bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sodium Chloride Cotransporter Upregulation in Settings of Zinc Deficiency Offers New Insight into Blood Pressure Dysregulation in Chronic Diseases
Authors: Williams Clintoria R. , Mistry Monisha , Mallick Rickta , Mistry Abinash , Ko Ben , Gooch Jennifer L. , Hoover Robert S. 
Published: November 2023

BackgroundZn2+ deficiency is a common comorbidity with numerous chronic diseases including type II diabetes and chronic kidney disease. Experimental data show that Zn2+ deficiency exacerbates hypertension in these settings. Moreover, Zn2+ deficiency alone may be sufficient to alter blood pressure; however, the mechanisms involved are unknown.ObjectiveThe renal distal convoluted tubule sodium chloride cotransporter (NCC) plays a critical role in blood pressure regulation and hypertension. The objective of this study was to determine if Zn2+ deficiency alters NCC expression and/or activation in vivo and in vitro.Experimental DesignTo this end, WT mice were pair‐fed a diet with 50 ppm Zn2+ (Zn2+ adequate [ZnA]) or a diet with 1 ppm Zn2+ (Zn2+ deficient [ZnD]) for 6 weeks. To reduce intracellular Zn2+ levels in vitro, WT mouse distal convoluted tubular (mDCT‐15) cells were cultured in 1 nM N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN)‐ or vehicle (DMSO)‐containing medium for 24 hours. To replete intracellular Zn2+, TPEN‐exposed cells were then cultured in 1 nM Zn2+‐supplemented medium for 24 hours. NCC mRNA and protein expression were examined in vivo and in vitro via qRT‐PCR, western blot and immunohistochemistry. NCC activation was assessed by cellular localization (via biotinylation and immunofluorescence) and Na+ uptake activity (via thiazide‐sensitive assays).ResultsIn ZnD mice, NCC protein expression is increased in DCT cells compared to mice receiving a ZnA‐diet. In the in vitro model of Zn2+ deficiency, Zn2+ chelation with TPEN stimulates NCC mRNA and protein expression compared to vehicle‐treated cells. Furthermore, TPEN promotes NCC surface localization and Na+ uptake activity. Notably, restoration of intracellular Zn2+ levels by supplementation reverses TPEN effects on NCC.ConclusionsOur findings indicate that NCC is a Zn2+‐sensitive transporter and is upregulated in settings of Zn2+ deficiency.SignificanceThis sodium transporter represents a novel mechanism by which Zn2+ deficiency may alter blood pressure and contribute to hypertension in chronic disease settings. Future experiments are needed to determine what additional factors are involved in Zn2+ deficiency‐induced upregulation of NCC and to test if protecting NCC from Zn2+ regulation has a positive impact on hypertension.
Novel Cardiorenal Effects of the Lactate Receptor GPR81 Activation
Authors: Jones Natalie K , Wallenius Kristina , Menzies Robert I , Stewart Kevin , Mullins John J , Bailey Matthew A 
Published: November 2023

G protein‐coupled receptor 81 (GPR81) is highly expressed in adipocytes and activation of the receptor by the endogenous ligand lactate inhibits lipolysis. The receptor is also expressed in non‐adipose tissue but the function of GPR81 at these sites is not known. In the present study we first used end point PCR to screen for GPR81 expression in the kidney and vasculature isolated from male Sprague‐Dawley rats (n=4). GPR81 was expressed in the whole kidney, aorta, mesenteric, renal and interlobar arteries. We next used a specific GPR81 agonist, AZ13415538, to assess the function of the receptor on renal haemodynamics in vivo. Male Sprague Dawley rats (n=14) were anesthetised with Inactin (120mg/kg) and a Doppler ultrasound probe placed round the left renal artery. Mean arterial blood pressure was measured via a cannula inserted into the femoral artery. Data are expressed as mean±sem. The agonist was injected into the jugular vein as a bolus in a 5 point cumulative dose response curve from 0.5μmol to 5μmol. Injection of AZ13415538 caused a rapid, dose‐dependent rise in blood pressure and a reduction in renal blood flow. At the highest dose of 5μmol, the increase in mean arterial blood pressure over the pre‐injection baseline was 20.4±1.6 mmHg; the nadir of renal blood flow was 3.0±0.5 mL/min. Both effects were transient and recovered after 75 seconds. The 5% mannitol vehicle (0.5ml) also caused an increase in blood pressure (11.4±0.8 mmHg) and a reduction of renal blood flow (1.8±0.2 mL/min) but these effects were not sustained. The effect of AZ13415528 was therefore expressed as area over and under the curve for blood pressure and renal blood flow, respectively. A significant dose‐dependent effect for both blood pressure and blood flow was identified by One‐Way Anova (P<0.0001). Post hoc comparisons against the effect of vehicle showed significant increases in blood pressure at 2–4μmol (P<0.05) and 5μmol (P<0.01). Renal blood flow was significantly reduced at 4μmol AZ13415528 (P<0.05) and 5μmol (P<0.01). The increase in renal vascular resistance observed after GPR81 activation shows novel cardiovascular effects of this receptor. The mechanisms underlying these actions have not been investigated.Support or Funding InformationSupported by The British Heart Foundation (fellowship number ‐ FS/15/63/32033) and AstraZeneca
Sodium Butyrate Protects against Renal Injury in DOCA Salt Hypertensive Rats
Authors: Zhu Qing , Wang Lei , Zhang Linlin , Lu Aihua , Yang Tianxin 
Published: November 2023

We previously found that infusion of sodium butyrate (NaBu) into the renal medullary suppressed angiotensin II‐induced hypertension and improved renal injury. The present study was to test whether systemic administration of NaBu protects the kidneys from damage in DOCA‐salt rats. Uninephrectomized male Sprague‐Dawley rats were treated with a subcutaneous DOCA pellet (200mg/rat) and 1% NaCl drinking water for 2 weeks. Sham rats were uninephrectomized and drank tap water. Animals received an oral gavage of NaBu (0.1g/kg) or vehicle once per day. Hypertension induced by DOCA‐salt was significantly attenuated in rats treated with NaBu. Urinary albumin excretion increased from 0.24 ± 0.05 mg/24h in sham rats to 12.01 ± 5.13 in DOCA‐salt rats, which was significantly reduced to 2.32 ± 0.94 in NaBu‐treated DOCA‐salt rats (p<0.05). Morphological examination showed that the glomerular damage indices were 1.04 ± 0.1, 2.87± 0.08 and 1.69 ± 0.12 in sham, DOCA salt‐treated, and NaBu‐treated DOCA‐salt rats, respectively. There was no difference in creatinine clearance rate in sham or NaBu‐treated DOCA‐salt rats compared with only DOCA‐salt rats. These results suggest that sodium butyrate may protect against renal injury in DOCA‐salt hypertension and that NaBu could serve as a novel therapeutic approach for the treatment of chronic renal injury.Support or Funding InformationNational Natural Science Foundation of China Grant No. 91439205, No. 31330037 and No. 81600322; Fundamental Research Funds of Sun Yat‐sen University 16ykpy47; Veterans Affairs Merit Review, and National Institutes of Health Grant DK094956.
Inactivation of Hv1 proton channels is not responsible for bicarbonate‐mediated reductions in tubular cast formation in Dahl salt‐sensitive rats
Authors: Ray Sarah , Ocasio Hiram , Sun Jingping , O'Connor Paul M 
Published: November 2023

We have shown that oral bicarbonate promotes a marked reduction in tubular cast formation and fibrosis in the Dahl salt‐sensitive (SS) rat model when fed a high salt diet, independent of arterial blood pressure, glomerular sclerosis, and proteinuria. Rats lacking the voltage‐gated proton channel (Hv1), which is highly expressed in mTAL, denoted a similar phenotype. In the current study, we examined whether bicarbonate intake protected from cast formation in the Dahl SS rat model by inactivation of the voltage‐gated Hv1 proton channel. Wild‐type Dahl SS (WT) and Hv1 knock‐out (KO) Dahl SS rats were treated with either vehicle (0.1M NaCl; n=9) or bicarbonate (0.1M NaHCO3; n=11) in drinking water for the length of the study. Following a 4 day baseline period in which both groups were fed a 0.4% NaCl diet (Dyets AIN76A), rats were placed on an 8% NaCl diet for 14 days. Urine was collected at day 4 of baseline, day 7, and day 14 high salt; telemetry was used to measure blood pressure. On day 14, rats were anesthetized, and the right kidney taken for histology. Mean arterial pressure (mmHg) showed no difference between the WT and KO groups regardless of vehicle or bicarbonate treatment. Tubular casts were significantly decreased (pSTRAIN=0.008) in KO rats when compared to WT. Importantly, bicarbonate treatment had an additive effect, further decreasing tubular cast formation and by a similar magnitude in both KO and WT rats (PTREATMENT=0.02; PTREATMENT*STRAIN=N.S). In conclusion, bicarbonate‐induced inactivation of Hv1 proton channels does not mediate the reduction of cast formation in Dahl SS rats. Further investigation will be required to determine whether a common downstream mechanism is responsible for limiting tubular cast formation in the absence of blood pressure changes or glomerular injury in bicarbonate treated and Hv1−/− Dahl SS rats.Support or Funding InformationThe authors would like to acknowledge funding from the National Institute of Health: NIDDK R01099548
Large conductance potassium (KCa1.1) channel mediated potassium secretion provides the driving force for water secretion in organoids from rat distal colon
Authors: Rottgen Trey , Nickerson Drew , Minor Emily , Rajendran Vazhaikkurichi M 
Published: November 2023

Organoid culture has been adapted as a method to recapitulate colonic physiology, especially epithelial electrolyte transport in an in vitro model. We tested the hypothesis that K+ secretion can provide a novel pathway for water secretion. Colonic crypts, containing Lgr5+ cells, were isolated from male Spraque‐Dawley rats (n=4) and plated in an extracellular support (Matrigel) supplemented with crypt culture media containing specific niche factors necessary for proliferation and organoid formation. Utilizing an activator (BMS‐204352, 10μM) of large‐conductance of potassium (KCa1.1) channels, we were able to induce swelling of organoids and increase the luminal to total volume ratio that was significantly different from controls (63.3±4.7 vs. 33.5±5.2, x<0.05). Paxilline (KCa1.1 blocker, 1μM) completely inhibited the BMS‐204352 induced swelling and was not significantly different from unexposed organoids (34.4±3.8 vs. 33.5±5.2, ns). CFTR‐172 inhibitor did not prevent BMS‐204352‐induced swelling, which was not significantly different from BMS‐204352 alone (55.8±5.6 vs. 63.3 ns). We conclude from these data that large conductance of potassium (KCa1.1) channels mediate potassium secretion that contribute to a novel mechanism of water secretion that could be clinically impactful in certain pathophysiological states.Support or Funding InformationNIH/NIDDK DK‐018777
Regulated Expression of the Na+/K+‐ATPase Pump in Colonic Epithelium by Bile Acids
Authors: Borg Johan Fredrik , Yde Jonathan , Wu Qi , Lajczak Natalia , Keely Stephen , Fenton Robert A , Moeller Hanne 
Published: November 2023

Bile acid malabsorption (BAM) is a common cause of chronic diarrhea. The mechanism underlying the development of bile acid induced diarrhea is unclear. We aimed to determine if expression levels of mRNA and protein, as well as localization of Na+/K+‐ATPase (NKA) are influenced by bile acids (BA) in colonic epithelial cells during BAM.Rats were fed a 1% Na‐cholate containing diet for three days. Colonic epithelium was isolated using Ca2+‐chelation and prepared for protein analysis and RT‐qPCR. Colonic tissue was prepared for immunohistochemistry (IHC). T84 cells were treated with deoxycholic acid (DCA) to examine the direct effect of this bile acid on epithelial cells.The treated rats developed diarrhea with an increased feces water content compared to controls. In a previous experiment with similar results we used liquid chromatography tandem mass spectrophotometry (LC‐MS/MS) on colonic epithelial tissue from the animal model of BAM to create a protein profile. We found that that the Na+/K+‐ATPase subunit β1 (NKA β1) was significantly down‐regulated in the colon during BAM. In our BAM model, western blotting confirmed the down‐regulation of the NKA β1 in colon. Na+/K+‐ATPase subunit α1 (NKA α1) abundance was also decreased in proximal, but not distal colon. RT‐qPCR analysis showed no regulation of the genes Atp1b1 or Atp1a1 at the mRNA level. IHC showed no morphological differences in cross‐sections from Na‐cholate treated rats, nor any change in NKA β1 or α1 localization.T84 cells treated with 150 μM DCA for 48 hrs showed a similar down‐regulation of NKA β1 at protein level. mRNA levels were also down‐regulated for both the ATP1B1 and ATP1A1 gene. Transepithelial electrical resistance decreased on average by 65% after 48 hrs of DCA treatment.In conclusion, we have found the NKA β1 to be down‐regulated at the protein level in the colonic epithelium of rats after 3 days of Na‐cholate treatment. This down‐regulation was also found in T84 cells treated with DCA for 48 hrs. Additionally, mRNA levels of ATP1B1 and ATP1A1 in T84 cells were down‐regulated by DCA. Due to the central role for NKA in the absorption and secretion of water and electrolytes in colon, this regulation could be central in development of diarrhea during BAM.
Dietary K+ and Cl− independently regulate basolateral conductance in the principal and intercalated cells of the collecting duct
Authors: Zaika Oleg , Tomilin Viktor , Mamenko Mykola , Pochynyuk Oleh 
Published: November 2023

Kidneys play a central role in maintaining systemic homeostasis by matching dietary variations of electrolyte intake with their excretion in urine. Final adjustments take place in the collecting duct where transport rates are highly sensitive to electrolyte intake. The collecting duct comprises of electrically uncoupled principal and intercalated cells mediating Na+/K+ exchange and Cl− reabsorption, respectively. Basolateral membrane conductance sets up an electrochemical driving force governing electrolyte transport. Previous work established that potassium Kir4.1/5.1 channels underlie basolateral conductance in principal cells, whereas ClC‐K2 channels are essential for basolateral Cl− flux in intercalated cells. However, little is known whether variations in dietary K+ and Cl− affect activity of these channels. Using patch clamp electrophysiology at the single channel and whole cell levels in freshly isolated mouse collecting ducts, we found a marked upregulation of the basolateral K+‐selective current and hyperpolarization of basolateral membrane potential in principal cells of mice subjected to a high (5%) K+ diet for one week compared to the control. This stimulation stemmed from the significantly increased functional levels of Kir4.1/5.1 but not from channel open probability. When high K+ was accompanied with Cl−, this led to drastically decreased basolateral Cl− ‐selective current, the number of active ClC‐K2 and reduced channel open probability in intercalated cells. In contrast, when high K+ was given as citrate, ClC‐K2 macroscopic current and channel activity were significantly increased. Consistently, dietary Cl− restriction with regular K+ augmented ClC‐K2‐dependent conductance but had little effect on Kir4.1/5.1 activity. Overall, our results suggest independent regulation the basolateral Kir4.1/5.1 and ClC‐K2 in principal and intercalated cells by dietary K+ and Cl−. We propose that this mechanism contributes to precise tuning of urinary excretion in response to variable dietary electrolyte intake.Support or Funding InformationThis research was supported by NIH‐NIDDK DK095029 (to O. Pochynyuk), AHA‐15SDG25550150 (to M. Mamenko)
A novel Na+ transport system in the A‐type intercalated cells of the CCD that mediates Na+ secretion through a H/K‐ATPase type 2‐dependent pathway
Authors: Edwards Aurelie , Morla Luciana , Walter Christine , Doucet Alain , Crambert Gilles 
Published: November 2023

In the last few years, our understanding of the manner in which the cortical collecting duct (CCD) transports Na+ has significantly evolved. Indeed, in addition to the classical ENaC‐mediated Na+ reabsorption by principal cells, it has been shown that an electroneutral coupled transport system expressed in B‐type intercalated cells involving pendrin and a Na+/Cl−/HCO3− exchanger (NDBCE) also contributes to renal Na+ reabsorption (1). In the present study, we used in vitro microperfusion experiments in the presence of the transepithelial ion concentration gradients that prevail in vivo (asymmetric conditions), combined with the predictions of a mathematical model, to fully characterize the relevant Na+ transport systems in the CCD of Na+‐depleted mice.Under these asymmetric conditions, we first assessed the contribution of the paracellular route to Na+ transport by inhibiting all known and possible transcellular pathways with amiloride, hydrochlorothiazide (HCTZ), Schering 28080, and bumetanide. Under these conditions, the CCD excreted Na+ (JNa = − 16.8 ± 0.8 pmol/min/mm; n = 5) whereas the Cl− flux was negligible (JCl = − 3.4 ± 2.4 pmol/min/mm), and the transepithelial voltage (DVte) was – 12.8 ± 1.8 mV. To estimate the contribution of each cell type, we then removed one or more inhibitors. Removal of amiloride abolished JNa showing that Na+ reabsorption by principal cells counterbalances Na+ secretion via the paracellular route. Conversely, removal of all inhibitors except amiloride increased Na+ secretion (JNa = −32 ± pmol/min/mm,) in wild‐type mice but not in H/K‐ATPase type 2 (HKA2) knock‐out mice or in the presence of bumetanide.Altogether our results indicate that type A intercalated cells mediate the secretion of Na+ via basolateral Na+‐K+‐2Cl− (NKCC1) cotransporters in tandem with HKA2 pumps functioning as apical Na+ pumps. We have therefore identified a third CCD transcellular sodium transport pathway, in A‐type intercalated cells, which mediates Na+ secretion in the CCD.Support or Funding InformationSupported by a Nestlé Waters Grant
Intestine‐specific dual oxidase deficiency lacks ATP‐P2Y‐mediated luminal H2O2 production in mice
Authors: Maruta Koji , Akiba Yasutada , Kaji Izumi , Kaunitz Jonathan D 
Published: November 2023

The intestinal epithelium affects luminal microbial composition via anion and mucus secretion, and the secretion of anti‐bacterial peptides or chemorepellents such as H2O2. Dual oxidase‐2 (Duox‐2) an enzyme predominantly expressed in the intestinal epithelium among NADPH oxidase family generates luminal H2O2 via intracellular Ca2+ increase. We found that luminal ATP‐P2Y signals increased luminal H2O2 output in rat duodenum, which we hypothesized was due to the activity of Duox‐2. Lacking specific Duox inhibitors, we created mice selectively deficient for DuoxA1–A2 (accessory proteins essential for membrane trafficking of Duox‐1 and ‐ 2, respectively) by breeding villin‐Cre mice with mice bearing floxed DuoxA1–A2, needed since global Duox knockout (KO) causes hypothyroidism. We examined the effect of luminal ATP on H2O2 production in wild type (WT) and Duox KO mice.We measured H2O2 output using the fluorogenic substrate Amplex® and HCO3− secretion in an ATP‐perfused duodenal loop in vivo using flow‐through pH and CO2 electrodes. We also measured luminal H2O2 production in response to luminal ATP or ionomycin in Ussing chambered muscle‐stripped duodenum and cecum.Luminal perfusion of ATP (0.1 or 1 mM) augmented HCO3− secretion with increased luminal H2O2 output in WT duodenum, whereas ATP‐induced HCO3− secretion was reduced and ATP‐induced H2O2 output was abolished in KO duodenum. In Ussing chambered WT duodenum and cecum, basal H2O2 concentrations were higher in the cecum than in the duodenum. Luminal H2O2 production was similarly increased in the duodenum and cecum in response to luminal ATP (1 mM) and ionomycin (1 μM) in WT, whereas no additional H2O2 production was observed in KO intestine. Furthermore, luminal pretreatment with the non‐specific P2Y receptor antagonist suramin (1 mM) abolished ATP‐induced H2O2 production in WT.These results confirm that epithelial Duox‐2 generates luminal H2O2 via luminal ATP‐P2Y signals. We propose that H2O2 produced by Duox‐2 is a key factor governing the composition of the gut microbial flora.Support or Funding InformationSupported by VA Merit Review, NIH R01 DK54221
The Epithelial Sodium Channel (ENaC) α‐ and γ‐subunits are cleaved by furin in human kidney
Authors: Langkilde Rikke Zachar , Mikkelsen Maiken Krogsbæk , Skjødt Karsten , Zamani Reza , Marcussen Niels , Jensen Boye , Svenningsen Per 
Published: November 2023

The aldosterone‐sensitive Epithelial Sodium Channel (ENaC) plays an essential role in sodium homeostasis and blood pressure regulation. In vitro data suggest that during biosynthesis, the proprotein convertase furin cleaves the alpha and gamma subunit. Full activation takes place at the apical membrane by proteolytical excision of an inhibitory domain from the gamma subunit. The GPI‐anchored serine protease prostasin is a possible candidate to activate ENaC under physiological conditions, while aberrant filtrated plasmin cleaves under pathophysiological conditions. The aim of the study was to investigate proteolytical cleavage of ENaC in human kidney tissue and it was hypothesized that intracellular furin cleavage can be detected in the alpha and gamma subunit in human kidney; that apical plasmin/prostasin‐cleavage is associated with proteinuria and that cleaved products are detectable predominantly in human urinary exosomes.Monoclonal antibodies directed against αENaC (AA 436–44, constant region at the c‐terminal side of putative furin cleavage) and against epitopes in the cleaved γENaC (AA 138–147 (furin‐cleavage site, C‐terminal) and AA 173–190 (overlapping prostasin‐cleavage site)) were developed in balb/c mice and tested on human kidney tissue from nephrectomized patients and on urinary exosomes from healthy persons. A furin cleavage site‐specific γENaC antibody revealed protein at 75 kDa compatible with furin‐cleavage in a human kidney cortex pool. An antibody overlapping and therefore requiring intact prostasin/plasmin cleavage site showed bands compatible with full‐length (100 kDa) and furin‐cleaved (75 kDa) γENaC. No difference was detected in patients with proteinuria. The furin‐cleaved part was mainly detected within the membrane. The αENaC antibody showed a dominant 50kDa product compatible with predominance of furin‐cleaved α‐subunit in the kidney tissue. All three antibodies revealed labeling associated with collecting ducts in kidney tissue sections when immunostained. In urinary exosomes furin‐cleaved, but not intact γENaC were detected. αENaC was detected in both its intact (≈80kDa) and furin‐cleaved form in exosomes.The results indicate that proteolytic processing by furin of both alpha and gamma ENaC occurs in human kidney. The presence of prostasin‐cleavage was not clearly present. Cleavage products of both γENaC and αENaC and intact αENaC were detectable in urinary exosomes.Support or Funding InformationOdense University Hospital PhD Fund, University of Southern Denmark ‐ Faculty of Health, The Danish Diabetes Academy – supported by the Novo Nordisk Foundation, Leo Pharma, A.P. Møller and Hustru Chastine Mc‐Kinney Møllers Foundation.