bluuebunny/crossref_metadata_2025 huggingface dataset:
Perinatal taurine depletion increases oxidative stress in adult female rats
Authors: Lerdweeraphon Wichaporn , Wyss James Michael , Boonmars Thidarut , Roysommuti Sanya
Published: June 2021

Our previous studies and those of others demonstrate that perinatal taurine exposure affects adult physiology and susceptibility to disease in several animal models of cardiovascular and renal disease. Perinatal taurine depletion typically produces adult disorders, but perinatal taurine supplementation minimally influences normal physiology in adults. This study tests the hypothesis that perinatal taurine depletion leads to increased oxidative stress in adult female rats. Female Sprague‐Dawley rats were fed normal rat chow with 3% β‐alanine (taurine deficient, TD), 3% taurine (taurine supplemented, TS) or water alone (C) from conception to weaning. Their female offspring were fed normal rat chow and water alone throughout the experiment. At 7–8 weeks of age, body, kidney and heart weights were not significantly different among groups (n = 10/group). Neither did their plasma sodium, plasma potassium, mean arterial pressure and heart rate. Compared to control, plasma malondialdehyde levels (a bio‐marker of oxidative stress from lipid peroxidation) were significantly higher in TD and the TS group was intermediate between the other two groups, i.e., not significantly different than either. These results suggest that perinatal taurine depletion increases oxidative stress in adult female rats and could play a role in the morbidity of these animals as they mature through adulthood.
SHB deficient mice display an increased GFR and augmented renal arteriolar contractions to both Adenosine and Ang II
Authors: Gao xiang , Sällström Johan , Ma Zufu , Welsh Michael , Persson Erik
Published: June 2021

BACKGROUND

Src homology 2 domain‐containing protein B (Shb) is an adapter protein which regulates several signal transduction cascades and endothelial cell functions. Adenosine‐angiotensin II (Ado‐Ang II) interaction plays important roles in regulation of glomerular filtration rate (GFR), vascular resistance and tubuloglomerular feedback. We used Shb‐knockout (Shb−/−) and wild‐type (Shb+/+) mice to investigate GFR and effectiveness of Ado and Ang II to renal resistance vessels.

METHODS

GFR was measured in conscious Shb−/− and Shb+/+ mice using FITC‐inulin. Isolated and perfused renal afferent arteriolar responses to Ang II doses (10−12 to 10−6M) or low‐dose Ado (10−8 mol/l) alone, as well as Ado (10−8 mol/l) in combination with Ang II doses were studied from both genotypes.

RESULTS

GFR was increased significantly in Shb−/− (371 ± 12 μL/min) comparing to Shb+/+ (321 ± 11 μL/min). The maximal arteriolar contraction to Ang II was significantly larger in Shb−/− (87 %) than in Shb+/+ (54 %). Low‐dose Ado alone contracted afferent arterioles (6% in Shb−/−; 7% in Shb+/+) and significantly enhanced Ang II arteriolar constrictions (6% in Shb−/− ; 18 % in Shb+/+).

CONCLUSION

Low‐dose adenosine augments Ang II arteriolar constriction effectiveness, which indicates Ado‐Ang II interaction in both Shb−/− and Shb+/+ mice. The absence of Shb increases GFR and enhances renal arteriolar contractions to both Ado and Ang II.
Control of aldosterone release in vitro in the renin knockout rat
Authors: Bruder Eric D , Nunez Lizbeth , Hoffman Matthew J , Moreno Carol , Raff Hershel
Published: June 2021

There are local tissue renin‐angiotensin systems (RAS) throughout the body, in addition to the classic renal (peripheral) RAS that is responsible for generating plasma angiotensin II (AngII). An adrenocortical RAS may be involved in the control of aldosterone (Aldo) synthesis in the zona glomerulosa (ZG). In order to characterize the control of adrenal steroid production by a local RAS, we utilized adrenal glands from renin knockout (KO) rats and compared steroidogenesis in vitro to wild‐type (WT) controls. Adrenal capsules (ZG; Aldo production) and subcapsules (zona reticularis/fasciculata [ZFR]; corticosterone production) were separately dispersed, cell concentration was normalized, and cells studied in vitro (4 experiments). Basal and cAMP‐stimulated Aldo production was significantly reduced in renin KO ZG cells. Corticosterone production was not different between WT and KO ZFR cells. The reduction in Aldo from the ZG of the renin KO adrenal may be due to the absence of chronic stimulation of the ZG by circulating AngII or to a reduction in locally released AngII within the adrenal gland. Supported by NIH HL‐82798.
Developmental origins of renal disease: a translational approach
Authors: Ojeda Norma B. , Intapad Suttira , Carter Anthony J , Alexander Barbara T , Bhatt Abhay
Published: June 2021

Previous studies from my laboratory indicate that low birth weight (LBW) is a risk factor for renal disease; however, the exact mechanisms involved are unclear. The objective of this translational study was to determine whether leptin and aldosterone levels at birth are associated with the etiology of renal disease. The clinical protocol used a prospective study in newborns and infants with 1,100 participants form both sexes and all races enrolled at the University of Mississippi. The experimental protocol used a rodent model of LBW induced by placental insufficiency. Human LBW newborns showed an 8‐fold increase in plasma aldosterone and a 2.5‐fold reduction in plasma leptin at birth compared to normal birth weight (NBW) newborns. Glomerular filtration rate (GFR) was reduced (P<0.05) in LBW infants exposed to hypoperfusion between 6 and 12 months of age, with positive correlation with leptin and negative correlation with aldosterone levels at birth. Rodent LBW offspring showed increased aldosterone and decreased leptin levels at birth and reduced GFR when exposed to mild renal ischemia later in life. Perinatal antioxidant treatment in rodents completely abolished aldosterone, leptin and GFR disturbances in LBW offspring. Thus, the parallelisms between human and animal studies suggest that aldosterone and leptin levels at birth are associated with increased susceptibility for renal injury in LBW. Additionally, early interventions may prevent this enhanced susceptibility in the LBW individual; and monitoring of renal function should be indicated in this “at‐risk” population when exposed to hypoperfusion events.
Evidence for functional P2X receptors in a mouse cortical collecting duct cell line
Authors: Birch Rebecca , Scott‐Ward Toby , Unwin Robert J , Peppiatt‐Wildman Claire M , Schwiebert Erik , Wildman Scott S P
Published: June 2021

P2 receptors are potent modulators of key solute and water transport mechanisms in the renal collecting duct (CD) [1]. Controversy exists over the P2 receptor subtype(s) responsible; most studies focus on metabotropic P2Y receptors and arguably ionotropic P2X receptors are largely overlooked.

Using an M1 cell line derived from mouse CD and the excised outside‐out configuration of the patch‐clamp technique we have sought functional evidence for extracellular ATP‐gated P2X receptor ion channels. External bath solution contained 145 mM NaCl and the pipette solution contained 145 mM K+ (70 mM Cl−).

In outside‐out patches, application of external ATP (100 μM) transiently activated flickery single channel currents (tact ~3s, tdes ~20s). At −100 mV, the mean inward single channel current amplitude (i), open probability (Po) and chord conductance of channels were 0.71±0.12 pA, 0.66±0.12, and 7.7±0.8 pS (n=5), respectively.

We demonstrate that ATP directly activates receptors with singlechannel properties similar to those of P2X receptors. Consequently P2X receptors may regulate CD solute and water transport.

Research supported by the Wellcome Trust, BBSRC and DiscoveryBiomed.
Insights into cAMP signaling in polycystic kidney disease from depletion and interaction studies of Phosphodiesterase 1A and Polycystin 2 in zebrafish
Authors: Sussman Caroline R , Ward Christopher J , Leightner Amanda C , Harris Peter C , Torres Vicente E
Published: June 2021

Substantial evidence indicates the importance of elevated cAMP in polycystic kidney disease (PKD). Preclinical and clinical trials have shown the feasibility of therapies (vasopressin V2 receptor antagonists and somatostatin analogs) acting on G protein coupled receptors to inhibit adenylyl cyclase activity and production of cAMP. An additional or alternative approach could be to increase phosphodiesterase (PDE) mediated hydrolysis of cAMP. Of the large PDE superfamily, PDE1 is the only family activated by calcium, which is reduced in PKD cells. To assess the contribution of PDE1A to renal cyst formation, we depleted PDE1A levels using antisense morpholinos in zebrafish. We identified two splice isoforms with alternative starts corresponding to human PDE1A1 and PDE1A5. Two splice‐blocking morpholinos targeting the common hydrolase domain of both isoforms induced renal cysts. Two additional morpholinos, targeting each start, also induced renal cysts with dose dependence and additive effects. Co‐injection of the start 1 morpholino with pkd2 morpholino increased the severity of hydrocephalus, renal cysts, and body curvature, demonstrating functional interaction of PDE1A and PC2. Overall, data implicate PDE1A in PKD pathogenesis and validate further investigation of PDE1A as a therapeutic target in PKD.
Renalase protects against cisplatin acute kidney injury in mice
Authors: Desir Gary V , Wang Ling , Velazquez Heino , Moeckel Gilbert , Safirstein Robert
Published: June 2021

Acute kidney injury (AKI) is an important clinical syndrome for which effective therapies are currently unavailable. Renalase, a novel flavoprotein secreted by the kidney, participates in catechols metabolism protects against ischemic AKI by reducing tubular necrosis, inflammation and apoptosis.

The aims of the current study were to investigate the efficacy of renalase in treating cisplatin‐induced AKI, and the mechanisms of its protective action. Three days post treatment with cisplatin (20 mg/kg), plasma creatinine was significantly higher in renalase KO mice compared to WT (1.82±0.56 vs 0.71±0.02 mg/dl, n= 6, P=0.021), as was the renal injury score (Jablonski scale: KO=3.5 vs WT=1.3) indicating that renalase deficiency worsens cisplatin mediated AKI. HK‐2 cells, (human proximal tubular line) exposed to cisplatin for 24 hours showed a 50% decrease in cell viability, and a marked reduction in renalase expression. The addition of exogenous renalase fully restored cellular renalase expression, and increased cell survival. This was associated with decreased BAD, BAX and cleaved caspase 3, and increased BCL2 expression.

These data show that renalase protects against cisplatin AKI by decreasing pro‐aopoptotic, and increasing pro‐survival signals. Renalase could provide a useful therapeutic option for cisplatin AKI.
Aldosterone Alters Chromatin Structure of the Murine Endothelin‐1 Gene
Authors: Welch Amanda Kuhns , Jacobs Mollie E. , Lynch I. Jeanette , Gumz Michelle L. , Cain Brian D. , Wingo Charles S.
Published: June 2021

Aldosterone (Aldo) increases sodium (Na) reabsorption in the renal collecting duct (CD) and systemic blood pressure. We have shown that Aldo induces transcription of the endothelin‐1 (Edn1) gene and increases protein (ET‐1) levels (JBC 2009;294:30087). Since ET‐1 inhibits the action of Aldo on CD Na reabsorption, we hypothesize that ET‐1 acts in a negative feedback loop on Na retention. Whereas transcriptional regulation of the Edn1 gene has been investigated, the importance of epigenetic Edn1 gene regulation is largely unexplored. Here we examine the effect of Aldo on chromatin structure of the Edn1 gene. We used a novel quantitative PCR‐based DNaseI hypersensitivity assay of chromatin structure in 4 regions of the Edn1 gene known to affect transcription in murine inner medullary collecting duct‐3 cells. The calcium‐responsive element (NFAT) (JBC 2010;285:28520) at −1263 bp has low chromatin accessibility under all conditions tested. However, the NFAT element at −1563 bp upstream has low accessibility and is significantly more accessible in cells exposed to Aldo.

The distal Aldo hormone response element (HRE2) is highly accessible under all conditions tested. However, Aldo caused a significant increase in chromatin accessibility of the more proximal promotor including HRE1. The results extend our previous observations and suggest that Aldo activation of the mineralocorticoid receptor (MR) results in MR binding at HRE2 to open chromatin structure of specific regions of the Edn1 gene.

This work was funded by National Public Health Service Grant no. R01 DK82680 to CSW and BDC.
Kelch‐like 3 (KLHL3) controls WNK4 ubiquitination and degradation
Authors: Peng Ji‐Bin , Wu Guojin
Published: June 2021

Mutations in with‐no‐lysine (K) kinase 4 (WNK4) and ubiquitin E3 ligase components KLHL3 and Cullin 3 (CUL3) cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. WNK4 regulates Na‐Cl cotransporter (NCC) and so does KLHL3. However, it is unclear whether NCC or a regulator of NCC is a substrate of the KLHL3‐CUL3 E3 ligase complex. The objective of this study is to identify which protein is the true substrate of KLHL3‐CUL3 E3 ligase. We expressed KLHL3 and/or CUL3 together with Na‐K‐2Cl cotransporter 2 (NKCC2) and WNK4 in Xenopus oocytes. KLHL3 significantly decreased NKCC2‐mediated Na uptake and the positive effect of WNK4 on NKCC2. However, the protein abundance of NKCC2 was not decreased, but the level of WNK4 was robustly decreased by KLHL3. KLHL3 also did not substantially reduce the protein abundance of NCC or oxidative stress‐responsive 1 (OSR1) in a similar setting. KLHL3 significantly increased the ubiquitination and degradation of WNK4 protein. Although CUL3 alone didn't have significant effect on WNK4, a dominant negative CUL3 construct alleviated the effect of KLHL3 on WNK4, suggesting that CUL3 works together with KLHL3 to regulate WNK4 stability. All five PHAII mutations in KLHL3 tested decreased the ability of KLHL3 to reduce WNK4 protein abundance. We conclude that WNK4 is a substrate of KLHL3‐CUL3 ubiquitin E3 ligase complex and PHAII mutations in KLHL3 affect electrolyte transporters indirectly via WNK4.
BK‐beta4 increases apical BK‐alpha expression in collecting duct intercalated cells (IC)
Authors: Wen Donghai , Cornelius Ryan , Hatcher Lori , Sansom Steven
Published: June 2021

The calcium‐activated potassium channel (BK) is comprised of a pore‐forming alpha (BK‐a) and ancillary beta 4 subunit (BK‐b4) in the collecting duct where it has a role to promote urinary K excretion when mice are maintained on a high K, alkaline diet (HK‐alk). We previously showed that both BK‐a and BK‐b4 are up‐regulated in the kidneys of mice on HK‐alk, but not HK‐acid diet. Here we examined the role of BK‐b4 in the regulation of apical BK‐b expression. Immunofluorescence with anti‐BK‐a showed that BK‐a localized mostly in the cytoplasm of intercalated cells (IC) in the mouse cortical collecting ducts (CCD) and was perinuclear in both principle cells (PC) and IC of medullary collecting ducts (MCD). Using anti‐V‐ATPase as a marker for IC subtypes, we found that wild type mice (WT) fed HK‐alk exhibited increased BK‐a expression at the apical membrane of a‐IC, b‐IC, and non‐A, non‐B IC of the CCD and MCD; however, no significant change in the localization of BK‐a was found in WT on HK‐acid. BK‐b4 knock‐out mice (b4‐KO) did not exhibit apical BK‐a expression when consuming HK‐alk. When WT mice on HK‐alk were treated with spironolactone (aldosterone inhibitor) the apical BK‐a expression in the CCD and MCD was significantly reduced. These data suggest that aldosterone mediates the HK‐alk‐induced increase in expression of BK‐b4, which helps trafficking of BK‐b to the apical membrane, thereby promoting urinary K excretion.
Expression and function of the Ca2+‐dependent SK3 K+ channel in mouse cortical collecting duct: Regulation by TRPV4
Authors: Berrout Jonathan , Mamenko Mykola , Zaika Oleg L , Pochynyuk Oleh , O'Neil Roger G
Published: June 2021

The cortical collecting duct (CCD) is a major site of flow‐induced K+ secretion that is thought to be predominantly mediated by the large conductance Ca2+‐dependent K+ channel (BK). We now report the expression of the small conductance, Ca2+‐dependent, SK3 K+ channel in mouse CCD. Expression of SK3 in fixed kidney sections was detected by immunohistochemistry in both principal cells (PC, Aquaporin 2‐positive cells) and intercalated cells (IC) with dominant staining at the luminal cell border (modest staining at basolateral border). Membrane potential measurements demonstrated that activation of TRPV4, using the selective TRPV4‐agonist (GSK1016790A), induced calcium influx and activation of SK3 (and BK) in isolated, split‐open CCD. High flow rates (a 10‐fold increase in flow) induced calcium influx and revealed activation of both SK3 and BK channels in CCD, effects that were abolished by application of the selective TRPV4‐ antagonist (HC‐067047). We conclude that SK3 is expressed in both PC and IC cells of the CCD, and that TRPV4 mediates activation of SK3 and BK. Further, these findings suggest that SK3 channels may play an important role, alongside BK channels, in mediating K+ secretion under conditions of high luminal flow rate.
Sphingosine‐1‐phosphate Modulates Aldosterone‐induced Epithelial Sodium Channel Subunit Trafficking in Renal Cortical Collecting Duct Cells
Authors: Han Wei‐qing , Zhu Qing , Hu Junping , Li Pin‐Lan , Li Ningjun
Published: June 2021

We reported previously that sphingosine‐1‐phosphate (S1P) inhibited amiloride‐sensitive epithelial Na+ channels (ENaC) in the renal medulla. The present study aimed to investigate the underlying molecular mechanisms using cultured M1‐cortical collecting duct cell line. Transepithelial voltage and resistance were measured with a volt ohm meter. Equivalent current (Ieq) was calculated as the ratio of voltage to resistance. Results showed that S1P significantly inhibited aldosterone (ALDO)‐induced Ieq increase. In the presence of S1P1 receptor antagonist W146, however, the inhibitory effect of S1P was blocked (Control, 1.79 ± 0.11; ALDO, 3.57 ± 0.32; ALDO + S1P, 2.42 ± 0.05; ALDO + S1P + W146, 3.22 ± 0.30). In contrast, S1P2 or S1P3 antagonists had no affect. Furthermore, protein kinase A inhibitor 8‐ bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) attenuated ALDO‐induced Ieq increase, but it had no additive inhibitory effect with S1P (Control, 1.82 ± 0.10; ALDO, 3.69 ± 0.30; ALDO + 8‐Br‐cAMP, 2.41 ± 0.39; ALDO + 8‐Br‐cAMP + S1P, 2.35 ± 0.34). In addition, S1P blocked ALDO‐induced cAMP production. Finally, S1P prevented ALDO‐induced α‐ENaC subunit translocation to apical membrane. It is suggested that S1P reduces epithelial Na+ transport via S1P1 receptor‐mediated inhibition of cAMP production and ENaC translocation. (NIH grants HL89563, HL106042 and DK54927).
Mechanoprotection by Polycystins Against Apoptosis is Mediated Through the Opening of Stretch‐Activated K2P Channels
Authors: Duprat Fabrice , Peyronnet Remi , Sharif‐Naeini Reza , Folgering Joost H.A. , Arhatte Malika , Jodar Martine , El Boustany Charbel , Gallian Claire , Tauc Michel , Duranton Christophe , Rubera Isabelle , Lesage Florian , Pei York , Peters Dorien , Somlo Stefan , Sachs Frederick , Patel Amanda Jane , Honoré Eric
Published: June 2021

How cells sense and adapt to mechanical stress remains poorly understood. The TREK two‐pore K+ (K2P) subunits underlie the stretch‐activated K+‐selective channels (SAKs). Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 genes, encoding the polycystins PC1 and PC2. Cystogenesis results mainly from an increase in tubular epithelial cells proliferation. Nevertheless, how a few cysts (typically less than 1% of the nephrons) may impair the function of the majority (99%) of non‐cystic nephrons, ultimately leading to kidney failure, is still an unresolved question. An increase in intra‐renal mechanical stress leading to tubular cells apoptosis and formation of atubular nephrons is a proposed mechanism.

In our study, Pkd1 knock out or expression of a PC2 pathogenic mutant enhances tubular cells apoptosis induced by mechanical stress. We show that polycystins protect renal epithelial cells against mechanical stress and this effect is mediated through the opening of SAKs. We establish for the first time, both in vitro and in vivo, a functional relationship between mechanotransduction and mechanoprotection (Peyronnet et al. 2012 Cell Rep 1(3): 241–250).

Funding: ANR, FRM, EEC, FRHTA, FRC, Human Frontier, Fondation de France, AFM, AIRG, Région PACA, SFHTA.
REGULATION OF GLOMERULOTUBULAR BALANCE IV: IMPLICATION OF AQUAPORIN 1 IN FLOW‐DEPENDENT TRANSPORT AND CELL VOLUME
Authors: Du Zhaopeng , Yan Qingshang , Weinbaum Sheldon , Weinstein Alan M , Wang Tong
Published: June 2021

Underlying glomerulortubular balance (GTB) is the impact of axial flow to regulate Na+ and HCO3− transport by modulating luminal NHE3 and H‐ATPase, and presumably other peritubular transporters, since there is little flow‐dependent change in cell volume. Aquaporin (AQP‐1) serves as a water channel and is the principal water pathway for luminal and peritubular membranes of proximal tubule. We investigated flow‐mediated fluid (Jv) and HCO3− (JHCO3) absorption in proximal tubules (S2) of mouse kidney by microperfusion in vitro in wild‐type (WT) and AQP‐1 KO mice.

Perfusion
Tubule No.
Jv (nl/min/mm)
JHCO3 (pmol/min/mm)
Cell Volume (μm3)

(nl/min)
WT
KO
WT
KO
WT
KO
WT
KO

5
31
9
0.79±0.04
0.57±0.06†
56.3±3.1
61±5.9
1027
1034

20
10
18
1.37±0.06*
0.38±0.03*†
116±7.5*
133±7.8*
1001
946

P<0.05 compared from low flow rate;

P<0.05 compared between WT and KO at same flow rate.

An increase in perfusion rate from 5 to 20 nl/min increased Jv by 73% and JHCO3 by 106% in proximal tubules of WT mice (P<0.001). Compared with WT, AQP‐1 KO significantly decreased Jv at both low and high flow rates (P<0.05), and there was actually a decrease in volume flux in the transition from low to high flow (P<0.05). In contrast, the AQP‐1 KO mice showed no difference from WT in JHCO3 at either low or high flow rates. Cell volume showed no significant difference between either low or high flow rates or between WT and AQP‐1 KO mice. These results confirm the role of AQP‐1 as the principal pathway for water reabsorption in proximal tubule. The decrease in Jv with increasing perfusion rate is consistent with a model in which proximal Na+ reabsorption produces a transepithelial osmotic gradient, which then drives volume reabsorption. Whether axial flow increases AQP‐1 activity, remains uncertain. Flow‐stimulated Na+ and HCO3− absorption is independent of intact AQP‐1.
The membrane cytoskeletal crosslinker ezrin is essential for the regulation of phosphate and calcium homeostasis
Authors: Asano Shinji , Hatano Ryo , Matsumoto Yosuke , Segawa Hiroko , Miyamoto Ken‐ichi , Matsubara Mitsunobu
Published: June 2021

Ezrin is a cross‐linker between the plasma membrane proteins and actin cytoskeleton. In the kidney, ezrin is mainly localized at brush border membrane of proximal tubules with a scaffolding protein, Na+/H+ exchanger regulatory factor (NHERF) 1. NHERF1 interacts with Na+/phosphate (Pi) cotransporter Npt2a. We focused on the physiological role of ezrin in the renal Pi reabsorption by using ezrin knockdown mice (Vil2kd/kd). The Vil2kd/kd mice exhibited hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma concentration of Pi was attributed to the defective urinary Pi reabsorption. Immunofluorescence staining and immunoblotting revealed that the Npt2a and NHERF1 expressions at apical membrane of proximal tubules were markedly reduced in the kidney of Vil2kd/kd mice. On the other hand, urinary loss of Ca2+ was not observed in Vil2kd/kd mice. Plasma concentration of 1,25‐dihydroxyvitamin D was elevated following reduced plasma Pi level. Furthermore, bone mineral density in tibiae was significantly lowered in the adult and young Vil2kd/kd mice. These results suggest that ezrin is required for the regulation of systemic Pi homeostasis in vivo.
Thiazide induces hypocalciuria independent of sodium‐calcium exchanger 1
Authors: Li Wenjun , Bonny Olivier
Published: June 2021

Thiazide diuretics are known to decrease renal calcium excretion. The mechanisms and renal segments involved in thiazide‐induced hypocalciuria remain debated. In the distal tubule, thiazide inhibits Na‐Cl cotransporter and may in turn stimulate Na‐Ca exchanger 1 (NCX1) to promote Ca reabsorption. Here, we examined the role of NCX1 in thiazide‐induced hypocalciuria using kidney‐specific NCX1‐knockout (KO) mice. A single dose of 25 mg/kg hydrochlorothiazide (HCTZ) or vehicle was injected to KO and wild‐type (WT) mice and urines were collected 0, 1, 2, 3, and 6 h afterwards. NCX1‐KO and WT mice showed similar diuretic responses to HCTZ with peaks of urinary Na/creatinine within 1 h. HCTZ‐induced hypocalciuria was observed by the decreases in urinary Ca/creatinine at 2 and 3 h after HCTZ and the extent of reduction being greater in KO than in WT at 3 h. Fractional excretion of lithium (FELi) in WT mice was reduced to 30% of baseline level 2 h after HCTZ. Whereas in KO mice, FELi was 3‐ times lower than WT at baseline and was not further reduced after HCTZ. The sustained ability of NCX1‐KO mice to lower Ca excretion after HCTZ indicated minimal roles of NCX1, and probably distal tubule, in thiazide‐induced hypocalciuria. Changes in FELi suggested that proximal tubule and thick ascending limb may be the major contributors for the hypocalciuric effect. Supported by Swiss National Science Foundation and IKPP (FP7) program.
Interactions between sorting protein‐related receptor and calcineurin to activate renal Na‐K‐2Cl cotransporter
Authors: Mutig Kerim , Borschewski Aljona , Willnow Thomas , Dathe Christin , Paliege Alexander , Ferreri Nicholas , Bachmann Sebastian
Published: June 2021

Activity of the Na‐K‐2Cl cotransporter (NKCC2) of thick ascending limb (TAL) depends on its phosphorylation. The sorting protein‐related receptor SORLA facilitates this process, but the underlying mechanism is still elusive. We hypothesized that SORLA may interfere with kinases or phosphatases relevant for NKCC2 phosphorylation.

We have studied the regulation of the proline/alanine‐rich kinase (SPAK), oxidative stress responsive kinase 1 (OSR1), and calcineurin phosphatase A (CnA) in wildtype (WT) and SORLA‐deficient (SORLA−/−) kidneys in vivo and in cell culture.

Knockdown of SPAK/OSR1 in cultured rat TAL cells decreased, whereas inhibition of calcineurin with cyclosporin A (CsA) increased NKCC2 phosphorylation thus confirming their relevance for the transporter. SORLA‐deficiency was associated with decreased NKCC2 phosphorylation (−84%), unchanged abundance and distribution of SPAK and OSR1 kinases, and increased abundance of CnAβ isoform in the apical compartment of TAL epithelia (+201%). Short term administration of CsA to SORLA−/− mice restored their decreased NKCC2 phosphorylation. Coimmunoprecipitations from rat kidney revealed interactions between CnAβ, NKCC2, and SORLA.

Our results suggest that SORLA participates in calcineurin degradation in TAL thereby supporting activating phosphorylation of NKCC2.
Role of Myosin1a in regulated exocytosis of CFTR in villus enterocytes
Authors: Hoekstra Nadia , Kravtsov Dmitri , Mooseker Mark , Ameen Nadia
Published: June 2021

Insertion of CFTR channels into the intestinal brush border membrane (BBM) is necessary for anion secretion, but the mechanisms of CFTR delivery into the BBM are poorly understood. BB myosins move cargo and regulate membrane trafficking. Myosin1a (Myo1a) regulates CFTR BBM localization and function in villus enterocytes. cAMP and cGMP regulate CFTR traffic into the enterocyte BBM from subapical endosomes. We hypothesized that Myo1a regulates cAMP/cGMP traffic of CFTR into the BBM. Myo1a KO and control mouse intestine were treated with cAMP or cGMP agonists. CFTR distribution was examined by confocal microscopy and surface biotinylation. Immunolocalization revealed absence of BBM CFTR and its accumulation in subapical endosomes in Myo1aKO villus enterocytes. Surface biotinylation confirmed no change in surface CFTR in stimulated Myo1aKO tissues. Specificity of Myo1a was confirmed by lack of changes in BBM localization of ALP, SLC26A6 (PAT‐1), and NHE3. Surface biotinylation of cAMP‐stimulated polarized Caco2BB‐e cells lacking Myo1a expression (shRNA silencing) confirmed CFTR specificity. Surface CFTR was reduced by 98% in Myo1a knockdown (KD) cells compared to control, while surface PAT1 was unchanged in control and Myo1a KD cells. These data support Myo1a as the motor for regulated exocytosis of CFTR. Future studies will seek to dissect the molecular mechanisms regulating Myo1a‐dependent CFTR traffic.
ATP regulates Cl− and K+ channels in human pancreatic ducts
Authors: Novak Ivana , Wang Jing , Hayashi Mikio
Published: June 2021

Pancreas is one of the major organs affected by cystic fibrosis. Purinergic receptors may provide novel strategies for up‐regulation of pancreatic secretion. Our aim was to elucidate how purineric receptors regulate ion transport in human pancreatic ducts and identify Cl− and K+ channels. Transepithelial transport was monitored in monolayers of Capan‐1 and CFPAC‐1 cells in Ussing chambers. Apical ATP/UTP stimulated CFTR and Ca2+‐activated Cl−channels, which were inhibited by CFTRinh‐172 and niflumic acid, respectively. Basolateral ATP stimulated CFTR. In CFPAC‐1 cells (with mutated CFTR), basolateral ATP/UTP had negligible effects. In Capan‐1 cells, effects of DC‐EBIO and clotrimazole indicated functional expression of the intermediate conductance K+ channels (IK, KCa3.1). Apical effects of ATP/UTP were greatly potentiated by IK opener DC‐EBIO. On mRNA and protein level we showed that Capan‐1 cells expressed TMEM16A/ANO1 and KCa3.1. We conclude that in human pancreatic duct cells, ATP/UTP regulates via P2 receptors both Cl− channels (ANO1 and CFTR) and K+ channels (KCa3.1). The K+ channels provide the driving force for Cl− channel‐dependent secretion, and luminal ATP provided locally or from acini potentiate secretory processes.

Funded: the Lundbeck Foundation;The Danish Council for Independent Research | Natural Sciences
SKA‐111, a positive KCa channel gating modulator with selectivity for KCa3.1
Authors: Coleman Nichole , Brown Brandon , Jenkins Paul David , Köhler Ralf , Wulff Heike
Published: June 2021

Intermediate‐conductance KCa3.1 and small‐conductance KCa2.3 channels are co‐expressed in vascular endothelium, where both channels contribute to endothelium‐derived hyperpolarization (EDH) vasodilator responses. However, despite having very similar biophysical properties, KCa3.1 and KCa2.3 seem to play different roles in EDH. While KCa3.1 channels are predominantly localized to endothelial cell projections and are activated by Ca2+ released from the ER, KCa2.3 channels co‐localize with TRP channels and have been shown to be involved in sheer‐stress induced vasodilation. It therefore is perceivable that selective activation of either channel could differentially affect endothelial functions and blood pressure. However, the currently available KCa channel activators like EBIO, NS309 and SKA‐31 at most display 5–10 fold selectivity for KCa3.1 over KCa2.3 making it difficult to interpret in vivo observations made with these compounds. We here report the design of a new benzothiazole, SKA‐111, which displays 160‐fold selectivity for KCa3.1 (EC50 = 30 nM) over KCa2.3 (EC50 = 5 μM) in whole‐cell patch‐clamp experiments. Similar to the prototypical KCa2/3 channel activator EBIO, SKA‐111 is a positive‐gating modulator, which shifts the calcium‐dose response curve of KCa3.1 to the left, making the channel apparently more sensitive to calcium activation. We propose SKA‐111 as a novel pharmacological tool to study the role of KCa3.1 in EDH responses.

Supported by R21 NS072585 from the National Institute of Health
The ClC‐5 N‐terminus confers plasma membrane Cl−/H+ transport capacity to ClC‐3
Authors: Rohrbough Jeffrey , Nguyen Hong‐Ngan , Lamb Fred S
Published: June 2021

ClC family Cl−/H+ exchangers are primarily endosomal proteins that support vesicle acidification and reactive O2 production. When expressed in HEK293 cells, WT ClC‐5 localizes to the plasma membrane and produces outwardly rectifying whole‐cell ion currents, whereas WT ClC‐3 localizes intracellularly and ion currents are absent. We utilized N‐terminus mutations and ClC‐5/3 chimeras in combination with confocal imaging and whole‐cell patch clamp recordings, to show that this region regulates ClC‐5 and ‐3 trafficking and functional ion currents in HEK cells. Disruption of an N‐terminal clathrin‐ binding motif (LLDLLD to LLALLA) localized ClC‐3 to the plasma membrane, but yielded no ion current. By contrast, replacing the ClC‐3 N‐terminus (M1‐ E59) with that of ClC‐5 (M1‐A46) resulted in both plasma membrane localization of “N‐ClC‐5/3”, and rapidly activated, strongly rectifying (ClC‐5‐like) ion currents. Cellular pH changes were quantified with BCECF fluorescence and revealed coupled Cl−/H+ transport during both ClC‐5‐ and N‐ClC‐5/3 currents. These findings directly demonstrate for the first time that ClC‐3 is a functional Cl−/H+ exchanger, and support the hypothesis that the N‐terminus of these proteins directs membrane localization and modulates ion current gating.
Mechanisms Involved in an Acidic pH‐conditioned NOXmediated Chloride Conductance in Nodose Sensory Neurons
Authors: Wang Runping , Lu Yongjun , Whiteis Carol A , Chapleau Mark W , Benson Christopher J , Abboud François M
Published: June 2021

We recently reported a novel prolonged low pH‐conditioned current in nodose sensory neurons following brief exposures to acidic extracellular pH. This current is similar to the Swell‐ Activated Chloride current, and mediated by NADPH oxidase (NOX) dependent hydrogen peroxide. Since NOX is known to be activated by intracellular (IC) alkaline pH, we hypothesized that low extracellular pH conditioning activates NOX by inducing IC alkalinity. We first buffered the IC pipette solution with 40mM HEPES, and found that the pH‐conditioned current was inhibited from 11.8±2.0 pA/pF to 3.4±0.7 pA/pF (n=6, p<0.01), indicating that an IC pH change is required for the current activation. The pH sensitive fluorescence dye 2′‐7′‐bis(carboxyethyl)‐5(6)‐ carboxyfluorescein showed significant but transient decreases in IC pH during the brief exposures to extracellular low pH (n=12). However, sustained reductions of IC pH to 7.0 and 6.0 did not elicit the current. Instead, increases in IC pH to 7.5, 7.6 and 8.0 dose‐dependently induced a similar current averaging 9.2±1.8, 16.3±4.1 and 30.6±9.7 pA/pF. Finally, inhibition of proton extrusion by blocking the Na+‐H+ exchanger with amiloride and proton channels with Zn2+ inhibited the low pH‐conditioned current (n=6). We therefore conclude that transient extracellular pH reductions can be transmitted intracellularly and activate Na+‐ H+ exchanger and proton channels. Our data suggest that the resulting activation of NOX and increase in chloride conductance involve IC alkalinization (NIH HL 14388).
Hydrogen peroxide‐induced inhibition of L‐type Ca channel current in A7r5 cells: Effect of ebselen, dithiothreitol and N‐acetylcysteine
Authors: Ochi Rikuo , Chettimada Sukrutha , Gupte Sachin A.
Published: June 2021

L‐type Ca2+ channel current (ICa,L) is increased by H2O2 in some cardiac and smooth muscle cells by phosphorylation of the channel but is inhibited in some cell types. We studied the mechanism of the inhibition by recording whole cell Ba2+ current from A7r5 cells derived from fetal rat aorta. We examined the effects of ebselen (Ebs), a scavenger of H2O2, dithiothreitol (DTT, 2 mM), a reducing agent, and N‐acetylcysteine (NAC, 10 mM), a precursor of glutathione (GSH) on ICa,L. Ebs is a glutathione peroxidase (GPx) mimic that converts H2O2 and GSH to H2O and GSSG which induces protein glutathionylation. Ebs induced a dose‐dependent inhibition of ICa,L resulting in complete suppression at 3 μM in a few minutes. The inhibition induced by sub‐maximal concentrations was not reversible by washing out and was enhanced by H2O2. H2O2 induced ~40% inhibition of ICa,L at 1 mM with weak voltage‐dependence without affecting ICa,T. Both DTT and NAC markedly reduced inhibitory effect of Ebs and H2O2 and they induced partial recovery of the inhibition when applied after washout of H2O2 and Ebs. We propose that H2O2‐induced inhibition of ICa,L is due to glutathionylation of CaV1.2. NAC did not aggravate but reduced the inhibition probably by facilitating enzymatic reduction of GSH‐protein mixed disulfides via glutaredoxin‐catalyzed pathway. Supported by National Heart, Lung, and Blood Institute Grant RO1‐HL‐085352.
The deltaF508‐CFTR mutation alters the membrane distribution of DMT1 and ferroportin in human cystic fibrosis airway epithelial cells
Authors: Moreau‐Marquis Sophie , Coutermarsh Bonita A , Stanton Bruce A
Published: June 2021

Iron homeostasis is abnormal in patients with cystic fibrosis (CF). Previously, we found that human CF bronchial epithelial cells expressing ΔF508‐CFTR (CFBE+ΔF508 cells), the most common mutation in CF, secrete more iron into the airway surface liquid than WT‐CFTR cells (CFBE+WT cells). Here we report that CFBE+ΔF508 cells express 2.5‐fold more DMT1, which transports iron into cells, and 6‐fold more FPN1, which transports iron out of cells, than CFBE+WT cells. In addition, selective cell surface biotinylation revealed that the ΔF508‐CFTR mutation leads to significant changes in the localization of these two major iron transporters. In CFBE+WT cells, DMT1 and FPN1 were distributed equally in both the apical and basolateral membrane. By contrast, in CFBE+ΔF508 cells, DMT1 polarized to the basolateral membrane (70/30%‐basolateral/apical) while FPN1 polarized to the apical membrane (86/14% apical/basolateral). This polarization of DMT1 and FPN1 can account for the increased transepithelial secretion of iron into the airway surface liquid of CF airway cells, and contribute to the enhanced iron levels in CF airways. To our knowledge, this is the first study reporting a link between the ΔF508‐CFTR mutation and iron transporters involved in iron homeostasis.

Supported by NIH R01‐HL074175 and the CF Foundation (STANTO07R0).
SGK1 Increases Plasma Membrane CFTR in Human Airway Epithelial Cells by Inhibiting Its Endocytic Retrieval
Authors: Bomberger Jennifer , Sato J. Denry , Chapline Christine , Coutermarsh Bonita , Barnaby Roxanna , Stanton Bruce
Published: June 2021

Chloride secretion by CFTR located in the apical membrane of airway epithelial cells (AECs) plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of respiratory pathogens and debris. Previously, others and we have shown that the serum and glucocorticoid‐inducible kinase1 (SGK1) increases the amount of wt‐CFTR protein in the plasma membrane of Xenopus oocytes by an unknown mechanism. The goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of CFTR in human AECs. We report that elevated levels of SGK1, induced by dexamethasone, increases plasma membrane abundance of CFTR. Reduction of SGK1 expression by siRNA (siSGK1) or inhibition of SGK1 activity by GSK‐650394 abrogated the ability of dexamethasone to increase plasma membrane CFTR. By contrast, overexpression of a constitutively active SGK1 (SGK1–422D) increased plasma membrane abundance of CFTR. To examine the mechanism whereby SGK1 increased plasma membrane CFTR the effects of siSGK1 and SGK1‐S442D on the endocytic retrieval of CFTR was examined. Whereas siSGK1 increased CFTR endocytosis, SGK1‐ S442D inhibited CFTR endocytosis. By contrast, neither siSGK1 nor SGK1‐S442D altered the recycling of endocytosed CFTR back to the plasma membrane. This work demonstrates that SGK1 increases CFTR in the plasma membrane of human airway epithelia by inhibiting its endocytic retrieval.
Softening of hyperoxia treated alveolar epithelial cells reduces stretch‐induced injury
Authors: Wilhelm Kristina Rebecca , Roan Esra , Gosh Maik , Waters Christopher M.
Published: June 2021

Objectives

Acute respiratory distress syndrome (ARDS) patients are administered high concentrations of oxygen during mechanical ventilation; hyperoxia and mechanical ventilation can independently cause injury. The combination may even accelerate the injury, but how this injury occurs is still not fully understood. Here we show that stretch‐induced injury following hyperoxia is a function of cell elasticity (E).

Methods

We utilized an atomic force microscope (Asylum) in the indentation‐mode to measure E in cultured alveolar epithelial cells, and a cell‐stretch device to mimic mechanical ventilation. Cell detachment was determined following pre‐treatment with hyperoxia and subsequent cell stretching.

Results

Hyperoxia increased cell elasticity of lung epithelial cells, thus making them more brittle and more susceptible to stretch‐induced injury (Roan et. al). Decreasing E (softening) the cells after hyperoxia treatment by inhibiting RhoA Kinase (Y‐27632), by decreasing f‐actin (cytochalasin D), or by reducing acto‐myosin contractility (blebbistatin) reduced subsequent stretched‐induced injury.

Conclusion

Stretch‐induced injury in ARDS patients may be reduced by lowering the stiffness of cells.
Transient receptor potential cation channel vanilloid (TRPV) 4 in ventilator‐induced lung injury (VILI)
Authors: Michalick Laura , Mertens Michael , Liedtke Wolfgang , Kuebler Wolfgang Michael
Published: June 2021

Study objective

Ca2+ entry via TRPV4 has been implicated in stretch‐induced barrier failure in isolated lungs. Here, we assessed the relevance of TRPV4 in VILI in vivo, and probed for the contribution of serum/glucocorticoid‐regulated kinase (SGK) 1, which phosphorylates TRPV4, and of Ca2+‐activated K+ channels (SK, BK) as putative signalling mechanisms upstream and downstream of TRPV4, respectively.

Methods

Male, anesthetized mice were mechanically ventilated for 2 h by low (7 ml/kg) and high (20 ml/kg) tidal volumes (LVT/HVT) in wild type and TRPV4‐deficient mice, and following pharmacological inhibition of SGK1, TRPV4, SK or BK channels.

Results

TRPV4 deficiency or inhibition by HC‐067047 attenuated VILI as demonstrated by reduced lung edema, protein leak, MPO activity, inflammatory cytokine levels, and histological features of lung injury. Inhibition of SK or BK channels by apamin or charybdotoxin attenuated lung edema in HVT mice, yet only charybdotoxin had concomitant anti‐inflammatory effects. Lung edema formation in HVT mice was also largely reduced by the SGK1 inhibitor GSK650394.

Conclusions

Here, we demonstrate the critical role of TRPV4 for VILI in vivo, and identify potential upstream (SGK1) and downstream (SK, BK) signalling events that may present putative pharmacological targets for the prevention or treatment of ventilator‐associated lung disease.
Synchronized activation of alveolar macrophages determined by live alveolar imaging
Authors: Westphalen Kristin , Islam Mohammad Naimul , Bhattacharya Jahar
Published: June 2021

Alveolar macrophages (AMs) play a major role in lung defense against pathogens. However, it is not known whether AMs intercommunicate signals in response to pathogen challenge. To determine AM signals in situ, we crossed CD11cCre mice with R26‐LSL‐EYFP floxed mice to obtain mice containing EYFP‐expressing AMs (AMYFP). We gave i.n. endotoxin (1 mg/kg) or buffer (control) to AMYFP expressing mice, then after 24 h, we excised and blood‐perfused their lungs at constant pulmonary artery, left atrial and alveolar pressures. We viewed the lungs by live confocal microscopy, to image 30–40 alveoli containing a total of 10–15 AMYFP. To detect cytosolic Ca2+ signals as a marker of activation, we microinfused alveoli with the Ca2+‐sensitive dye, fluo‐4 (10μM). In control lungs, AMYFP cytosolic Ca2+ levels were stable. By contrast in endotoxin‐treated lungs, AM activation was indicated in that 45±5% of the AMYFP evoked slow‐frequency Ca2+ spikes (2±1 spikes/20 min) of which 67±3% occurred synchronously amongst the activated AMYFP. We show here for the first time, that AMs can be viewed in situ in real time, and that following endotoxin treatment, subsets of AMs evoke synchronized Ca2+ signaling, suggesting that endotoxin‐challenged AMs develop network responses. AM networks may orchestrate lung defense against endotoxin challenge (HL78645, HL64896).
Rac1 inhibition decreases branching in embryonic mouse lung explants
Authors: Danopoulos Soula , Warburton David , Kim Richard , Bellusci Saverio , Al Alam Denise
Published: June 2021

Epithelial mesenchymal interactions, cell proliferation, polarity, migration and differentiation are essential for proper lung branching morphogenesis. Rac1, a small Rho GTPases, plays a key role in cell polarity and cell migration. Therefore, we hypothesize that Rac1 controls branching morphogenesis during embryonic lung development. We used an in vitro lung culture model to assess the effect of NSC23766, a Rac1 inhibitor, on lung branching. Using 50μM (IC50) of Rac1 inhibitor, we observed a decrease in branching and necrosis of the tissue at 48 hrs. At 25 μM, Rac1 inhibitor decreased branching without necrosis. This decrease was accompanied with a reduction in cell proliferation. Furthermore, using Wnt reporter, TOPGAL, Rac1 inhibition reduced the expression of LacZ. In addition, using Flk1‐LacZ reporter, we observed a decrease in LacZ expression in presence of Rac1 inhibitor. Our data suggests that Rac1 controls epithelial and vascular morphogenesis through inhibition of Wnt signaling. Hence, dissecting the molecular pathways underlying Rac1 activity could lead to the discoveries of new therapeutic targets for arrested lung development.
PDE6s generate cGMP microdomains in native endothelial cells
Authors: Blanchette Alexandre , Toussaint Fanny , Charbel Chimène , Comtois Philippe , Ledoux Jonathan
Published: June 2021

Cyclic GMP (cGMP) is a key messenger for cellular functions including ion channel regulation and gene expression. Nucleotides microdomains have a significant impact on cellular function. Phosphodiesterases (PDEs) play a prime role in the regulation of these functions through hydrolysis of cGMP and thus regulation of cGMP levels. Endothelial PDEs were studied for their impact on local cGMP levels. PDE5 and PDE9 are cGMP‐specific PDEs expressed in endothelial cells. Interestingly, the retina‐confined PDE6 have also been found in freshly isolated native endothelial cells by qPCR and immunostaining. Although PDE5 and PDE6γ are homogeneously distributed, PDE6α and PDE6β are mainly found in the perinuclear region. While PDE6β forms a continuous ring, PDE6α are distributed in clusters within the same region. Mathematical modeling revealed the dissimilar role of PDE5 and PDE6s in the spatial regulation of local cGMP following a bolus of NO. The presence of PDE6s near the nucleus suggests a potential role in the regulation of transcription factors. Therefore, endothelial PDEs and more specifically PDE6, might play a significant role in the control of cGMP‐mediated cellular functions through the fine‐tuning of cGMP microdomains. Supported by FRQS, HSFC and NSERC.