bluuebunny/crossref_metadata_2025 huggingface dataset:
Exaggerated Salt‐Sensitive Hypertension in the ALMS1 (Alstrom syndrome 1) Knockout Rat
Authors: King‐Medina Keyona N. , Jaykumar Ankita , Ortiz Pablo
Published: November 2023

We recently found that a protein named ALMS1 (Alstrom syndrome 1) is expressed in the kidney thick ascending limb (TAL) where it mediates endocytosis of the renal Na/K/2Cl cotransporter termed NKCC2. To study the role of ALMS1 in renal physiology we generated ALMS1 knockout (KO) rats in a Dahl salt‐sensitive genetic background via zinc‐finger nuclease targeting. We previously found that the amount of NKCC2 in the apical surface is higher in the TALs from ALMS1 KO rats compared to WT‐ Salt‐Sensitive (SS) rats. In order to determine the role of NKCC2 on blood pressure (BP) we utilized both noninvasive tail cuff measurements and invasive radio‐telemetry to study the effects of dietary sodium (.22% Na chow and 4% Na chow) on the systolic blood pressure (SBP) of the ALMS1 KO rats and WT‐SS rats. We hypothesized that deletion of the ALMS1 gene will increase SBP and enhance salt‐sensitivity of BP, in part due to higher NKCC2‐mediated Na reabsorption. First, we used tail cuff measurements to obtain the SBP in both groups of rats. We found that with normal Na intake (0.22% Na chow), the ALMS1 KO rats had a higher SBP than the WT‐SS rats (KO: 136±3 and WT‐SS: 125±3 mmHg, p=0.0461). To further explore the salt‐sensitive response of these rats we utilized radio‐telemetry monitoring for 4 weeks. We found the ALMS1 KO rats to have a higher baseline SBP on .22% Na chow compared to the WT‐SS rats (KO: 145±2 and WT‐SS: 134±1 mmHg, p=0.0009). After 2 weeks of high Na intake (4% Na chow) the SBP in ALMS1 KO rats increased to 181±1 mmHg, a 35±3 mmHg increase, whereas the SBP increased to 159±2 mmHg in WT‐SS rats, a 25±1 mmHg increase (p<0.01 vs ALMS1 KO). Thus, the SBP was higher in ALMS1 KO rats fed a high salt diet (p=0.0001). We then explored the involvement of NKCC2 in the hypertension observed in ALMS1 KO rats. A daily dose of the loop diuretic bumetanide (3mg/kg), an NKCC2 inhibitor, decreased the SBP in both groups (KO: −23±4 and WT: −30±6 mmHg, p=0.40). After 7 days of treatment with bumetanide, the SBP was normalized only in the WT‐SS rats while the SBP remained elevated in the ALMS1 KO rats (KO: 131±3 and WT: 115±4 mmHg, p<0.025). Therefore, we conclude that the ALMS1 KO rats have a higher salt‐sensitivity of BP than the WT‐ (Dahl) SS rats and this in part is mediated by enhanced renal sodium reabsorption in the TAL. Furthermore, our data suggest that additional mechanisms are likely involved in the hypertension observed in ALMS1 KO rats fed a high salt diet.

Support or Funding Information

NIH F31 Diversity Grant Number: 5F31HL123285‐02
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 Information

British 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 Information

This 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
Stimulation of sweet taste receptors expressed in the kidney enhance surface NKCC2 levels in thick ascending limbs (TALs)
Authors: Ares Gustavo , Caceres Paulo , Kassem Kamal M , Ortiz Pablo A
Published: November 2023

The thick ascending limb (TAL) reabsorbs 30% of the filtered NaCl via the Na/K/2Cl cotransporter, (NKCC2). NKCC2 activity is regulated by trafficking into and out of the apical membrane. We previously found that the monosaccharide fructose, acutely (20 minutes) increases surface NKCC2 levels and NKCC2 activity in isolated TALs. In the tongue, fructose and other monosaccharides are sensed by the G‐protein coupled sweet taste receptors (T1R2/T1R3). However, the expression and function of T1R2/T1R3 in the kidney or the TAL specifically is unclear. We hypothesize that T1R2/T1R3 are expressed in TALs and their stimulation increases surface NKCC2 levels. We used surface biotinylation and Western blot to measure surface NKCC2 levels in rat or mouse TAL suspensions. By Western blot we observed a single band for T1R2 and T1R3 expression in TALs. We then used gurmarin (GUR), a selective inhibitor of T1R2/T1R3 in rodents, to inhibit fructose‐stimulated surface NKCC2 expression. We found that treating TALs with GUR completely blocked fructose‐stimulated surface NKCC2 expression (Baseline= 100%; fructose= 132 ± 3%; GUR= 89 ± 9%; GUR + fructose= 93 ± 6% n.s.). To directly stimulate T1R2/T1R3 we used the non‐caloric sweetener acesulfame‐K (AceK). In TALs, 20 min treatment with AceK increased surface NKCC2 expression (Baseline= 100%; AceK 0.5 mM= 134 ± 7%, AceK 1mM= 157 ± 15%, p<0.05). In GUR‐treated TALs, AceK did not stimulate surface NKCC2 levels (GUR= 100%; GUR + AceK= 94 ± 5%, n.s.). To understand the role of T1R2/T1R3 in renal function we obtained knockout mice (KO) with dual deletion of the T1R2/T1R3 genes. Under baseline conditions surface NKCC2 levels was decreased in T1R2/R3 KO TALs (WT: 100, Tas1R2/3 KO: 59 ± 15%, p<0.05) whereas total NKCC2 was not changed (n=4). We concluded that stimulation of sweet taste receptors T1R2/T1R3 increases surface NKCC2 expression in TALs. Our data show for the first time a renal function for stimulation of T1R2/T1R3 receptors, and suggest that some of the deleterious effect of fructose could be due to activation of renal sweet taste receptors.
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 Information

Supported 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 Information

R01DK098145, P30DK079307, T32DK061296
Studying Na‐K‐2Cl cotransporter 1 membrane dynamic in mouse colon in vivo
Authors: Bouyer Patrice G , Tang Xu , Bridgmon Kenneth , Smallwood Jesse , Matlin Karl S , Matthews Jeffrey B
Published: November 2023

The Na‐K‐2Cl cotransporter 1 (NKCC1) plays an important role in transepithelial chloride secretion in the colon. Previously we have shown that activation of PKCδ and PKCɛ in T84 cells causes a rapid internalization of NKCC1 thereby blunting chloride secretion. Whether similar mechanisms are at play in vivo remain unexplored. To test this hypothesis, we have generated a transgenic (Tg) mouse expressing eGFP‐NKCC1 under the control of the villin promoter to study NKCC1 membrane dynamic in vivo using confocal microscopy. In the Tg mice, eGFP‐NKCC1 is expressed at the basolateral membrane of colonic cells. Using the short‐circuit current to estimate chloride secretion in mouse colons mounted in Ussing chamber, we found that forskolin elicits a similar current in wild type (WT) and in Tg mice. Similarly results were obtained with carbachol. Next, we used confocal microscopy to image eGFP‐NKCC1 trafficking during activation of PKC in vivo. To do so a piece of colon is externalized and a small incision is performed on the opposite side of the mesenteric to preserve innervation and blood supply. The open lumen is apposed on a glass coverslip of a Petri dish filled with a physiological solution. The mouse and the Petri dish are mounted on the stage of a confocal microscope in a temperature‐controlled box. We found that adding phorbol 12‐myristate 13‐acetate (PMA) to activate PKC causes eGFP‐NKCC1 internalization in colonic cells. Similar results were observed with addition of carbachol in the Petri dish. Finally, we have tested the effect of rottlerin a PKCδ inhibitor on PMA‐induced eGFP‐NKCC1 internalization and found that rottlerin blocks internalization of NKCC1.
Acute Aldosterone‐mediated Signaling Networks in Distal Convoluted Tubules
Authors: Cheng Lei , Wu Qi , Olesen Emma TB , Peng Li , Pisitkun Trairak , Fenton Robert
Published: November 2023

The kidney distal convoluted tubule (DCT) plays an important role in modulating body sodium balance and blood pressure. Long‐term effects of aldosterone to increase sodium reabsorption in the DCT are well described. However, potential effects of aldosterone to acutely modulate DCT function via non‐genomic effects and the signaling cascades responsible remain largely uncharacterized. In mpkDCT cells, 30 min of aldosterone (1nM) caused significant increases in both cellular cAMP and inositol phosphate (IP) levels. RT‐PCR and immunohistochemistry identified GPR30 and the EGFR in mpkDCT cells and mouse DCT, providing a possible mechanism for rapid aldosterone effects. 30 min aldosterone treatment resulted in activation of various kinases/kinase substrates in the EGFR/PI3K/AKT or cAMP/PKA pathways. Stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics was used to map global changes in protein phosphorylation in mpkDCT cells following 30 min aldosterone treatment. A total of 8136 unique proteins, containing 14838 different phosphopeptides were identified. Of these, 148 phosphopeptides were significantly up regulated, and 96 phosphopeptides were downregulated in abundance following aldosterone treatment. The EGFR, ERK1/2, AKT, GSK3B and P70S6K were predicted to be important pathway nodes based on the quantitative proteomics data using network analysis. Ex vivo studies in isolated mouse cortical tubules demonstrated an increase in phosphorylated (active) NCC following 30 mins of aldosterone treatment. In summary, the early effects of aldosterone in the DCT may occur via a GRP30‐EGFR pathway to “prime” the DCT cells for future transcriptional and translational regulation, but activation of NCC under similar conditions suggest a novel non‐genomic mechanism for aldosterone‐mediated alterations in DCT NaCl transport.
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 Information

This 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‐TAL

Support or Funding Information

This 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 Information

Funding: 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 Information

This work was supported by a K01 DK099617 to AAA and by the University of Florida College of Medicine
Alternative splice variant of the thiazide‐sensitive NaCl cotransporter: the dominant role of the novel phosphorylation site S811
Authors: Tutakhel Omar A. Z. , Hoederop Joost G.J. , Bindels René J.M.
Published: November 2023

Introduction

The thiazide‐sensitive sodium‐chloride cotransporter (NCC) is located in the apical membrane of epithelial cells lining the distal convoluted tubule of the kidney, and plays an instrumental role in blood pressure regulation by fine‐tuning the renal sodium excretion. The human SLC12A3 gene, encoding NCC, gives rise to three isoforms, of which only the 3rd isoform (NCC3) has been extensively investigated in patho(physiological) conditions. All three human NCC isoforms contain phosphorylation sites at threonine 55 (T55) and 60 (T60) that are essential mediators of NCC activity. Recently, we identified a new phosphorylation site located at S811 in NCC isoform 1 and 2 (NCC1/2).

Objective

The aim of the present study was, therefore, to i) investigate the role of the new S811 phosphorylation site in NCC1/2 function; ii) determine the interaction of NCC1/2 with NCC3; iii) study the effect of NCC1/2 S811 phosphorylation on the function of NCC3.

Methods

First, with the use of immunoblot analysis the phosphorylation of T55 and T60 was investigated in two NCC1/2 S811 mutants (phosphorylation‐deficient S811A and constitutively phosphorylated S811D) compared to wild type NCC1/2 and NCC3. The NCC wild types and mutants were transiently transfected in human embryonic kidney 293 (HEK‐293) cells and incubated for 30 minutes in hypotonic low Cl− buffer, a well‐known stimulator of NCC T55/T60 phosphorylation. Second, to investigate the dimerization of NCC1/2 with NCC3, co‐immunoprecipitation analysis of NCC was performed. Finally, to determine the effect of NCC1/2 S811 mutants on NCC3 T55/T60 phosphorylation, NCC3 was co‐transfected with either NCC1/2 wild‐type, NCC1/2 S811D or NCC1/2 S811A. In the co‐transfection experiments, transiently transfected HEK293 cells were also incubated for 30 minutes in hypotonic low Cl− buffer.

Results

Our results demonstrate that i) in the presence of hypotonic low Cl− buffer the S811A mutant of NCC1/2, but not S811D, prevents the phosphorylation at T55 and T60 of NCC1/2; ii) NCC1/2 and the two NCC1/2 S811 mutants form dimers with NCC3; iii) S811A mutant, not only abolishes the hypotonic low Cl− induced phosphorylation at T55/60 phosphorylation sites of NCC1/2, but also prevents the phosphorylation at T55 and T60 of NCC3.

Conclusion

The present study highlights that the phosphorylation of S811 in NCC1/2 is likely to act as a dominant regulatory site for phosphorylation of T55 and T60. A better understanding of the regulation of NCC function in the kidney is critical to elucidate both the molecular events underlying the pathogenesis of essential hypertension, and to subsequently design and utilize effective therapeutic strategies to combat the dysregulation of blood pressure.

Support or Funding Information

Omar A.Z. Tutakhel is funded by the Dutch Kidney Foundation (PHD12.14).
NADPH oxidase 4 mediates ROS‐induced NKCC2 activity in thick ascending limbs
Authors: Saez Fara , Garvin Jeffrey
Published: November 2023

Luminal flow through the nephron is highly variable, and it is increased in pathological conditions including diabetes and hypertension. Luminal flow increases thick ascending limb superoxide production by NADPH oxidase 4. Exogenously added superoxide increases NaCl reabsorption in this segment. We hypothesize that NADPH oxidase 4‐dependent flow‐induced superoxide stimulates Na/K/2 Cl cotransporter (NKCC2) activity. We measured NKCC2 activity in isolated thick ascending limbs perfused at 20 nl/min in the presence and absence of a superoxide dismutase mimetic and a NADPH oxidase 1/4 inhibitor. During the control period NKCC2 activity was 3.1±0.2 arbitrary fluorescent units (AFU/s). After the superoxide dismutase mimetic MnTBAP (30μM) was added to the bath, NKCC2 activity fell to 2.3±0.2 AFU/sec (n=8, p<0.03), a 30% reduction. Next we tested the effect of a selective NADPH oxidase 1/4 inhibitor. During the control period NKCC2 was 8.4±1.4 AFU/s. After treating thick limbs with GKT137831 (5μM), NKCC2 activity decreased to 3.9±0.7 AFU/sec (n=4, p<0.03), a 54% reduction. Time controls showed no significant change in NKCC2 activity with time (4.1±0.4 vs. 4.3±0.4 AFU/s; n=8). We conclude that flow‐induced superoxide generated by NADPH oxidase 4 stimulates NaCl reabsorption by activating NKCC2 in thick ascending limbs.

Support or Funding Information

This work was supported by a grant from the National Institutes of Health (HL 070985) to J.L. Garvin and a fellowship from the American Heart Association (15POST22650010) to F. Saez.
Segmental and Cellular V1a Receptor Distribution in the Mammalian Kidney
Authors: Giesecke Torsten , Giesecke Yvonne , Kawahara Katsumasa , Koshimizu Taka Aki , Gimber Niclas , Schmoranzer Jan , Smorodchenko Alina , Bachmann Sebastian , Mutig Kerim
Published: November 2023

Vasopressin (AVP) promotes urinary concentration via its V2 receptors in the distal nephron and collecting duct of the kidney. Parallel activation of V1a receptors (V1aR) has been implicated in renal acid‐base handling, but cell type‐related information on this receptor type is still scarce. We have analyzed segmental and cellular distribution of V1aR in mouse, rat, and human kidneys using new antisera. Localization data were supported by functional studies.

An anti‐V1aR was generated and its specificity verified using V1aR‐knockout tissues. Immunofluorescence and super resolution imaging (SIM) were applied for localization studies. AVP‐deficient Brattleboro were analyzed using the V1aR‐specific agonist, AO‐4‐67.

The V1aR antibody showed basolateral membrane‐associated and punctate cytoplasmic signal in alpha‐type intercalated cells of connecting tubules and collecting ducts in rodent and human kidneys. In contrast, beta‐type intercalated cells showed only punctate perinuclear and subapical V1aR signal. Double‐labeling of V1aR with markers for flotillin‐1 (lipid rafts), GM130 (trans‐Golgi network protein), Rab9 (endosomal marker), or LAMP1 (lysosomal marker) suggested broad distribution of the receptor in the respective cellular structures in both types of intercalated cells. Administration of AO‐4‐67 to Brattleboro rats for 4h resulted in luminal trafficking of V‐ATPase in alpha‐type intercalated cells, whereas basolateral V‐ATPase or luminal pendrin of beta‐intercalated cells were not affected by the stimulation.

In summary, divergent cellular V1aR distribution in alpha‐ vs. beta intercalated cells may explain their distinct responsiveness to the V1aR agonist. These results corroborate and extend data on the role of AVP in renal acid‐based handling.
Flow Stimulation of Endothelin‐1 Production by the Collecting Duct: Role of Nitric Oxide and Reactive Oxygen Species
Authors: Kohan Donald E , Wheatley Will
Published: November 2023

Collecting duct (CD)‐derived endothelin‐1 (ET‐1) is an important autocrine inhibitor of CD Na+ and water reabsorption; CD‐specific ET‐1 deletion causes marked salt‐sensitive hypertension. Volume expansion increases CD ET‐1 production thereby facilitating elimination of the volume load. We have previously shown that increased luminal flow, as occurs in salt and/or water loading, stimulates CD ET‐1 production via Ca2+‐dependent pathways; however, how this flow effect occurs is incompletely understood. To examine this, a mouse inner medullary CD cell line (IMCD3) was exposed to shear stress in flow chambers and ET‐1/GAPDH mRNA assessed (ET‐1 mRNA very accurately reflects ET‐1 protein and ET‐1 protein levels are too low to measure in this system). A shear stress of 9.8 dyne/cm2 for 2 h was used based on previous studies (N=6–12 for all of the following data). Perfusion with HBSS + 0.25 mM L‐Arg (used in all subsequent studies) increased ET‐1 mRNA by 4‐fold compared to no flow (ET‐1 flow response). Exposure to 1 μM L‐NAME reduced the ET‐1 flow response to 67% compared to vehicle. Inhibition of NOS1 (1 μM SMTC or 200 μM TRIM) or NOS2 (0.1 nM 1400W) did not alter the ET‐1 flow response. NOS3 siRNA (no specific NOS3 antagonists exist) reduced NOS3 levels by 70%, however cells detached on exposure to flow (likely due to lower cell density needed for transfection). Inhibition of NO signaling pathways, including PKG (1 μM KT5823), guanylyl cyclase (10 μM ODQ) or NFkB (50 μg/ml NFkB inhibitory peptide), did not affect the ET‐1 flow response. These findings suggested NO may not be involved, but rather NOS (at least in part)‐generated reactive oxygen species (ROS) may play a role in the ET‐1 flow response. Inhibition of ROS with 5 mM tempol reduced the ET‐1 flow response to 67% vs. vehicle, no further inhibition was seen with tempol + L‐NAME. Superoxide dismutase (SOD) inhibition (50 μM MnTBAP or 100 U/ml PEG‐SOD) reduced the ET‐1 flow response to 65% of vehicle, while 1000 U/ml PEG‐catalase was without effect. Apocynin (100 μM) also reduced the ET‐1 flow response to 60% of vehicle, suggesting NAPDH oxidase (NOX) might be involved. Inhibition of NOX1/4 with 5 μM GKT137831 also reduced the ET‐1 flow response to 70% of vehicle. Taken together, these data suggest that both NOS and NOX contribute to flow stimulated superoxide‐mediated stimulation of CD ET‐1 production. Since ROS have been reported to augment ENaC activity, the current studies suggest that ROS‐stimulated ET‐1 may mitigate their Na+‐retaining effects in the CD. Finally, to our knowledge, this is the first report of flow‐stimulated reactive oxygen species in the CD, as well as the first report of such flow‐stimulated ROS exerting a biologic effect in the CD.

Support or Funding Information

Supported by NIH R01 DK107454.
ROMK is Required for the Net K Secretion in the Thick Ascending Limb of Mice on a Low Na High K Diet
Authors: Wang Bangchen , Li Huaqing , Wang‐France Jun , Bales Cayla , Wen Donghai , Sansom Steven
Published: November 2023

Understanding the effect of a cardio‐ and reno‐protective low Na, high K diet (LNaHK) on renal K handling is crucial to choosing diuretics and anti‐hypertensive agents for patients on such diets. We previously showed that furosemide, a K‐wasting diuretic that inhibits the Na‐K‐Cl‐cotransporter‐2 (NKCC2) in the thick ascending limb (TAL), increased urinary K clearance in mice on a control diet but decreased that in mice on LNaHK. We hypothesized that there is a net K secretion in the TAL of mice on LNaHK via the renal‐outer‐medullary‐K channel (ROMK) located in the apical membrane of TAL. Wild‐type (WT) and ROMK knockout mice (ROMK KO) were given either a control diet or LNaHK (0.01% Na, 5% K) for 4–7 days. Free‐flow micropuncture was performed using K‐selective microelectrodes to measure the K concentration in the early distal tubule (EDT [K]) before and after intravenous administration of vehicle or furosemide (5mg/kg). Urine was collected via a bladder catheter. Na concentration was measured by a flame photometer. Within 5 minutes of administration, furosemide increased the EDT [K] of WT on a control diet (Δ[K] = 2.33 ± 0.46 mM; n = 6) but decreased that of WT on LNaHK (Δ[K] = −3.07 ± 0.61 mM; n = 11). However, furosemide did not affect the EDT [K] of ROMK KO on LNaHK (Δ[K] = −0.09 ± 0.23 mM; n = 3). Vehicle had no effect on EDT [K] of mice on either diet. The furosemide‐sensitive Na excretion was significantly greater in mice on LNaHK (1298 ± 354 nmol/min; n = 5) than those on a control diet (362 ± 132 nmol/min; n = 5). Furthermore, single‐channel patch clamp analysis was used to evaluate the activity of ROMK in the apical membrane of split‐open TALs from mice on a control diet or LNaHK. The 70‐pS ROMK channel exhibited a higher open probability (Po) in mice on LNaHK than those on a control diet (Po = 0.65 ± 0.11 vs 0.35 ± 0.13, p = 0.07). However, the density of the 70 pS ROMK was not different in LNaHK compared with control diet. No significant difference was found in the number or Po of the 30‐pS K channels between the two groups. These results indicate ROMK‐dependent net K secretion in the TAL of mice on LNaHK, and it is associated with increased NKCC2 and ROMK activities.

Support or Funding Information

This project is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01‐DK‐092474, R01‐DK‐071014 and F30‐DK‐108456.
Impact of Exercise on Stress Responses and Memory in an Undergraduate Student Population
Authors: Carranco Ana I. , Monthony Justine M. , Mumtaz Shiraz A. , Morton Lindsay C. , Burns Daniel J. , Cohen Brian D.
Published: November 2023

When the body perceives a stressor, the the hypothalamic‐pituitary‐adrenal (HPA) axis is activated leading to the synthesis and release of cortisol from the adrenal cortex. Exercise is a physical stressor that increases cortisol levels in the body. Studies have considered exercise as a self‐regulatory strategy to adapt to stressful events. People who exercise regularly have higher baseline cortisol levels and condition their body to higher levels of cortisol but lower self‐reported stress. Many studies focus on professional athletes who exercise vigorously and more frequently than college athletes or college students in general. The aim of this study is to examine the relationship between exercise and physiological responses to stress in college students who exercise compared with students who don't exercise on a regular basis. Similarly, the study also aims to investigate if exercise modulates the impact of stress on memory performance.

Stress was induced in the undergraduates (with the approval of the Union College Human Subjects in Research Committee) using the Trier Social Stress Test (TSST). Saliva samples were collected and heart rate and blood pressure were measured four times during the study; before the TSST, immediately after the TSST, and then 5 and 30 minutes after the TSST. Salivary cortisol was measured using an enzyme‐linked immunosorbent assay. Salivary amylase was measured using an enzymatic assay as a proxy for activation of the sympathetic nervous system. In addition to physiological measures, participants completed the brief mood introspection scale, the revised undergraduate hassles scale, and a self‐reported exercise survey describing the amount and kind of exercise they perform. They also reported how stressed they felt before and after the TSST. A negative correlation was observed between salivary cortisol concentrations and salivary amylase activity when comparing the pre and immediately post TSST samples. Surprisingly, most participants had a decrease in amylase levels and an increase in cortisol levels. In contrast, participants who reported a higher number of hours of vigorous activity in the last 7 days had a greater increase in amylase and decrease in cortisol compared to participants who reported a lower number of hours of vigorous activity. Together these data indicate the need for specific stress‐reduction programs for college students that include physical activity as one aspect of the intervention.