bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel mitochondria‐targeted hydrogen sulfide delivery molecule prevents uraemia and diabetes‐induced renal cell oxidative stress
Authors: Stevens Megan , Payne Molly , Innes Elayne , Torregrossa Roberta of , Wood Mark , Stevens Maarten Koeners of , Whiteman Matthew of , Oltean Sebastian of 
Published: June 2021

Chronic Kidney Disease (CKD) is a disturbingly fast growing global public health problem affecting an estimated 9–16% of the world population. We and others have confirmed that cardiovascular disease is often a state of hydrogen sulfide (H2S) deficiency, coinciding with mitochondrial dysfunction and increased oxidative stress. Mitochondria‐specific H2S‐delivery molecules such as AP39 have been shown to protect the kidney during ischaemia‐reperfusion injury and during organ transplantation. Although the primary effect of endogenous and pharmacological H2S on mammalian cells appears to be overwhelmingly mitochondrial, the pathophysiological importance of mitochondrial H2S deficiency, and the potential therapeutic benefit of H2S in CKD models of uremic stress and diabetic nephropathy have not yet been explored.To elucidate the pathophysiology of CKD‐induced uremic stress, we first investigated the effect of indoxyl sulfate (IS), a representative uremic toxin, on the production of reactive oxygen species (‘ROS’) in multiple immortalised glomerular cell lines (glomerular endothelial cell [GEnCs], podocytes [PODs], and proximal tubule epithelial cells [PTECs]). Furthermore, we investigated the effects of mitochondria‐targeted H2S delivery molecules on IS‐induced ‘ROS’ production. Preliminary data showed that IS increased ‘ROS’ production in PODs and PTECs (100 μg/ml; P<0.05) and that the mitochondria‐targeted H2S delivery molecule AP39 (100nM) at least partially prevented this. We also investigated the effect of IS treatment on cell death, and whether AP39 was able to prevent this. IS (100ug/ml) induced significant cell death after 24 hrs treatment in both GEnCs and PODs, which was prevented by treatment with AP39 (100nM) (P<0.05). In addition to this, we found that AP39 increased the phosphorylation (Ser453) of the cytoprotective AKT in GEnCs exposed to diabetic and uremic stress. AP39 also prevented the mitochondria‐cytoplasmic translocation of the pro‐apoptotic factor cytochrome c in diabetic and uremic‐stressed GEnCs (P<0.05).These preliminary data indicate that uremic states may deteriorate renal function via mitochondrial dependent oxidative stress, which could be prevented by therapeutic H2S delivery. Indeed, the mitochondrial ROS generation, cell death, and cytochrome c release into the cytoplasm by uremic toxins and a diabetic environment in renal cells were, at least in part, reversed by AP39. Further studies are currently underway to determine whether mitochondrial H2S deficiency could be a novel clinically applicable target for treatment in (chronic) kidney disease.Support or Funding InformationBritish Heart Foundation and MRC funded research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Alamandine on the NHE3 Exchanger in in vivo Proximal Tubule of Spontaneously Hypertensive Rats.
Authors: Castelo‐Branco Regiane Cardoso , Crajoinas Renato Oliveira , Malnic Gerhard 
Published: June 2021

The action of Alamandine on the NHE3 exchanger contributes to understanding the etiology of hypertension in spontaneously hypertensive rats (SHR) and their controls, Wistar‐Kyoto rats (WKY). Angiotensin‐(1–7) [Ang‐(1–7)], a heptapeptide member of the renin‐angiotensin system, acts in opposition to the vasoconstrictor, proliferative, and profibrotic actions of Angiotensin II (Ang II) in the cardiovascular system and proximal renal tubule. In normotensive Wistar rats, the dose‐dependent biphasic action of Ang‐(1–7) on NHE3 of renal proximal tubular epithelium is also in opposition of the dose‐dependent biphasic action of Ang II. The presently reported effects of Alamandine were similar to those obtained with Ang‐(1–7), including vasodilation, antifibrosis, reduction of blood pressure in spontaneously hypertensive rats and control of cardiovascular functions. This study was designed to determine the effects of Alamandine on reabsorptive bicarbonate flow (JHCO3−) in in vivo middle proximal tubules. This action was evaluated by bicarbonate reabsorption (JHCO3−) of in vivo proximal renal tubules, using microperfusion with H+ ion‐sensitive microelectrodes. By means tail‐cuff plethysmography the mean values of systolic pressure were obtained in WKY [136 ± 1 mmHg (N=15)] and SHR [181± 3 mmHg (N=9)]. In WKY rats, the mean control value of JHCO3− was 2.34 ± 0.13 nmol.cm−2.s−1 (N= 14). Alamandine [10−12 M] in luminally perfused tubules caused a significant decrease of the JHCO3− to 1,10 ± 0,07 nmol.cm−2.s−1 (28); the following perfusions also significantly decreased JHCO3−: Alamandine [10−9 M] to 1.20 ± 0.13 nmol.cm−2.s−1 (22); Alamandine [10−6 M] to 1.58 ± 0,10 nmol.cm−2.s−1 (17). In SHR rats, the mean control value of JHCO3− was 2.31 ± 0.15 nmol.cm−2.s−1 (12). Alamandine [10−12 M] in luminally perfused tubules causes a significant decrease of the JHCO3− to 1.38 ± 0.07 nmol.cm−2.s−1 (18); the following perfusions also significantly decreased JHCO3−: Alamandine [10−9 M] to 1.42 ± 0.16 nmol.cm−2.s−1 (15); Alamandine [10−6 M] to 1.58 ± 0.13 nmol.cm−2.s−1 (20). So, our data are showing that in WKY and SHR rats, the inhibitory dose‐dependent effect of Alamandine on JHCO3−, demonstrates a possible inhibitory beneficial effect of Alamandine in hypertensive rats opposite to the deleterious stimulatory effects of Ang II. It is therefore reasonable to assume that, in the intact animal, the interaction of the opposite dose‐dependent effects of Alamandine and Ang II on the Na+/H+ exchanger may represent an important physiological regulation in terms of intra‐ and extracellular volume and/or pH changes.Support or Funding InformationFAPESP; CNPq and CAPESThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of vasopressin on cell‐autonomous expression of membrane transport proteins in rat distal nephron
Authors: Smorodchenko Alina , Mutig Kerim , Bachmann Sebastian 
Published: June 2021

Transcellular reabsorption of NaCl in kidney thick ascending limb (TAL) is controlled by Na,K,2Cl cotransporter (NKCC2) and involves the action of luminal (ROMK) and basolateral (Kir4.1) potassium channels, a basolateral calcium sensing receptor (CaSR); paracellular transport controlled by the claudin family of proteins. A morphological cell heterogeneity has previously been described, and mosaic expression of ROMK and Kir4.1 was reported. Here we have studied TAL EM morphology and zonal as well as cell‐autonomous heterogeneity of the transport proteins at steady state and under stimulation by vasopressin (AVP; V2R agonist dDAVP for 7 or 72 h) using AVP‐deficient Brattleboro (BB) and Long Evans (LE) control rats. NKCC2, ROMK, Kir4.1, CaSR, and Claudin (Cldn)‐10 and ‐16 signals were analyzed using immunohistochemistry, in situ hybridization, EM and Western blot (WB).TAL morphological cell heterogeneity was obvious between kidney zones, but not at the cell‐to‐cell level within a zone. NKCC2 was continuously expressed in all TAL cells throughout, whereas ROMK vs. CaSR and Kir4.1 expression showed conspicous heterogeneity in a mutually exclusive pattern. Nearly all tight junctions (TJ) of inner stripe TAL were Cldn‐10 positive. In the outermost inner stripe and outer stripe, Cldn‐10‐ and ‐16 signals were coexpressed; ROMK‐positive cells had Cld‐10‐positive TJ, whereas ROMK‐negative cells had Cldn‐16‐positive TJ. 72h dDAVP administration significantly increased the number of ROMK‐positive cells which then coexpressed Kir4.1 signal. In parallel, Cldn‐10 expressing TJ became more numerous at the expense of Cldn‐16‐positive TJ. WB analysis showed 2.2‐fold increase for ROMK, 1.3‐fold for Kir4.1, but down‐regulation for CaSR to 60%. ISH revealed no differences in mRNA abundances. No significant changes were detected after 7h.The present results demonstrate expressional cell heterogeneity in TAL with respect to individual cell ROMK, Kir4.1, CaSR, and Cldn‐10 and ‐16 signals and reflect an AVP‐dependent adaptive mechanism in renal solute handling in the outer medulla.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Radiofrequency Renal Denervation Decreases Fibrosis in Kidney Cortex and Medulla in Spontaneously Hypertensive Rats (SHR)
Authors: Gao Juan , Denys Ian Bart , Del Valle Luis , Kapusta Daniel R. 
Published: June 2021

There are over 8.3 million people in the US that have chronic kidney disease (CKD), which ultimately can progress to end stage renal fibrosis. Despite an increasing prevalence of CKD, there are no new therapies targeting fibrosis. Our previous studies showed that removal of the renal nerves by radiofrequency renal denervation (RF‐RDN) decreases blood pressure, sympathetic activity, and renal inflammation in hypertensive SHR. Renal fibrosis is a common consequence of chronic kidney inflammation. Therefore, these studies investigated whether RF‐RDN will attenuate renal fibrosis in hypertensive SHR. 22‐week old SHR (n=7/group) received bilateral RF‐RDN or Sham‐RDN (Biosense Webster Stockert 70 generator and RF‐probe). Four weeks later, rat kidneys from all animals were harvested and paraffin sections were stained for fibrosis by Masson's trichrome staining. A pathologist, who was blinded to treatment groups, evaluated each kidney section for fibrosis. The results showed that Sham‐RDN treated SHR had extensive fibrosis demonstrated by moderate thickening of Bowman's capsule, collagen deposition in glomerulus, extensive tubulointerstitial fibrosis, and segmental glomerulosclerosis. In contrast, RF‐RDN significantly reduced each of these pathological components of fibrosis in kidney cortex and medulla. Chemokines play a critical role in mediating the infiltration of immune cells and subsequent fibrosis formation. Therefore, in other studies we measured (ELISA) expression levels of Chemokine ligand 5 (CCL5), CCL2 and CXCL16 in kidney homogenates from RF‐RDN or Sham‐RDN SHR. RF‐RDN decreased CCL5 expression in kidney of SHR compared to Sham‐RDN (78.9±1.7 vs 87.9±2.7 pg/mg protein). RF‐RDN did not alter CCL5 expression in normotensive WKY (68.0±2.2 vs 68.4±3.3 pg/mg protein). These findings suggest that RF‐RDN may offer a novel therapy for renal fibrosis and CCL5 may serve as a potential therapeutic target to treat CKD.Support or Funding InformationNIGMS of NIH U54 GM104940 LA CaTS Roadmap Scholar to JG. NIH P30GM106392 to DRK & Pilot project to JG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic changes in sodium handling in the distal nephron during sympathetic stimulation in healthy males
Authors: Petersen Johan Casper Grove , Holstein‐Rathlou Niels‐Henrik , Sørensen Charlotte Mehlin , Petersen Lonnie Grove , Jonassen Thomas 
Published: June 2021

AimHypovolemia, functional or by blood loss, is associated with decreased venous return and stroke volume. Baroreceptor mediated sympathetic reflexes are activated to increase heart rate to maintain cardiac output, while additional neuro‐humeral responses supress renal sodium excretion. By application of lower body negative pressure (LBNP) to induce graded central hypovolemia, we tested the hypothesis, that low‐pressure cardio‐pulmonary baroreceptors are the main efferent renal neural pathway to sodium retention in the distal part of the nephron.MethodsWe simulated mild and/or severe hypovolemia in twenty‐four healthy males (age 23 ± 3 yrs, height 182 ± 7 cm, weight 75 ± 9 kg; mean ± SD) by application of LBNP at 0 mmHg, −15 mmHg or −30 mmHg. All subjects followed a 3‐day regimen of supplemental sodium ingestion prior to the trial and ingested 600 mg Li+ p.o. 10 hours prior to the trial. Urine samples were collected every 15 minutes during 1.5 hours of LBNP application and 1 hour of recovery. The ratio of excretion, CNa/CLi, was used as a measure for changes in sodium handling in the distal part of the nephron.ResultsLBNP of −15 mmHg and −30 mmHg caused a decrease in CNa/CLi of −3.53±0.69% (P<0.0001) and −2.79±0.49% (P<0.0001), respectively. There was no significant difference in the decrease in CNa/CLi between the two interventions. Heart rate was unchanged in the −15 mmHg group, but significantly elevated in the −30 mmHg group. Stroke volume and pulse pressure were both significantly lower in the −30 mmHg group.ConclusionWe have shown, that the significant decrease in distal sodium clearance is similar during LBNP of both −15 and −30 mmHg even though the increased arterial baroreceptor unloading is pronouncedly increased during −30 mmHg.Our data support an efferent sympathetic neural pathway from the low‐pressure pulmonary baroreceptors in regulation of distal renal sodium excretion, which is suppressed even at mild central hypovolemia. Moderate volume depletion including activation of arterial baroreceptors did not cause further suppression of distal renal sodium excretion.
Quantitative Cellular Anatomy of the Rat Kidney
Authors: Reece Takisha , Knepper Mark 
Published: June 2021

The understanding of renal physiology at a cellular level is progressing rapidly as a result of the recent introduction of techniques for measurement of mRNA levels (RNA‐Seq) and protein levels (protein mass spectrometry) in very small samples consisting of single tubules and single cells. An important question is: How can we use single‐cell and single‐tubule data to predict results of analyses carried out at a whole kidney level, e.g. western blotting? The purpose of this work is to estimate the number of each cell type present in renal tubules of a kidney from a typical 250‐g adult rat (from proximal tubule [PT] to inner medullary collecting duct [IMCD]). For this, we used quantitative structural data gleaned from various 20th century publications. These studies identify the length of each tubule segment, the number of nephrons/collecting ducts, the number of cells per mm and the protein content per mm of each renal tubule segment. The calculations account for differing lengths of loops of Henle, junctions in the connecting tubule (CNT) and IMCD, and different populations of nephrons (short‐looped [71%] and long‐looped [29%]). The calculations reveal that the 38,000 nephrons and their collecting ducts have an aggregate length of 840 meters. The total number of cells from PT to IMCD is 207 million of which 52% are PT cells, 17% are thick ascending limb (TAL) cells, 10% are distal convoluted tubule (DCT) cells, 5% are collecting duct intercalated cells (ICs) and 10% are aquaporin‐2‐expressing cells (PCs). The calculations reveal that the total protein mass of all renal tubule epithelial cells is 119 mg or 40% of the dry weight of the kidney. In terms of protein mass, percentage of individual cell types are: PT, 72%; TAL, 10%; DCT, 7%; ICs, 2%; and PCs, 5%. The average protein masses per cell (pg/cell) are: PT, 804; medullary TAL, 402; cortical TAL, 287; DCT, 425; CNT, 288, cortical collecting duct PC, 212 and IMCD, 300. These calculations provide an illustration of how old data can take on a new significance in light of modern systems biology.Support or Funding InformationThis work was supported by the APS 2017 Frontiers Research Community Leader Fellowship to TR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Control of intracellular chloride signaling by IRBIT‐mediated recruitment of multiple kinase and phosphatase pathways
Authors: vachel laura , Shcheynikov Nikolay , Yamazaki Osamu , Fremder Moran , Ohana Ehud , Nakazawa AI Yamazaki , Shin Dong Min , Son Aran , Yang Chin‐Rang , Knepper Mark , Muallen Shmuel 
Published: June 2021

Cl− is the major anion and important osmolyte in most living cells and has a prominent role in cellular homeostasis and intracellular chloride (Cl−in) affect the activity of several transporters. IRBIT is a multifunctional protein that controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters by unknown mechanism. In the case of the ubiquitous NBCe1‐B, IRBIT interacts with the NBCe1‐B N terminus autoinhibitory domain (AID), exposing two cryptic Cl‐interacting sites to confer regulation of NBCe1‐B by Cl−in. Here, we report that IRBIT do so by recruiting a panel of phosphatases and kinases that both regulate signaling by Cl−in and modulate NBCe1‐B conformation to open the transport pathway. Thus, IRBIT recruits the phosphatase PP1 and the kinase SPAK to control phosphorylation of S65 to control regulation of NBCe1‐B by Cl−in at a 32GXXXP36 site. IRBIT recruits the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of S12 to control regulation of NBCe1‐B by Cl−in at a 195GXXXP199 site. IRBIT then acts on S232, S233, S235 to generate multiple conformations with either dephosphorylated S232/S233, S232/S235 or S233/S235. Each conformation has different activation by IRBIT and regulation by Cl−in. S232/S233/S235 are conserved in several members of the NBC transporters. Mutation of serine to alanine resulted in a fully activated conformation, while mutation of serine to phosphomimetic aspartate inhibited transport and eliminated IRBIT mediated activation, suggesting that S232/S233/S235 determine whether NBC transporter is in an opened or closed conformation. These findings together with structural modeling of NBCe1‐B uncover an intricate mechanism by Cl−in signals through IRBIT‐mediated recruitment of kinases and phosphatases to regulate key transporters involved in epithelial fluid and electrolyte secretion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Vasopressin‐Responsive Regulators of Aqp2 Gene Transcription
Authors: Jung Hyun Jun , Knepper Mark 
Published: June 2021

The peptide hormone vasopressin regulates water excretion largely through control of the water channel protein, aquaporin‐2 (AQP2). Vasopressin regulates AQP2 in part by increasing Aqp2 gene transcription in collecting duct principal cells. Dysregulation of Aqp2 gene transcription has been implicated in a number of water balance disorders. However, how vasopressin regulates Aqp2 gene transcription is unclear. Here, we used next generation sequencing (NGS) techniques as well as prior published data to identify vasopressin‐responsive transcriptional regulators for Aqp2 gene transcription.CTCF is a DNA‐binding protein that homodimerizes with upstream or downstream DNA‐bound CTCF molecules to create ‘insulated loops'. Genes in these insulated loops are regulated mainly via enhancer or repressor interactions within the same loop. From the CTCF ChIP‐Seq database in mouse ENCODE, we found that three genes (Faim2, Aqp2 and Aqp5) are within the same insulated loop (99 kb‐length) defined by two strong CTCF‐binding sites. To identify regulatory elements for Aqp2 gene transcription in mpkCCD cells, we carried out ATAC‐Seq to identify genomic regions accessible to Tn5 transposase (analogous to DNase sensitivity footprinting). We identified 12 transposase‐accessible regions within the Faim2‐Aqp2‐Aqp5 insulated loop, which mark likely locations of enhancer or repressor elements controlling Aqp2 gene transcription.ChIP‐Seq was carried out for two candidate transcription factors, namely cyclic AMP‐responsive element‐binding protein (CREB) and CCAAT/Enhancer‐binding protein β (C/EBPβ) in mpkCCD cells. Vasopressin resulted in increased phosphorylation of CREB at Ser133, confirming a physiological response. Surprisingly, over multiple replicates, ChIP‐Seq identified no CREB‐binding sites within the Faim2‐Aqp2‐Aqp5 insulated loop, while CREB‐binding sites were found in the promoters of 29 transcription factors including AP‐1 transcription factors (Fos and Junb). The latter are recognized to play roles in regulation of Aqp2 gene transcription. In contrast, a strong C/EBPβ‐binding site was found just downstream from the Aqp2 gene within one of the transposase‐accessible regions identified by ATAC‐Seq. Vasopressin treatment increased C/EBPβ‐binding at this and other sites. Overall, the data are consistent with roles of C/EBPβ and CREB in regulation of Aqp2 gene transcription, although CREB effects are likely to be indirect through regulation of other transcription factor genes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kir5.1 Is Associated with NEDD4‐2 And Is Required for High K+ Intake Induced Inhibition of Kir4.1 and NCC
Authors: Lin Dao‐Hong , Wu Peng , Gao Zhong‐Xiuzi , Su Xiao‐Tong 
Published: June 2021

Dietary K+ intake plays an important role in regulating NCC such that a high K+ intake (HK) decreases while a low K+ intake increases NCC. Our previous study has demonstrated that Kir4.1 plays an important role in mediating the effect of dietary K+ intake on NCC expression and activity since the deletion of Kir4.1 not only caused K+ wasting but also abolished the effect of dietary K+ intake on NCC. While Kir4.1 forms the heterotetramer with Kir5.1 and is responsible for providing K+ conductance of the basolateral membrane in the DCT, the function of Kir5.1 in the heterotetramer is not completely understood. GST‐pull‐down and immunoprecipitation have shown that Kir5.1 is associated with the Nedd4‐2 with its second WW domain on phosphothreonine‐binding motif (TPVT) of Kir5.1 at its c‐terminus (AA249–252). Ubiquitin assay and patch‐clamp experiments demonstrate that co‐expression of Nedd4‐2 decreases the expression of Kir4.1 and its currents in HEK293 cells transfected with Kir4.1/5.1 but not in the absence of Kir5.1. We now test the hypothesis that Kir5.1 is required for the effect of HK intake on the basolateral K+ channel activity of the DCT and apical NCC function. Immunostaining and western blot show that Kir4.1 expression is upregulated in Kir5.1−/− mice. Moreover, the patch‐clamp experiments demonstrate that the deletion of Kir5.1 increases the basolateral K+ conductance of the DCT and increased the negativity of DCT membrane potential (hyperpolarization). Renal clearance study has also demonstrated that thiazide‐induced natriuretic effect in Kir5.1−/− mice is significantly larger than in WT mice, indicating the upregulation of NCC in Kir5.1−/− mice. HK intake significantly decreases the basolateral K+ conductance in the DCT, depolarized the DCT membrane and inhibited NCC activity in WT mice. However, HK intake fails to decrease the basolateral K+ conductance in the DCT in Kir5.1−/− mice. Moreover, renal clearance experiments demonstrate that HK intake attenuates thiazide‐induced natriuresis only in WT mice but not in Kir5.1 knockout mice, suggesting that Kir5.1 is important in mediating HK intake‐induced inhibition of NCC. We conclude that Kir5.1 is a binding partner of Nedd4‐2 which plays a role in mediating the effect of HK intake on the basolateral Kir4.1/5.1 activity in the DCT. We speculate that HK intake may stimulate the Kir5.1/Nedd4‐2‐dependent down‐regulation of the basolateral Kir4.1 in the DCT thereby facilitating the down‐regulation of NCC.Support or Funding InformationNIH‐DK115366This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.