bluuebunny/crossref_metadata_2025_split huggingface dataset:
Important role for the mammalian‐target‐of‐rapamycin (mTOR) in the collecting duct as a determinant of transporter/channel profile and function
Authors: Brown Aaron L , Lee Hwal , Tiwari Swasti , Li Lijun , Ecelbarger Carolyn M. 
Published: November 2023

mTOR, a serine‐threonine‐specific kinase, is known as a central energy integrator of a number of signaling pathways and may have a role in renal transport in the collecting duct (CD), including facilitating ENaC activation by insulin. To further elucidate the role of mTOR in the CD principal cell, we generated a CD‐principal‐cell‐select mTOR KO mouse by Cre‐lox recombination using aquaporin‐2 (AQP2) promoter to drive Cre‐recombinase. Mice were of normal appearance and body weight. Basal urine volume (24 hr) and kidney weights were not different. A benzamil‐sensitivity test showed that KO mice had significantly reduced urine chloride (Cl−) concentrations (mM), as well as, absolute excretion of both sodium (Na+) and Cl− (μmol/4 hours), as compared to the WT suggesting reduced ENaC activity. To further explore this finding, urine (24‐hr) was collected in male mice under normal (NS) and low‐Na+ (LS) diets (< 0.05% Na+) over the course of a week (n = 14 mice/genotype/diet). Urine Na+ concentrations under LS diet were higher in the KO on day 1, but were reduced to a similar level by the end of the week. Surprisingly, urine aldosterone excretion was slightly, but significantly, lower in the KO mice in the basal period and after 1 day LS, but rose to similar level as the WT mice by day 7. After a recovery week, mice were split into two groups and randomly assigned to receive one‐of‐the‐two diets for another week, then euthanized. Blood analyzed by iSTAT showed modest alkalosis in the KO, especially under normal Na+, i.e., slightly elevated pH, HCO3, base excess, and reduced pCO2 (all significant differences). Plasma Na+ was slightly lower in the KO under NS, but not different from WT under LS. No differences in plasma K+ or Cl− were found. Western blotting conducted on cortex homogenates showed mTORKO had significantly reduced β‐ and γ‐subunits of the epithelial sodium channel (ENaC), aquaporin‐2 (AQP2), and rhesus protein family b‐glycoprotein (Rhbg) relative to WT under both dietary conditions. In the inner medulla, AQP2 was about 50% lower in the KO mice under NS, but actually 20% higher than WT under LS. We predict this may represent compensation for down‐regulation in cortical CD. Overall, these results support a central role for CD mTOR in homeostatic modeling of the CD and expression of key transport proteins.Support or Funding InformationGeorgetown University Department of Medicine; CTSA Georgetown/Howard; Department of Biochemistry (AB)
Reversible modulation of the proximal tubule apical endocytic pathway by fluid shear‐stress
Authors: Shipman Katherine E , Long Kimberly R , Rbaibi Youssef , Baty Catherine J , Weisz Ora A 
Published: November 2023

Epithelial cells that line the proximal tubule (PT) of the kidney are responsible for the reabsorption of low molecular weight proteins and other small molecules from the glomerular ultrafiltrate. A limitation in understanding the regulation of receptor mediated apical endocytosis of filtered ligands has been the lack of a highly differentiated cell culture model for the proximal tubule. Because PT cells in vivo are continually exposed to flow, we have cultured cells under continuous fluid shear stress (FSS) to better recapitulate this environment. Opossum kidney cells were plated on permeable filter supports, and the following day were either exposed to orbital FSS or maintained under static conditions for an additional four days. Ultrastructural analysis revealed striking differentiation of the apical brush border and endocytic pathway in these cells, similar to that observed in PT cells in vivo. Endocytic capacity per cell, quantified based on uptake of fluorescently‐labeled albumin, was also dramatically increased in cells cultured under FSS. Endocytic capacity was rapidly decreased upon reducing FSS. Consistent with enhanced endocytosis, we found a significant increase in expression and a change in distribution of Rab11a, a marker of recycling endosomes, in cells cultured under FSS. Batch deconvolution of confocal images will be implemented prior to quantitation of colocalization of megalin, its ligands, and known components of the endocytic machinery to measure the effect of FSS on the apical endocytic pathway. We are currently working to identify changes in other trafficking proteins that will be used to construct a testable model for how the endocytic pathway is acutely regulated in differentiated PT cells.Support or Funding InformationNational Institutes of Health R01‐DK101484, R01‐DK100357, P30‐DK079307
Comparative proteomic analysis of kidney distal convoluted tubule and cortical collecting duct cells following long‐term hormonal stimulation
Authors: Wu Qi , Møller Hanne , Rosenbæk Lena Lindtoft , Pisitkun Trairak , Fenton Robert A. 
Published: November 2023

The distal convoluted tubule (DCT) and the cortical collecting ducts (CCD) are portions of renal tubule that are partly responsible for maintaining the systemic concentrations of potassium, sodium, calcium and magnesium. Despite being structurally similar, DCT and CCD cells have different transport capabilities due to a variety of different membrane‐associated transport proteins. However, DCT and CCD cells appear to be modulated via the same hormones. The objective of this study was assess the differential response of DCT and CCD cells to long‐term exposure to the hormones vasopressin or angiotensin II, both of which modulate DCT and CCD cells differently. Mass spectrometry based quantitative proteomics was used to profile the differential proteome between DCT and CCD. Mouse kidney distal convoluted tubule cells (mpkDCT) were cultured in heavy SILAC medium (Lys+6, Arg+10) while cortical collecting duct cells (mpkCCD) were cultured in light SILAC medium (Lys+0, Arg+0). Four passages of labelled cells were used to generate four biological replicates for statistical analysis. In addition to control conditions, cells were also stimulated for 4 days with the vasopressin type II receptor agonist 1‐desamino‐8‐D‐arginine vasopressin (dDAVP, 1nM) or angiotensin II (ANGII, 1nM). Cells were harvested, equally pooled and subjected to offline high‐pH fractionation based two dimensional LC‐MS/MS analysis (Q‐Exactive). Identification and quantification of proteins was performed by MaxQuant. Proteins that had at least three ratios out of four biological replicates were defined as quantifiable proteins. Proteins that had at least three iBAQ values out of four biological replicates in light or heavy channel and zero iBAQ values in the other channel were defined as unique proteins in a particular cell type. Out of the some four thousand quantifiable proteins under basal, dDAVP and AngII conditions, 1101, 1566 and 1294 proteins passed the quantitative Benjamini‐Hochberg (BH) FDR 1% threshold, respectively. The mpkDCT or mpkCCD specific proteins were defined based on being an up‐regulated protein that passed the 1% BH FDR threshold in one cell type plus the unique proteins in this cell type. These 1025 mpkDCT specific proteins and 1211 mpkCCD specific proteins under the three conditions were subjected to further bioinformatics analyses including Panther and DAVID gene ontology analyses, E3 ligase and deubiquitinating enzyme identification, and kinase and transcription factor predictions, with the aim to identify cell specific proteins that define tubule‐specific biological processes. Preliminary data suggests that one specific gene CHIP in mpkCCD might be involved in the regulation of AQP2.Support or Funding InformationDanish Council for Independent Research, Lundbeck Foundation
Nuclear Factor‐kB Mediates Zinc Deficiency‐Induced Sodium Chloride Cotransporter Upregulation in Mouse Distal Convoluted Tubular Cells
Authors: Naraine Meagan K , Li Mirandy S , Williams Jasmine , Mistry Monisha , Hoover Robert S , Williams Clintoria R 
Published: November 2023

Zn2+ deficiency (ZnD), a common disorder in chronic kidney disease, contributes to hypertension. However, the mechanism is unknown. The Sodium Chloride Cotransporter (NCC) plays a critical role in blood pressure homeostasis. Recently, our lab found that ZnD leads to hyperactivation of NCC that was reversed with Zn2+ supplementation, suggesting a novel mechanism by which ZnD contributes to hypertension. Other studies have shown that Nuclear Factor‐kB (NFkB) is activated in ZnD. The purpose of this study was to determine the role NFkB plays in ZnD‐induced NCC upregulation. We hypothesize that ZnD activates NFkB, which leads to an upregulation of NCC. To investigate this mechanism, mouse distal convoluted tubular (mDCT‐15) cells were treated with the pharmacological agent Caffeic acid phenethyl ester (CAPE), a NFkB inhibitor, for 24 hours prior to inducing ZnD with the Zn2+ chelator TPEN for an additional 24 hours. NCC expression (via Western blot, qRT‐PCR, and immunofluorescence) and localization (via biotinylation) were examined. Compared to vehicle, TPEN increases NCC mRNA and protein expression. Additionally, NCC surface localization was enhanced. However, NFkB inhibition with CAPE prevents TPEN‐induced NCC upregulation. Based on these results, it is apparent that NFkB plays a role in ZnD‐induced NCC upregulation in mDCT‐15 cells. Understanding the mechanisms by which ZnD contributes to hypertension may have an important impact on the treatment of blood pressure dysregulation.Support or Funding InformationFunding: NIH‐T32 (CRW); NIH‐ROI (RSH); VA‐MERIT (RSH); SUPERR (MKN)
NBCe1‐A Is Not The Only NBCe1 (SLC4A4) Isoform Expressed in the Mouse Kidney
Authors: Verlander Jill W. , Chen Chao , Lee Hyun‐Wook , Osis Gunars , Fang Lijuan , Chen AnPing , Holmes Heather L , Romero Michael F. , Weiner I. David 
Published: November 2023

The A‐splice variant of Na+‐dependent, electrogenic bicarbonate transporter‐1 (SLC4A4, NBCe1‐A) is generally believed to be the only SLC4A4 isoform expressed in the mammalian proximal tubule, and to have critical roles in filtered bicarbonate reabsorption, proximal tubule ammonia metabolism and proximal tubule organic anion metabolism. The purpose of the current study was to examine expression of other splice variants in the mouse kidney. We used recently generated mice with TALEN‐generated deletion of A and D NBCe1 splice‐variants, but with intact expression of the B, C and E isoforms. RT‐PCR using primers specific to the B, C and E isoforms showed mRNA of non‐A,D splice variants was expressed in the kidney of mice with intact NBCe1‐A expression and with NBCe1‐A‐specific deletion. We then used an antibody that recognizes portions of NBCe1 present in all splice isoforms and examined immunolabel in the kidney of NBCe1‐A‐deletion mice. There was basolateral immunolabel in the proximal convoluted tubule in the cortex, and lesser intensity basolateral immunolabel in the proximal tubule straight tubule in both the cortex and outer medulla. Specificity of this antibody was demonstrated by identifying that mice with deletion of all splice variants had no detectable immunolabel. An antibody specific to the A‐splice variant showed basolateral immunolabel throughout the entire proximal tubule of mice with intact NBCe1‐A expression, but no detectable immunolabel in mice with NBCe1‐A‐specific deletion. Thus, an NBCe1 splice variant other than NBCe1‐A appears to be expressed in the mouse proximal tubule. In acid‐loaded NBCe1‐A‐deletion mice, immunolabel observed using the pan‐specific anti‐NBCe1 antibody showed increased immunolabel in the proximal straight tubule, particularly in the outer medulla. These studies show that splice variants of NBCe1 other than NBCe1‐A/D are present in the mouse kidney and that their expression appears to be increased with acid‐loading. We conclude that these splice variants may have important roles in proximal tubule filtered bicarbonate reabsorption, ammonia metabolism and organic anion metabolism.Support or Funding InformationThe studies were supported by funds from the NIH (R01–DK045788 to IDW, R01‐DK107798 to IDW and JWV, and R01‐EY017732 to MFR) and the Department of Veterans Affairs (1I01BX000818 to IDW).
Measuring intracellular protein concentration: why and how?
Authors: Rana Priyanka 
Published: November 2023

In discussions of cell volume regulation, an assumption is often made that a cell can be characterized by its “natural” volume determined by some intrinsic set point. Although this may be true for stable and differentiated tissues, it is obviously wrong for actively dividing cell populations, where the volume doubles between divisions. In this case, it seems more natural to talk about a set point for protein concentration C, rather than for volume V. For example, the majority of data on regulatory volume increase or decrease have been collected within a brief period following the osmolarity change, presumably to avoid complications resulting from cell growth. By using the intensive property of protein/water concentration, rather than the extensive property of volume, this complication can be avoided. Another example comes from apoptosis. Apoptotic shrinkage can be caused by dehydration or by fragmentation. To distinguish between these two processes, one has to know both V and C. Thus, we have developed two simple methods based on light transmission microscopy, to measure these parameters.Cell volume is measured by adding a strongly absorbing extracellular dye to the medium. If a transmission image is taken at a wavelength of maximal dye absorbance, cells appear brighter than the background in direct quantitative relationship to their thickness. This method gives accurate information on V and has been used extensively in our lab. At the same time, one can collect brightfield images away from the absorbance peak. There is a theory that relates transmission image intensity to the phase delay by means of the transport‐of‐intensity equation (TIE). We have further improved the accuracy of TIE by introducing additional calibration, so that protein concentration can now be expressed as C(g/ml) = 1.0·∑ϕ(radians·mm2)/V(mm3), where ϕ is the output of TIE and 1.0 is an empirical coefficient. This approach gives a new insight into cell adaptation to anisosmotic environment and into apoptotic shrinkage.
High sFlt‐1 Concentrations During Pregnancy Modulate the Ppara Promoter Methylation in the Fetal Liver
Authors: Stojanovska Violeta , Holwerda Kim , van der Graaf Anne Marijn , Boekschoten Mark , Faas Marijke , Scherjon Sicco , Plösch Torsten 
Published: November 2023

BackgroundAn adverse prenatal environment significantly increases the risk of chronic metabolic diseases in the offspring. The mechanisms underlying such programming are unclear but may involve epigenetic modifications that lead to altered fetal gene expression. It was demonstrated that poor maternal nutrition affects the DNA methylation status of important transcriptional factors in the liver. However, little attention has been given to other pathological events during pregnancy e.g. increased concentrations of soluble fms‐like tyrosine kinase‐1 (sFlt‐1). Our working hypothesis is that increased sFlt‐1 concentrations in the mother can influence the developmental plasticity and the phenotype of the offspring via resetting of DNA methylation marks.MethodsPregnant Sprague‐Dawley rats were injected with control or adenovirus overexpressing sFlt‐1 on gestational day (GD 8). Fetal plasma, body and organ weight were assessed on GD 19. Microarray analysis was performed to assess the offspring liver gene expressions. The candidate gene methylation status was verified by pyrosequencing.ResultsPlasma concentrations of sFlt‐1 peaked 48 h post injections in the treated animals, and the high concentrations were maintained until the end of the pregnancy. The fetal sFLt‐1 concentrations were increased and body weight and length were decreased in the sFlt‐1 group. There were no significant differences in the brain weight between the groups, but the livers were smaller in comparison to the respective control group. Microarray analysis revealed 623 differentially expressed genes in the fetal liver. The functional analysis indicated a significant effect on lipid metabolism and in particular fatty acid beta‐oxidation. Moreover, Ppara, a transcriptional factor and key regulator of hepatic lipid metabolism, was one of the most induced genes by the high sFlt‐1 concentrations present in the mother and in the offspring. In addition, we performed pyrosequencing, to confirm whether Ppara overexpression is accompanied by changes in the methylation of the respective promoter. The promoter region of Ppara revealed decreased methylation in the high sFlt‐1 group in comparison to the controls, which is in accordance to the observed gene overexpression.ConclusionOur data show that increased sFlt‐1 concentrations in the mother and the offspring lead to growth restriction, brain sparing and Ppara upregulation in the liver. This is accompanied by Ppara promoter hypomethylation. Overall this suggests that epigenetic mechanism may induce the altered phenotype of the offspring via high sFlt‐1 maternal concentrations.Support or Funding InformationThis work was supported by the Netherlands Organization for Health Research and Development (ZonMW, grant number 91211053).
Early Life Stress (ELS) induces Arginase 1 Gene Expression in the Renal Cortex of Adult Male Rats Under High Salt Diet
Authors: Obi Ijeoma E. , Pollock Jennifer S. 
Published: November 2023

Early Life stress (ELS) increases cardiovascular disease risks. Human studies and animal models show that ELS is characterized by altered physiological function and immune dysregulation. Utilizing the rat model of ELS, maternal separation (MatSep), we previously showed that MatSep kidneys are in a pro‐inflammatory state. In this study, we aim to determine the effects of ELS on renal expression of inflammatory markers when challenged with a high salt diet for 7 days. We hypothesize that in response to a high salt challenge, rats would be more susceptible to a dysregulated inflammatory gene expression. MatSep involves separating the pups from the dam from postnatal day 2 (PD2) to PD14. Non‐separated pups were used as controls. At 12 weeks old, male control and MatSep rats were placed in metabolic cages under normal salt diet for 2 days and then switched to a high salt diet for 7 days. Food consumption, water intake and urine excretion were measured daily for the duration of the experiment. On day 7, kidney function was determined by creatinine clearance, sodium and potassium excretion was assessed using Easylyte plus analyzer, urine osmolality was measured using vapor pressure osmometer, and changes in inflammatory gene expression were determined in the renal cortex by quantitative real‐time polymerase chain reaction (qRT‐PCR). No significant differences were recorded for food intake, water intake, urine excretion, sodium and potassium excretion, and urine osmolality in MatSep rats compared to control rats. Likewise, creatinine clearance was similar in control and MatSep rats (control vs. MatSep; p>0.05; 3.53±0.41 mL/min vs. 3.48±0.19 mL/min). Interestingly, gene expression of arginase 1 (Arg1) was significantly increased in the renal cortex of MatSep rats compared to control rats (control vs. MatSep; fold change; 1±0.122 vs. 2.08±0.29; p=0.0122). Similar gene expression levels were observed for toll‐like receptor (TLR) 2 (control vs. MatSep; fold change; P>0.05; 1±0.31 vs. 1.40±0.341), TLR4 (1±0.44 vs. 1.44±0.49), and CD68 (1±0.46 vs. 2.14±0.86). No gene expression was detected for inducible nitric oxide synthase and interleukin 10. These findings suggest that high salt does not induce a difference in renal function of rats exposed to MatSep, although differences in Arg1 may reflect differences in renal injury.Support or Funding InformationFunded by William Townsend Porter Pre‐doctoral Fellowship from the American Physiological Society to IEO and P01HL69999 to JSP
High fat diet exacerbates adipose tissue‐derived adipokines and hypertension in female C57BL/6 mice exposed to early life stress (ELS)
Authors: Loria Analia , Murphy Margaret O , Herald Joseph , Cohn Dianne 
Published: November 2023

Early life stress (ELS) exposure has been recognized as an independent risk factor for obesity and cardiovascular disease. Maternal separation and early weaning (MSEW) during postnatal life show long‐lasting neuroendocrine and behavioral disturbances thereby is used as a mouse model of ELS. The goal of this study was to determine whether exposure to MSEW is associated with sex differences in adiposity, vascular function and blood pressure in response to high fat (HF) diet. MSEW was performed in C57BL/6 mice from postnatal days 2–16. Undisturbed litters served as control (C) group. Mice were placed on a low‐fat (LF, 10% kcal from fat) or HF (60% kcal from fat) diet for 16 weeks upon weaning. MSEW did not influence body weight gain in male and female mice fed a LF diet; however, MSEW increased fat mass with no changes in lean mass vs. C (p<0.05). In response to HF diet, despite significant increases in fat mass in both sexes, only female MSEW displayed higher levels of adiposity vs. C (44±2 vs. 31±2 %, p<0.05). In addition, MSEW exacerbated 24 hour mean arterial pressure (MAP) in both sexes; however, this increase in MAP was significantly greater in female (MSEW= 115.0±0.9 mmHg and C= 106.6±1.6 mmHg) than in male (MSEW= 121.2±1.3 mmHg and C=117.3±0.4 mmHg) mice (p sex<0.05, p MSEW p<0.05, p interaction <0.05). Yet, heart rate was increased only in male MSEW vs. C (p<0.05). Vascular reactivity assessed by wire myography showed increased serotonin‐induced vasoconstriction in female MSEW mice vs. C (73±15 vs. 36±3 %maximal constriction, p<0.05) but not in male MSEW mice vs. C (59±8 vs. 51±3 %maximal constriction, p<0.05). Plasma metabolic profile revealed that female, but not male, MSEW mice display hyperinsulinemia (1.3±0.1 vs. 0.5±0.1 ng/ml, p<0.05), hyperleptinemia (37.4±1.9 vs. 26.5±1.4 ng/ml, p<0.05), higher levels of plasma resistin (8.1±0.4 vs. 5.7±0.5 ng/ml, p<0.05) and PAI‐1 (1.7±0.2 vs. 0.5±0.0 ng/ml, p<0.05). Taken together, these data indicate that female mice exposed to MSEW are particularly susceptible to display fat expansion thereby increasing fat‐derived adipokines involved in vascular dysfunction and hypertension in response to HF diet.Support or Funding InformationHL111354
Corticotropin‐Releasing Hormone Regulates Spontaneous Excitatory Postsynaptic Currents on the Nucleus of the Solitary Tract
Authors: Jia Shuping , Mifflin Steve 
Published: November 2023

Corticotropin‐releasing hormone (CRH) plays a critical role in responses to stress. Two major subtypes of CRH receptors (CRHRs), type 1 receptors (CRH1Rs) and CRH type 2 receptors (CRH2Rs), have been identified in the medial nucleus tractus solitarius (mNTS). The goal of this study was to characterize whether CRH alters glutamatergic transmission to NTS neurons receiving arterial chemoreceptor afferent inputs. Whole‐cell recordings of NTS second‐order neurons identified by DiA labeling of carotid bodies were obtained in caudal NTS slices (250μM thick). Slices were incubated with 100nM CRH in aCSF for 20 minutes and spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded under voltage clamp 1–3 hours later. Control slices were treated with aCSF alone. Pre‐treatment with CRH (100nM) significantly increased mEPSCs amplitude (p<0.01) from 20.7 ± 1.0 pA to 25.1 ± 1.1 pA, with no change in frequency (control: 2.2 ± 0.2 Hz; CRH: 3.1 ± 0.4 Hz, p>0.05). Pretreatment with the CRH antagonist astressin (100nM–1μM) for 10 minutes and then in combination with CRH for another 20 minutes attenuated the CRH‐induced increase in mEPSC amplitude to 19.3 ±1.3 pA (p<0.01). mEPSCs observed following application of CRH were abolished by the non‐NMDA glutamate receptor antagonist CNQX (20μM). These studies indicate that CRH has a direct effect on NTS neurons through postsynaptic CRHRs.Support or Funding InformationThis work supported by PO1 HL88052