents in the DCT. Finally, application of 14,15-EET but not AA was able to inhibit the basolateral 40-pS K + channel in the DCT of Cyp2c44 -/- mice. We conclude that Cyp-epoxygenase-dependent AA metabolites inhibit the basolateral Kir4.1/Kir5.1 in the DCT and that Cyp2c44-epoxygenase plays a role in the regulation of the basolateral K + channel in the mouse DCT.

Background: Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown., Methods: We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice., Results: Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K + conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K + currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K + conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K + conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice., Conclusions: Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC., (Copyright © 2019 by the American Society of Nephrology.)

Kir4.1/5.1 heterotetramer participates in generating the negative cell membrane potential in distal convoluted tubule (DCT) and plays a critical role in determining the activity of Na-Cl cotransporter (NCC). Kir5.1 contains a phosphothreonine motif at its COOH terminus (AA249-252). Coimmunoprecipitation showed that Nedd4-2 was associated with Kir5.1 in HEK293 cells cotransfected with Kir5.1 or Kir4.1/Kir5.1. GST pull-down further confirmed the association between Nedd4-2 and Kir5.1. Ubiquitination assay showed that Nedd4-2 increased the ubiquitination of Kir4.1/Kir5.1 heterotetramer in the cells cotransfected with Kir4.1/Kir5.1, but it has no effect on Kir4.1 or Kir5.1 alone. Patch-clamp and Western blot also demonstrated that coexpression of Nedd4-2 but not Nedd4-1 decreased K currents and Kir4.1 expression in the cells cotransfected with Kir4.1 and Kir5.1. In contrast, Nedd4-2 fails to inhibit Kir4.1 in the absence of Kir5.1 or in the cells transfected with the inactivated form of Nedd4-2 (Nedd4-2C821A). Moreover, the mutation of TPVT motif in the COOH terminus of Kir5.1 largely abolished the association of Nedd4-2 with Kir5.1 and abolished the inhibitory effect of Nedd4-2 on K currents in HEK293 cells transfected with Kir4.1 and Kir5.1 mutant (Kir5.1T249A). Finally, the basolateral K conductance in the DCT and Kir4.1 expression is significantly increased in the kidney-specific Nedd4-2 knockout or in Kir5.1 knockout mice in comparison to their corresponding wild-type littermates. We conclude that Nedd4-2 binds to Kir5.1 at the phosphothreonine motif of the COOH terminus, and the association of Nedd4-2 with Kir5.1 facilitates the ubiquitination of Kir4.1, thereby regulating its plasma expression in the DCT.

Objective: To evaluate the expression of proteasome β5 subunit in human atherosclerotic plaque., Methods: The specimens of carotid endarterectomy were collected from 16 carotid stenosis patients. For the case group, they were divided into 2 groups: plaque (group A) and areas adjacent to plaque (group B). And carotid endarterium was obtained from 6 patients undergoing aortic replacement surgery. Relatively normal arterial intima (subclavian artery & innominate artery) was selected as the control group (group C). Content of proteasome β5 subunit was evaluated by hematoxylin & eosin (HE) staining, immunohistochemistry and Western blot., Results: (1) In patients with diabetes, hyperlipidemia and probable smoking, proteasome β5 subunit was enhanced in all groups; (2) Proteasome β5 subunit was demonstrated in both cytoplasm and nucleus of vascular smooth muscle cells, but mainly confined to cytoplasm; (3)The positive expression rate of β5 was 9/16 in group A, 11/16 in group B and 6/6 in group C. There was significant divergence in the expression of β5 in different sample sub-groups: group A (atherosclerotic plaque) < group B (areas adjacent to plaque) < group C (control group)., Conclusion: The expression of β5 decrease markedly in human atherosclerotic plaques.

Objective: To investigate the effects of Cox7a2 on the LH-induced testosterone production and the involved autophagy regulating signals in TM3 mouse Leydig cells., Methods: The Cox7a2-pEYFP-N1 fluorescent protein vector was constructed and transfected into TM3 mouse Leydig cells. The level of testosterone was determined by ELISA, and the effects of Cox7a2 on the expression of the steroidogenic acute regulatory protein (StAR) and the phosphorylation of the autophagy regulatory factor P70S6K were detected by Western blot., Results: LH stimulation increased the StAR protein expression and testosterone production, while Cox7a2 decreased P70S6K phosphorylation, reduced StAR expression and consequently inhibited LH-induced testosterone biosynthesis in the TM3 Leydig cells., Conclusion: Cox7a2 inhibits testosterone production by decreasing the StAR protein expression, which might be at least in part related with the activation of autophagy in TM3 mouse Leydig cells.

Objective: To investigate the relationship between mine environment and hypertension in miners., Methods: 1736 male miners who worked under the ground and 825 on the ground were recruited in this study. Prevalence of hypertension under the ground and on the ground miners was compared., Results: Prevalence of hypertension of miners under the ground was 23.91% and on the ground was 15.52% (chi(2) = 23.56, P < 0.001). Compared to miners on the ground, the relative risk of hypertension under the ground workers was 1.71 (95%CI 1.38 - 2.13). Prevalence of hypertension was correlated to the years of ground working (chi(2) = 37.00, P < 0.001). The binary logistic regression showed significant relationship between mine environment and hypertension under the ground miners (OR = 1.05, 95%CI 1.02 - 1.08)., Conclusion: The underground environment is an important risk factor hypertension to the miners.