bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temporal progression of salt‐sensitivity in Hsd11b2 Knockout rat
Authors: Mullins Linda Jane , Ivy Jessica , Bailey Matthew , Kenyon Christopher , Mullins John J 
Published: November 2023

Targeted knockout of the Hsd11b2 gene, using ZFNs, generated four founders, one of which carried multiple targeted alleles. The resultant Hsd2KO rat model faithfully recapitulated the syndrome of apparent mineralocorticoid excess (SAME). Adult animals were small, polydypsic and polyuric, and significantly hypertensive on 0.3% Na diet (mean arterial blood pressure (MABP) of 170 mmHg compared to controls of 120 mmHg). Plasma renin showed significantly reduced circadian rhythm in Hsd2KO animals on both 0.3% and 0.03% salt diets. On switching to the 0.03% Na diet, MABP fell dramatically to ~130 mmHg, suggesting extreme salt sensitivity. This study aimed to determine how the salt sensitive hypertension developed in young animals.Renal clearance studies revealed that Hsd2KO animals were already hypertensive (158 versus 119mmHg controls) and plasma volume contracted (Haematocrit 49% versus 42% control) at ~5 weeks of age and showed profound hypo‐kalemia (Hsd2KO 2mM versus Control 4.5mM plasma potassium), with increased fractional potassium excretion and urinary potassium. Glomerular filtration rate and renal plasma flow were normal compared to controls.MABP and haematocrit measurements between 2 and 5 weeks of age showed that, by 33 days, the Hsd2KO pups had increased MABP compared to controls. However, metabolic study revealed that Hsd2KO animals were already polydipsic and polyuric by 23‐days old.These data tease apart the developmental steps that lead to salt‐sensitive hypertension in the Hsd2KO rat, which could prove informative, given the habitually high dietary salt intake in most developed nations.Support or Funding InformationBritish Heart Foundation Center of Research Excellence Award
Novel Calcium Transport Pathways Mediate Intestinal Calcium Absorption Pre‐Weaning
Authors: Beggs Megan R , Lee Justin J , Alexander R. Todd 
Published: November 2023

Calcium is essential to many vital physiological functions including bone mineralization. Peak bone deposition rate occurs in infancy and maximal bone mineral content is achieved early in adulthood, thereafter bone mass is lost. Maintenance of a positive calcium balance during development is thus essential to achieving optimal bone mass. Calcium homeostasis is mediated by interactions between the intestine, kidneys, and bones. Intestinal absorption occurs by either an active transcellular or passive paracellular pathway. Currently, based on studies in adults the duodenum is thought to be a site of largely transcellular absorption whereas the jejunum and ileum are proposed to mediate exclusively paracellular calcium absorption. There are limited studies on young animals. We therefore set out to describe calcium absorption pathways pre and post weaning. Wildtype FVB/N mice at 7 ages from 1 day to 6 months old were examined. Levels of Trpv6 and CalbD9k mRNA, mediators of transcellular calcium transport, increased six‐fold between two weeks and one month of age in the duodenum with a corresponding increase in CalbindinD9k protein. In the jejunum and ileum, mediators of transcellular transport – Trpv6 (jejunum only), Cav1.3, and CalbD9k – are highly expressed prior to weaning, suggesting novel pathways of calcium absorption during development. Consistent with this, net calcium flux, measured in Ussing chambers, was not different from zero across ileum post weaning. However, we identified significant net absorption in the absence of an electrochemical gradient across ileum prior to weaning. Further, this net absorption was abolished by nifedipine, an L‐type calcium channel blocker. Abundance of Cldn‐2 and ‐15 mRNA, mediators of paracellular absorption, peak at 7 days in the duodenum. In the ileum, Cldn‐2 mRNA peaks at 14 days and decreases 10‐fold by 1 month, whereas Cldn‐15 expression increases 3‐fold from day 14 to 1 month of age. In conclusion, these observations suggest that weaning induces a significant shift from paracellular to transcellular calcium absorption in the duodenum and that there is significant, previously unreported, transcellular calcium absorption from the jejunum and ileum pre weaning mediated by Cav1.3.Support or Funding InformationThis research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Stollery Children's Hospital Foundation through the Women and Children's Health Research Institute
Rapid LPS transport during fat absorption in rat small intestine
Authors: Akiba Yasutada , Kaji Izumi , Maruta Koji , Kaunitz Jonathan D 
Published: November 2023

Lipopolysaccharide (LPS; endotoxin) is a lipophilic pathogenic factor derived from gut Gram‐negative bacteria. Increased serum LPS concentrations are associated with the metabolic syndrome, termed metabolic endotoxemia. Since the origin of circulating LPS is the gut lumen, we studied the mechanisms of LPS transport across the gut mucosa, hypothesizing that transport occurs via known lipid uptake pathways. Since glucagon‐like peptide‐2 (GLP‐2) reduces intestinal solute permeability but enhances fat absorption, we also hypothesized that GLP‐2 affects LPS transport during fat absorption. We thus examined LPS transport mechanisms and the effect of exogenous GLP‐2 on LPS transport in intact intestinal tissues in vitro and in vivo. FITC‐LPS absorption in vivo was quantitated by simultaneously measuring the appearance of FITC‐LPS into the portal vein (PV) and the mesenteric lymph, with or without luminal oleic acid with taurocholate (OA/TCA). We also examined fluorescent dextran 4000 (FD4) or LPS transport from mucosal to serosal solution (m‐to‐s) in Ussing chambered muscle‐stripped intestinal tissues.Bolus intraduodenal infusion of FITC‐LPS (50 μg/ml) with OA/TCA rapidly increased FITC‐LPS appearance into PV, followed by a gradual increase into the lymph, whereas FITC‐LPS alone did not appear on PV or lymph. In Ussing chambered jejunum, luminally‐applied LPS (10 μg/ml) rapidly appeared in the serosal solution with luminal OA/TCA, whereas LPS alone or LPS+TCA was not transported. OA/TCA‐induced LPS m‐to‐s transport in the jejunum was inhibited by luminal pretreatment with the lipid raft inhibitor methyl‐β‐cyclodextrin and the CD36 inhibitor sulfosuccinimidyl oleate, but was enhanced by serosal carbachol and luminal application of the competitive alkaline phosphatase inhibitor glycerol phosphate. Furthermore, FD4 m‐to‐s movement was also enhanced by luminal OA/TCA in the jejunum, whereas lesser effects were observed in the duodenum or ileum. Pretreatment with serosal GLP‐2 (100 nM) reduced OA/TCA‐induced LPS transport, whereas carbachol‐induced LPS transport was not affected.These results suggest that luminal LPS crosses the gut barrier physiologically during fat absorption via lipid raft‐ and CD36‐mediated transport mechanisms, similar to chylomicron transport, and possibly through emptied goblet cells. By reducing lipid raft‐, CD36‐, and chylomicron‐mediated LPS transport, GLP‐2 treatment may be a new therapy for metabolic endotoxemia.Support or Funding InformationSupported by VA Merit Review, NIH R01 DK54221
The Epsilon Subunit of Epithelial Sodium Channels in Xenopus laevis Prevents Channel Activation by Proteolytic Cleavage
Authors: Wichmann Lukas , Vowinkel Kirsty , Althaus Mike 
Published: November 2023

Epithelial Sodium Channels (ENaC) are sodium‐selective ion channels which facilitate the absorption of Na+ ions across epithelial tissues. The heterotrimeric channel is composed of three homologous subunits (α, β, γ) residing in the apical membrane of epithelial cells. Proteolytic cleavage of the α‐ and γ‐subunits is a major regulatory mechanism determining ENaC open probability. In Xenopus laevis, an additional epsilon (ɛ) subunit has been identified which can form functional ENaCs by substituting for the α‐subunit. Since channel properties as well as regulatory characteristics of ENaC containing the ɛ‐subunit are poorly understood, we aimed to investigate the regulation of ɛβγ‐ and αβγ‐ENaC by proteolytic processing.Heterologously expressed ɛβγ‐ and αβγ‐ENaCs in Xenopus laevis oocytes were functionally characterized using the two‐electrode voltage‐clamp technique. Oocytes expressing ɛβγ‐ENaC had larger amiloride‐sensitive transmembrane currents than oocytes expressing ENaC containing the α‐subunit. In contrast to αβγ‐ENaC, currents mediated by ɛβγ‐ENaC readily decreased in the presence of high extracellular Na+ concentrations due to enhanced sodium self‐inhibition. The employment of MTSET‐sensitive βS540C‐ENaC mutants as well as cell‐attached patch‐clamp recordings indicated that the presence of the ɛ‐ENaC subunit produced channels with a higher open probability than those containing the α‐subunit. Proteolytic processing of ENaC is a multi‐step mechanism that increases channel open probability by releasing inhibitory peptides from the α‐ and γ‐ENaC subunits. Proteolytic maturation is initiated through intracellular cleavage of both ENaC subunits by furin. At the cell surface the channels are further processed through cleavage of γ‐ENaC by extracellular proteases. Extracellular chymotrypsin increased currents mediated by αβγ‐ENaC but had no effect on ɛβγ‐ENaC. Channels containing the Xenopus ɛβ‐ENaC subunits together with human γ‐ENaC were also unaffected by chymotrypsin. Incubation of oocytes with a furin‐inhibitor potentiated the effect of chymotrypsin on αβγ‐ENaC expressing oocytes whereas it had no effect on ɛβγ‐ENaC mediated currents. Analysis of ENaC cleavage fragments by immunodetection of N‐terminally (HA) and C‐ terminally (V5) epitope‐tagged α‐, ɛ‐ and γ‐ENaC subunits suggested that there is no intracellular proteolytic processing of the ɛ‐ENaC subunit. Furthermore, the conserved inhibitory peptide sequence identified in α‐ENaC was absent in ɛ‐ENaC.In conclusion, these results demonstrate that incorporation of the ɛ‐ENaC subunit changes ENaC regulation by proteolytic processing and renders the channel resistant to cleavage by extracellular proteases. Hence, the subunit composition of ENaC might play a major role in the susceptibility of ENaC and transepithelial Na+ absorption to proteolytic regulation.
A Cysteine‐Rich Hydrophobic Motif in KS‐WNK1 Regulates WNK Body Formation
Authors: Boyd‐Shiwarski Cary R. , Shiwarski Daniel J. , Nkashama Lubika J. , Roy Ankita , Stolz Donna B. , Puthenveedu Manoj , Subramanya Arohan R. 
Published: November 2023

With‐No‐Lysine kinase 1 (WNK1) coordinates tubular NaCl and K+ transport through the dual action of two major isoforms: a ubiquitously expressed full‐length “Long” isoform with intact kinase activity (L‐WNK1), and an N‐terminally truncated kinase‐defective “Kidney Specific” isoform (KS‐WNK1) that is highly expressed in the distal convoluted tubule (DCT). These two isoforms are functionally different, presumably due to differences in their N‐termini, as their C‐termini are identical. In response to changes in dietary potassium, WNK signaling complexes concentrate in the DCT, forming discrete punctate foci of unknown function, which we call “WNK bodies”. The marked expression of KS‐WNK1 in DCT suggests that it may play role in the formation of these structures. We hypothesized that the unique N‐terminus of KS‐WNK1 is critical for WNK body formation in response to potassium stress. C‐terminal antibodies recognizing both L‐ and KS‐WNK1 recognized punctate structures in the DCT of mice maintained on either low or high potassium diets; surprisingly, WNK bodies were not present in mice on control diets. Mice exposed to low K diets developed subnuclear WNK bodies that were twice as abundant and larger than the subapical puncta that appeared in mice on high K diet. Since our WNK1 antibody recognizes both L‐ and KS‐WNK1, we moved to an in vitro system to determine whether L‐ or KS‐WNK1 is important for puncta formation. In cells, KS‐WNK1 collected in similar puncta, whereas L‐WNK1 was distributed diffusely throughout the cytoplasm. Live cell imaging and FRAP studies indicated that the puncta are mobile structures that slowly fill with eGFP‐tagged KS‐WNK1 protein and restrict KS‐WNK1 diffusion into the free cytosol. Mutagenesis studies revealed that KS‐WNK1 puncta formation is dependent on a cysteine‐rich hydrophobic motif harbored in its unique N‐terminus, as ablation of this motif shifted KS‐WNK1 diffusely. Immuno‐TEM and immunofluorescence confocal microscopy studies indicated that both the in vivo WNK bodies and in vitro KS‐WNK1 puncta are non‐membrane bound structures that co‐localize >90% with the 60s core ribosomal exit tunnel protein L22, suggesting that these puncta are similar in nature. Though L22 colocalization was detected under both high and low K stress conditions, low K WNK bodies additionally contained SPAK and WNK4. Coupled with our observations regarding the differences in WNK body positioning and size with different dietary potassium maneuvers, these findings suggest that WNK bodies contain different proteins and have different roles when the DCT is challenged with low or high K stress. Thus, the formation of WNK bodies in the DCT may reflect distinct WNK‐dependent stress responses that are coordinated by KS‐WNK1.Support or Funding InformationR01DK098145, P30DK079307, T32DK061296
Renal handling of potassium in the type 2 Bartter's mouse on a low Na, high K diet (LNaHK)
Authors: Wang‐France Jun , Wang Bangchen , Li Huaqing , Sansom Steven 
Published: November 2023

Mice on a low Na, high K diet (LNaHK; 0.01% Na, 5% K, 5% carbonate/citrate/Cl) maintain K balance because of the dietary effect to stimulate high distal flow and elicit a very high plasma aldosterone (P[aldo]) that maximally increases the activity of the epithelial Na channel (ENaC). Presumably, the high distal flow stimulates K secretion by the large, Ca‐activated K channel (BK) while the enhanced ENaC‐mediated Na reabsorption increases the driving force for K secretion via both BK and the renal outer medullary K channel (ROMK). However, the notion that ROMK plays a role in K secretion in the distal nephron of mice on LNaHK has not been examined in ROMK knockout mice (KO). To this end, we determined plasma aldosterone levels (P[aldo]; ELISA) and studied the renal handling of K in WT and KO (129/SvJ) mice on LNaHK (4–7 days) with metabolic cage experiments. Glomerular filtration rate (GFR) was measured with FITC‐inulin in conscious mice. On a control diet, there was no difference between WT and KO in the rate of K excretion (UKV; 516.1±63.4 and 521.3±64.1 mmol/day). On LNaHK, the UKV in KO was slightly but significantly lower than WT (1867±312.8 and 2483±92.1 mmol/day). Compared with WT on a control diet (0.3% Na, 0.6% K), KO exhibited a 4‐fold greater urine volume (KO = 5.4±0.6 and WT = 1.4±0.2 ml/day), a reduction in urine osmolality (658±63 and 3253±402 mOsm) but no change in plasma K (P[K]; 4.7±0.8 and 4.5±0.2 mM). Compared to WT on LNaHK, KO exhibited a higher urine volume (6.2±0.6 and 4.3±0.4 ml/day), a reduction in urine osmolality (660±43 and 1708±139 mOsm) but a reduced GFR (177.6±34.1 and 452.9±28.1 ml/day). The reduced GFR of KO was probably the result of a failure to retain Na and volume with a 2‐fold urinary Na loss (UNaV) compared with WT (64.9±11 and 30.8±4.1 mmol/day) and a three‐fold greater P[aldo] (9558±75 and 3717±470 pg/ml). However, KO on LNaHK still maintained K balance with a slightly, but not significantly, elevated P[K] (5.6±0.7 and 4.9±0.2 mM), a 2‐fold increased fractional excretion of K (193.8±52.1% and 97.7±8.0%) and a similar transtubular K gradients (TTKG; 27.4±2.8 and 26.3±1.4), compared with WT. These data indicate that reduced K excretion in KO on LNaHK is caused by reduced GFR due to inadequate Na and volume retention. ROMK of the aldosterone‐sensitive distal nephron does not contribute to K secretion of mice on LNaHK to maintain K balance.Support or Funding InformationThis project is supported by NIDDK grants 3451702067001 and 3451702071006.
The 70 pS Inward Rectifying K Channel Is Identified as Renal Outer Medullary K Channels (ROMK) on Rough, but not Smooth, Thick Ascending Limb Cells (TAL)
Authors: Li Huaqing , wang Bangchen , Wang‐France Jun , Bayles Cayla , Verlander Jill W. , Sansom Steven C 
Published: November 2023

Previous studies revealed that ROMK was represented by two inward rectifying K‐selective channels (Kir) in the TAL, with single channel conductances of approximately 30 pS and 70 pS (inward currents). However, the mouse TAL contains two types of cells; rough TAL (R‐TAL), with abundant apical microvilli and smooth TAL (S‐TAL), which are absent of microvilli. We performed patch clamping, immunohistochemistry, and electron microscopy with immunogold in attempt to determine whether the 30 pS and/or 70 pS channels were associated with R‐TAL or S‐TAL. Cell‐attached patches with 140 mM KCl in the pipette, applied to the split‐open TAL from WT or ROMK knock‐out mice (KO) (one tubule/mouse and 4 to 9 patches per tubule), revealed the 30 pS Kir (31.1 ± 1.2 pS) with an open probability of 0.60 ± 0.09 (at −Vp = −40 mV) and a density of 20 channels out of 73 patches. The 70 pS Kir (74.0 ± 3.4 pS) was observed with Po of 0.35 ± 0.13, and with a density of 12 channels out of 73 patches. We found no patches that contained both channels despite a mathematical probability that 5 patches would contain both the 30 pS and 70 pS channels. We found no 70 pS channels in 42 patches of TAL from ROMK−/−. However, 17 channels were identified as the 30 pS Kir (34.2 ± 1.0) with a Po of 0.56 ± 0.06 (−Vp = −40 mV). Co‐immunohistochemical staining with anti‐ROMK (from Dr. Paul Welling) and anti‐Tamm‐Horsfall protein (TAL marker) revealed ROMK on the luminal membrane of only 40 to 60% of the TAL cells. Using immunogold‐labeled anti‐ROMK and transmission electron microscopy, we found that ROMK was associated with the R‐TAL but not S‐TAL. We conclude that the 70 pS, but not the 30 pS Kir, represents ROMK and the 70 pS Kir is predominantly localized on R‐TALSupport or Funding InformationThis project is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01‐DK‐092474 and R01‐DK‐071014
Ex Vivo Quantification of pHi in Drosophila Malpighian Tubule Principal Cells Reveals Basolateral H+ Equivalent—Coupled Oxalate Transport through Likely Slc26a1 Ortholog
Authors: Rossano Adam J. , Anderson Jacob B. , Romero Michael F. 
Published: November 2023

Nephrolithiasis is a painful and costly healthcare complication. The most common kidney stones are composed of calcium oxalate and thus renal handling of oxalate is an important facet of understanding the pathogenesis of nephrolithiasis. Recently, the Drosophila melanogaster Malpighian tubule (MT) has emerged as a robust model of trans‐epithelial ion transport and nephrolithiasis as MTs readily form luminal calcium‐oxalate crystals in the presence of oxalate. Drosophila Prestin (dPrestin, Slc26a6) transports oxalate across the apical surface of the MT into the lumen but a full model of the trans‐epithelial movement of oxalate in the Drosophila MT has been lacking as the basolateral oxalate transporter has remain uncharacterized. The objective of this work was to identify and characterize the Drosophila basolateral oxalate transporter through ex vivo real‐time quantification of intracellular pH (pHi). pHi was monitored by using the GAL4/UAS system to selectively express pHerry, a pseudo‐ratiometric genetically‐encoded pH indictor (GEpHI) in the cytosol of the principal cells of the MT. Calibration of pHerry in extracted adult MTs yielded a pKa of 7.24. Basolateral perfusion of MTs in CO2/HCO3−‐buffered solution produced a large acidification followed by rapid recovery in all three functionally distinct segments of the anterior MT (initial, transitional, and main). Recovery was likely due to HCO3− loading by HCO3− transporters and this process could be interrupted by basolateral application of 1mM oxalate (in the initial and transitional segments) or 1mM SO42− (in all three segments). The response was most robust in the transitional segment and oxalate induced a more rapid acidification than SO42− (dpHi/dt = −0.60 ± 0.09 pH units min−1 compared to −0.06 ± 0.01). A putative ortholog of the mammalian renal basolateral oxalate transporter (SAT 1; Slc26a1) was identified through sequence homology. Tissue specific knock‐down of this protein with interference RNA (RNAi) reduced the rate of acid‐loading in the transitional segment of the MT with regard to oxalate but not SO42− (dpHi/dt = −0.34 ± 0.06 pH units min−1; p = 0.03 compared to controls, and −0.06 ± 0.01; p = 0.94 compared to controls for oxalate and SO42− respectively). These data indicate this protein is likely the Drosophila basolateral MT oxalate transporter and the basolateral movement of oxalate is functionally coupled to movement of acid equivalents, potentially as oxalate/HCO3− exchange, oxalate/OH− exchange, or oxalate/H+ co‐transport.Support or Funding InformationFunding: T32‐DK007013 to AJR, GM075148 (JBA), DK092408 and DK100227 to MFR.
Molecular analysis of exosomes from mouse kidney collecting duct cells
Authors: Dang Viet D , Jella Kishore K , Montgomery Darrice S , Denslow Nancy , Alli Abdel A 
Published: November 2023

Exosomes are a subtype of extracellular vesicles released by cells under physiological and pathophysiological conditions. These nano‐sized vesicles are found in biological fluids including urine and blood. In this study we conducted a proteomic and lipidomic analysis of exosomes originating from the apical plasma membrane and basolateral plasma membrane of mouse kidney collecting duct (mpkCCD) cells. NanoSight analysis was used to characterize exosomes by size and quantify exosome number. Proteins within the two types of exosomes were identified by liquid chromatography mass spectrometry analysis. There were several cytoskeleton associated proteins such as myosin‐9, clathrin heavy chain 1, alpha‐actinin‐1, moesin, 14‐3‐3 protein zeta/delta, annexin A5, annexin A6, and filamin‐A that were identified with ten or more signature peptides from the apical but not the basolateral plasma membrane exosomes. Lipid class and fatty acid composition analyzed by mass spectrometry revealed significant differences in the phospholipid classes between the two types of exosomes, with the apical plasma membrane exosomes showing higher levels of sphingomyelins and lower levels of cardiolipins, among other phospholipids compared to the basolateral plasma membrane exosomes. The comparative analysis of exosomes at the molecular level may allow for a better understanding of exosome biogenesis, release, and function.Support or Funding InformationThis work was supported by a K01 DK099617 to AAA and by the University of Florida College of Medicine