bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hyperpolarized 13C MRS reveals hypoxia accelerates pseudo hypoxia in the diabetic kidney (890.8)
Authors: Laustsen Christoffer , Lycke Sara , Palm Fredrik , Østergaard Jakob , Bibby Bo , Nørregaard Rikke , Flyvbjerg Allan , Pedersen Michael , Ardenkjær‐Larsen Jan 
Published: June 2021

In diabetic patients, reduced renal oxygen availability is associated with a higher risk of diabetic nephropathy (DN) [1]. Recently, dynamic nuclear polarization hyperpolarized [1‐13C]pyruvate has shown an increased lactate conversion (lactate dehydrogenase activity) in the diabetic kidney, as a consequence of increased nicotinamide adenine dinucleotide (NADH) substrate generated by glucose‐mediated elevated flux through the polyol pathway, while the pyruvate dehydrogenase activity (PDH) is unaltered (pseudo hypoxia) [2]. In this study, we evaluated the renal oxygenation level and energy metabolism in diabetes‐suffering rats in response to altered oxygen availability, using blood oxygen level dependent (BOLD) MRI and hyperpolarized [1‐13C]pyruvate MRI, in response to hypoxic, normoxic and hyperoxic conditions. Specifically, we investigated the enzymatic activity of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and pyruvate dehydrogenase (PDH). We show that reduced oxygen availability in the diabetic kidney alters the energy metabolism of pyruvate by increasing the lactate and alanine formation from pyruvate despite intact oxidative phosphorylation.Grant Funding Source: Supported by The Simon Spies Foundation, The Danish Kidney Foundation, Helen and Ejnar Bjørnows Foun
Deficiency in NBCe2 causes distal renal tubular acidosis (891.2)
Authors: Wen Donghai , Cornelius Ryan , Warner Paige , Boettger Thomas , Sansom Steven 
Published: June 2021

The pathogenesis of distal renal tubular acidosis (dRTA) is not fully understood. Here we show that the electrogenic Na/HCO3 cotransporter NBCe2 is involved in the pathophysiology of dRTA by reabsorbing HCO3 in the collecting duct (CD). NBCe2 knockout (KO) and wild type (WT) mice were fed with acid water for 7‐10 days and placed in metabolic cages. Immunohistochemistry (IHC) and Western blot were used to examine the expressions of NBCe2 and H‐ATPase B1 in the kidneys. IHC showed NBCe2 was expressed mostly at the apical membrane of Type A intercalated cells (α‐IC) of the CD. The H‐ATPase B1 expression was higher in the plasma membrane of α‐IC in NBCe2 KO than WT, suggesting compensation for NBCe2 defect. As compared with WT, NBCe2‐KO exhibited significantly lower plasma pH, [HCO3] and [K], while the urine pH, K clearance and transtubular K gradient were increased. NBCe2 expression was also examined in cultured MDCK‐C11 cells by immunocytochemistry (ICC), which showed that NBCe2 was expressed selectively at the apical membrane in C11 cells. The intracellular pH (pHi) recovery rate in C11 cells was recorded using BCECF. Knocking down of NBCe2 expression by siRNA significantly decreased the pHi recovery rate in C11 cells. In conclusion, NBCe2 plays an important role in reabsorbing HCO3 in the CD. NBCe2 deficiency leads to hypokalemic acidosis and is another form of dRTA.Grant Funding Source: NIDDK
Protein phosphatase 1 Inhibitor‐1 deficiency causes hypotension and reduces the phosphorylation of renal NaCl cotransporter (892.18)
Authors: Picard Nicolas , Trompf Katja , Yang Chao‐Ling , Miller R. , Carrel Monique , Loffing‐Cueni Dominique , Fenton Robert , Ellison David , Loffing Johannes 
Published: June 2021

The thiazide‐sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and blood pressure (BP). Gain‐of‐function in NCC‐regulating kinases or loss‐of‐function of NCC alters BP. The size and location of the DCT has hindered efforts to identify new molecular regulators of this protein. Here, we performed automated sorting of mouse DCTs and microarray analysis for identification of DCT‐enriched gene products, which may regulate NCC. We identified protein phosphatase‐1 inhibitor‐1 (I‐1) as a DCT enriched transcript. Immunolocalisation revealed I‐1 expression in mouse and human DCTs and thick ascending limbs. Coexpression of NCC with I‐1 increased thiazide‐ dependent Na+ uptake, whereas RNAi‐mediated knockdown of I‐1 reduced NCC phosphorylation. Likewise, levels of phosphorylated NCC decreased by approximately 50% in I‐1 knockout mice (I1‐/‐). Compared with WT mice, I1‐/‐ mice had lower arterial BP, but did not display other metabolic features of NCC dysregulation. Thus, I‐1 is a DCT enriched gene product that controls arterial BP, possibly through regulation of NCC.Grant Funding Source: Swiss National Fund
Beta‐adrenergic receptor stimulation enhances phosphorylation of NKCC2 at threonine96/101 via cAMP but independently of PKA (892.21)
Authors: Haque Mohammed , Ares Gustavo , Ortiz Pablo 
Published: June 2021

Beta‐adrenergic receptor (β‐AR) stimulation enhances phosphorylation of NKCC2 at threonine96/101 via cAMP but independently of PKA Mohammed Z. Haque, Gustavo R. Ares and Pablo A. Ortiz The thick ascending limb (TAL) reabsorbs 30% of the filtered sodium via the apical Na‐K‐2Cl cotransporter NKCC2. NKCC2 activity and trafficking to the apical surface are enhanced by β‐AR stimulation via cAMP. NKCC2 activity may also be stimulated by phosphorylation at threonine96/101 (Thr96/101) by SPAK/OSR1 kinases and serine126 (Ser126) by PKA. However, it is not known whether SPAK and NKCC2 phosphorylation (P‐NKCC2) at Thr96/101 are enhanced by cAMP and stimulation of β‐ARs in the TAL. We hypothesized that β‐AR stimulation increases Thr96/101 P‐NKCC2 via cAMP and SPAK. To maximally stimulate β‐ARs signaling we treated TALs (20 min) with 1µM isoproterenol and the phosphodiesterase4 inhibitor rolipram (ISO). ISO enhanced Thr96/101 and Ser126 P‐NKCC2 by 79±28% and 71±28% (p<0.05, n=6). ISO also enhanced phosphorylated SPAK at Ser373 by 50±4 % (p<0.05, n=6). Inhibition of PKA (H‐89,10 µM) did not inhibit β‐AR stimulated Thr96/101 P‐NKCC2. We then tested whether cAMP enhanced Thr96/101 P‐NKCC2 by stimulating endogenous cAMP with forskolin and the phosphodiesterase inhibitor IBMX (20 min). cAMP increased Thr96/101 by 30± 8% (p<0.05, n=4) and this was not blocked by PKA inhibition; whereas Ser126 P‐NKCC2 was enhanced by 32±6 fold (p<0.05) and PKA inhibition blocked this effect by 84±4%. We conclude that β‐adrenergic receptor stimulation enhances NKCC2 phosphorylation at Thr96/101 via cAMP and SPAK/OSR1 whereas PKA mediates the effect of cAMP on Ser126.Grant Funding Source: AHA/NIH
EGF/RhoGDIα/Rac1 pathway modulates ENaC activity and contributes to the development of salt‐sensitive hypertension (892.27)
Authors: Pavlov Tengis , Levchenko Vladislav , Ilatovskaya Daria , Palygin Oleg , Staruschenko Alexander 
Published: June 2021

Epithelial sodium channel (ENaC) performs sodium reabsorption in the aldosterone sensitive distal nephron. Amongst other hormonal agents, ENaC‐mediated sodium reabsorption is regulated by the members of epidermal growth factor (EGF) family; specifically, chronic treatment with the EGF‐family members downregulates ENaC activity in vitro. We have recently shown that EGF deficiency is involved in improper ENaC activity and contributes to the development of salt‐induced hypertension in Dahl salt‐sensitive (SS) rats. Investigation of signaling pathways potentially involved in regulation of ENaC by EGF, revealed that high salt diet in SS rats decreases abundance of Rho GDP‐dissociation inhibitor 1(RhoGDIα), a well characterized modulator of Rho‐family small GTPases. RhoGDIα deficiency in cultured mCCDcl1 principal cell line increases ENaC‐mediated current formation and causes enhanced response of ENaC activity to EGF treatment. Further experiments revealed that these effects are mediated via Rac1 small GTPase. We conclude that renal EGF deficiency in the SS rats on a high salt diet provokes excessive ENaC activity via RhoGDIα/Rac1pathway and contributes to the development of salt‐induced hypertension.Grant Funding Source: Supported by AHA, NHLBI
Signaling mechanisms for post‐transcriptional regulation of the type IIa sodium‐phosphate cotransporter mRNA by parathyroid hormone (892.35)
Authors: Murray Rebecca , Khundmiri Syed , Clark Barbara , Lederer Eleanor 
Published: June 2021

PTH is a critical regulator of serum phosphorus. We have previously shown that PTH decreases proximal tubule NpT2a mRNA expression through post‐transcriptional mechanisms, and that this effect can be reproduced by treatment with 8‐bromo‐cAMP, a PKA activator, but not phorbol myristate acetate, a PKC activator. We hypothesize that PKA is the primary mediator of NpT2a mRNA destabilization by PTH. To determine the contribution of each pathway to the PTH response, opossum kidney (OK) cells were treated with selective protein kinase inhibitors in the presence of PTH, and NpT2a mRNA expression was measured by qRT‐PCR. Pretreatment with 10µM PKC inhibitor peptide (PKCi) followed by 100nM PTH for 6h produced a 43.9% decrease in NpT2a mRNA versus PKCi alone. Pretreatment with 10µM H‐89, a PKA inhibitor, prevented the initial decrease in NpT2a mRNA by PTH but did not prevent the ultimate reduction. 8CPT‐2Me‐cAMP, a selective activator of Epac, failed to produce a decrease in NpT2a mRNA after 6h. We conclude that the initial effect of PTH on NpT2a mRNA is PKA‐dependent, whereas chronic regulation involves both the PKA and PKC pathways.Grant Funding Source: Support for this project is provided by VA to EDL.
Renal phenotype of Sortilin‐deficient mice (892.41)
Authors: Bikulowa Kamila , Borschewksi Aljona , Dathe Christin , Willnow Thomas E. , Bachmann Sebastian , Mutig Kerim 
Published: June 2021

Sortilin belongs to the family of the VPS10P‐domain receptors which mediate intracellular sorting of diverse ligands in various cell types. Sortilin is abundantly expressed in the kidney but little is known about its renal function. In this study we take advantage of sortilin‐deficient (Sort‐/‐) mice to test whether sortilin interferes with renal NaCl‐ or water reabsorption.To evaluate the role of sortilin in the kidney, sortilin‐deficient (Sort‐/‐) mice were analyzed for their physiologic kidney performance, kidney morphology, and biochemical profile of renal transporters.In wildtype (WT) mouse kidneys, sortilin has shown moderate expression in the distal nephron and strong expression in the principal cells of collecting duct. Sortilin deficiency had no major effects on kidney morphology. Physiologic evaluation of WT and Sort‐/‐ mice showed significantly increased urinary volume (+154%) upon sortilin disruption. Immunoblotting evaluation of distal epithelial salt transporters and water channels revealed that sortilin‐deficiency was associated with decreased levels of the Na‐Cl cotransporter (NCC) and of the phosphorylated, activated aquaporin 2.Our results suggest that sortilin facilitates the reabsorptive function of renal distal epithelia.
Distinct mechanisms of [Ca2+]i responses to flow and hypotonicity in the distal nephron (892.5)
Authors: Mamenko Mykola , Zaika Oleg , Boukelmoune Nabila , O'Neil Roger , Pochynyuk Oleh 
Published: June 2021

Changes in luminal fluid flow or osmolality elicit [Ca2+]i elevations in the distal nephron (DN) cells to regulate water‐electrolyte transport. Our group has recently identified a pivotal role of TRPV4 in mediating flow‐induced Ca2+ elevations in the DN cells, however little is known about molecular nature of the response to osmotic gradients. Here, using ratiometric Fura‐2 Ca2+ imaging in freshly isolated split‐opened DNs from wild‐type (WT) mice, we found that hypotonicity elicits a uniform and reproducible increase of [Ca2+]i in DN cells. This response is abolished by removal of extracellular Ca2+, but only moderately diminished upon depletion of intracellular Ca2+ stores. While genetic ablation of TRPV4 abrogates flow‐induced [Ca2+]i elevations, the response to hypotonicity, though significantly blunted, is retained in TRPV4‐/‐ mice. This points to additional mechanisms increasing [Ca2+]i in response to osmotic gradients. Indeed, pharmacological inhibition of Ca2+‐permeable TRPC3 channel markedly diminishes the amplitude of [Ca2+]i elevations induced by hypotonic stimuli in DN epithelium from WT mice. Moreover, the threshold of the response to decreased osmolality is elevated in TRPC3 knockouts. Thus, in contrast to flow‐induced [Ca2+]i elevations mediated by TRPV4, hypotonicity elicits a multifaceted Ca2+ response, at least in part due to coordinated activation of TRPV4 and TRPC3.Grant Funding Source: DK095029
Modulation of sodium channels by purine nucleotides in mouse vomeronasal sensory neurons (893.1)
Authors: Vick Jonathan , Rickner Hannah , Delay Rona 
Published: June 2021

The mouse vomeronasal organ (VNO) is a chemosensory structure that regulates many social and fear related behaviors. Social odorants, colloquially known as pheromones, are detected within the lumen of the VNO by bipolar vomeronasal sensory neuron (VSN)s. We have previously shown that adenosine 5’‐triphosphate (ATP), a potent neuromodulator in chemosensory systems, elicits an inward current and action potentials in VSNs through activation of ionotropic P2X receptors. However, it was unclear if G‐protein coupled P2Y receptors were involved in modulating the excitability of VSNs. Using RT‐PCR, we showed that P2Y1, 2, and 6 were expressed in the VNO. Immunohistochemical labeling with antibodies specific to P2Y1 and 2 revealed widespread expression of both receptors in the sensory and nonsensory epithelia. Using whole cell patch clamp, we observed that while ATP was without effect, adenosine 5’‐diphosphate (ADP) hyperpolarized the voltage dependence of sodium channel steady state inactivation in a subset of VSNs. Moreover, neither ATP nor ADP had any effect on the voltage dependence of sodium channel activation or fast inactivation. We hypothesize that following activation of P2X receptors by ATP, the rapid breakdown of ATP to ADP by ubiquitous ecto‐endonucleases would result in a reduction of sodium channel availability through activation of P2Y receptors.Grant Funding Source: NIH
Tumour necrosis factor alpha and interferon gamma induce transcriptional downregulation of aquaporin 3 RNA expression through distinct mechanisms (893.28)
Authors: Peplowski Michael , Vegso Andrew , MacNaughton Wallace 
Published: June 2021

Although aquaporins (AQP) are known to be involved in water movement, the role of AQP3 in the barrier dysfunction that characterizes inflammatory bowel diseases remains unknown. We hypothesized that TNFα and IFNγ were involved in the transcriptional inhibition of AQP3 RNA expression in intestinal inflammation. Methods: AQP3 expression was assessed in C57Bl/6 mice administered dextran sodium sulfate (DSS) to induce colonic inflammation. In vitro, the human adenocarcinoma cell line HT29 was treated with either TNFα or IFNγ and AQP3 pre‐mRNA and mRNA expression were assessed by real‐time RT‐PCR. Cytokine‐responsive transcriptional elements were elucidated using AQP3 promoter driven firefly luciferase expression.Results: AQP3 was decreased early in DSS colitis, with diminished basolateral membrane staining in epithelial cells lining colonic crypts. Similarly, TNFα or IFNγ‐treated HT29 cells had decreased AQP3 pre‐mRNA expression at 2 hr, followed by decreased mRNA expression at 6‐12 hr. IFNγ‐induced transcriptional inhibition of AQP3 expression was reversed using a broad‐spectrum JAK inhibitor (JAK Inhibitor I, 10 µM), but not a JAK2 specific inhibitor (JAK2 Inhibitor II, 10 µM). Truncated AQP3 promoter constructs revealed an IFNγ‐responsive transcriptional element in the 251bp region upstream of the TATA box and a transcriptional suppressor in the 251 ‐ 666bp region upstream of the TATA box. In contrast, TNFα‐induced transcriptional inhibition of AQP3 expression was not reversed by inhibitors of the PI3K, AKT, NF‐κB, ERK/MAPK and p38 MAPK pathways (LY294002, 10 µM; Triciribine, 1 µM; BAY11‐7082, 30 µM; U0126, 10 µM and SB203580, 10 µM respectively).Grant Funding Source: CCFC, CIHR & AIHS
Cleaved prolactin increases activity of amiloride‐sensitive sodium channels in mouse collecting duct principal cells (893.3)
Authors: Greenlee Megan , Chen Rose , Mitzelfelt Jeremiah , Eaton Douglas 
Published: June 2021

In the renal collecting duct, sodium reabsorption through the epithelial sodium channel (ENaC) creates an osmotic driving force for water reabsorption resulting in increased blood volume and pressure. Investigators have previously shown that prolactin (PRL) stimulated ENaC activity in bullfrog skin cells. However, studies in mammalian systems show no effect of PRL on ENaC function. PRL is present in two forms: a 23kDa full length form (PRL) and a 14‐16kDa cleaved form (PRL‐C). We hypothesized that these two PRL molecules may exert different effects on ENaC activity. To investigate this hypothesis, we used a cell culture model of principal cells from the cortical collecting duct (mpkCCD) which express ENaC subunits and exhibit amiloride‐sensitive current. We measured transepithelial voltage and resistance across cell monolayers before and after basolateral treatment with vehicle (water) or 1‐2 ug/mL of PRL or PRL‐C and calculated transepithelial current using Ohm’s law. We found that PRL produced only a small, variable effect on ENaC activity in mpkCCD cells. However, PRL‐C approximately doubled the amiloride‐sensitive current in these cells within 24 hrs. Compared to kidney lysates, mpkCCD cells exhibit a low abundance of the PRL receptor which may explain the need for large PRL concentrations in these cells. Our results indicate that cleaved PRL stimulates ENaC activity in mpkCCD cells. In future studies, we will examine the effect of PRL and PRL‐C on single channel properties of ENaC in isolated collecting ducts and the effect of these hormones on blood pressure in vivo.Grant Funding Source: Supported by NIH K12GM000680 and T32HL076118
Physiology and cell biology of clinical mutants of the human riboflavin transporter‐2: studies utilizing the human‐derived brain U87 cells (896.2)
Authors: Subramanian Veedamali , Kapadia Rubina , Ghosal Abhisek , Said Hamid 
Published: June 2021

Riboflavin (RF) is essential for normal cellular functions of all cells including those of the brain. RF is transported into human cells via three membrane transporters, hRFVT‐1,‐2 and ‐3. Mutation in hRFVT‐2 (which is predominantly expressed in brain cells) is believed to be the cause of the rare neurodegenerative disorder, Brown‐Vialetto‐Van Laere Syndrome (BVVLS). Little,however, is known about how such mutations affect the physiology and cell biology of hRFVT‐2. We addressed this issue using the human‐derived brain U87cells as model and live cell confocal imaging.Our results showed marked inhibition in RF uptake in U87 cells stably expressing clinical mutants L123P, L140P, G306R and L339P. Live cell confocal imaging of U87 cells stably expressing hRFVT‐2 clinical mutants showed the L123P and L339P mutants were retained within the endoplasmic reticulum, while the L140P and G306R mutants were expressed at the cell membrane though at reduced protein expression levels compared to wild‐type construct. These results show that some of the BVVLS associated mutations in hRFVT‐2 affect the transporter functionality and that this effect is mediated via alterations in membrane targeting and/or protein expression. (Supported by DVA and NIH grant DK 56061‐15).Grant Funding Source: DVA and NIH
Characterization of a human UT‐B urea transporter antibody (897.3)
Authors: Walpole Caragh , Winter Desmond , Baird Alan , Stewart Gavin 
Published: June 2021

Urea entry into the gastrointestinal tract is facilitated by the urea transporter UT‐B1 (Stewart 2011). This process is crucial to our intestinal wellbeing as it is the first step in the urea nitrogen salvaging mechanism, which is necessary for maintaining healthy gut bacterial populations. In a previous study using a bovine UT‐B antibody, a 35kDa human UT‐B1 protein was found to be highly expressed in the ascending colon (Collins et al., 2010). The aim of this study was to produce a reliable human UT‐B antibody to be used for further investigations of human UT‐B expression. Initial PCR analysis using primers designed along the UT‐B gene confirmed UT‐B1 as the major isoform expressed at the RNA level in all human gastrointestinal tissues. Three polyclonal human UT‐B antibodies ‐ one designed against the N‐terminal (UT‐B#N) and two against the C‐terminal (UT‐B#C and UT‐Bc19) ‐ were made according to the UT‐B1 amino acid sequence and extensive characterization was carried out using Western blot analysis. While both C‐terminal antibodies detected UT‐B1 in the membrane of erythrocytes as a large glycosylated signal at 45‐60kDa as expected, the UT‐B#N antibody failed to do so and was rejected. As UT‐B transporters are glycoproteins, deglycosylation experiments were performed on UT‐B1 protein using PNGaseF enzyme. While UT‐Bc19 successfully detected 36 kDa deglycosylated UT‐B1, UT‐B#C failed to do so and was also rejected. Next, β‐mercaptoethanol was used to reduce UT‐B1 from its native multimer to a monomer and UT‐Bc19 successfully detected a big shift from a smeared 40‐100kDa signal to a neat 40kDa signal. Finally, UT‐Bc19 also detected a strong 40‐70kDa glycosylated UT‐B1 signal in human ileum protein. In conclusion, these data have shown that UT‐Bc19 is the only consistent human UT‐B antibody. Future investigations of UT‐B1 expression will therefore utilise UT‐Bc19 with confidence and help establish the physiological significance of UT‐B1 in the human gastrointestinal tract.Grant Funding Source: Supported by The Wellcome Trust and Science Foundation Ireland.
NHE3 and Clostridium difficile infection (897.5)
Authors: Engevik Melinda , Yacyshyn Mary , Yacyshyn Bruce , Haslam David , Shull Gary , Worrell Roger 
Published: June 2021

Although changes in the intestinal microbiota have been linked to diabetes, obesity, Inflammatory Bowel Disease, and Clostridium difficile‐associated disease, it remains unclear how the intestinal environment, set by ion transport, affects luminal and mucosa‐associated bacterial composition. NHE3 plays an integral role in intestinal Na+ absorption, as indicated by chronic diarrhea in NHE3‐/‐ mice. In a renal cell lines C. difficile toxin B causes internalization of NHE3. We hypothesize that inhibition of NHE3, by genetic knockout or C. difficile toxin, promotes the proliferation of bacteria capable of growing in a high Na+, alkaline pH environment, altering the gut microbiota composition, which may enhance the C. difficile niche. qPCR was performed on 16S DNA extracted from NHE3‐/‐ mouse intestine and human CDI stool. Flame photometry, chloridometry and pH probe was used to measure ion composition and pH. Immunohistochemistry was used to analyze NHE3 localization and mucus oligosaccharide composition. Similar to distal colon samples from NHE3‐/‐ mice, fecal samples from humans with C. difficile infection was associated with decreased Firmicutes and increased Bacteroidetes. Fecal [Na+] was also similarly elevated being consistent with NHE3 inhibition in human CDI. Indeed, NHE3 appeared to be down regulated in biopsy’s from C. difficile infected patients, which likely contributes significantly to diarrhea. These changes also correlated with changes in host mucus oligosaccharide composition. Together these data demonstrate that loss of functional NHE3 occurs in CDI and the resulting alteration in the intestinal environment, may contribute to region‐specific changes in bacteria which serve to favor C. difficile niche development and maintenance.Grant Funding Source: Supported by NIH DK079979 to RTW