bluuebunny/crossref_metadata_2025 huggingface dataset:
Stem Cells Improved the Ability of the Contralateral Kidney to Excrete Sodium in the Renovascular Hypertension Induced by Renal Artery Stenosis
Authors: Varela Vanessa Araujo , de Oliveira‐Sales Elizabeth Barbosa , Shimoura Caroline Gusson , Maquigussa Edgar , Borges Fernanda Teixeira , Boim Mirian Aparecida
Published: November 2023

It was previously demonstrated that mesenchymal stem cells (MSC) improved renal function and renovascular hypertension in consequence of unilateral renal artery stenosis induced by 2 kidneys‐1clip model (2K‐1C) through immunomodulatory actions and by inducing neoangiogenesis. However, the urinary sodium loss was not corrected by MSC. Here, we investigated the tubular function of both stenotic and contralateral kidneys and the effect of MSC treatment by evaluating diuresis, natriuresis and the expression of the main water and sodium transporters. Adult Wistar rats were allocated in the following groups: Control (C, n=6), C+MSC (n=5), 2K‐1C (n=5), 2K‐1C+MSC (n=5). MSC were infused in the 3rd and in the 5th weeks after clipping through the tail vein (2×105 cells). Systolic blood pressure (BP) was monitored weekly by plethysmography. Six weeks after clipping, 24 hours urine and blood samples were collected for biochemical analysis. The glomerular filtration rate (GFR) and the renal plasma flow (RPF) of both kidneys were determined by inulin and PAH clearances respectively. Gene expression of the Na/H exchanger (NHE3), epithelial sodium channel (ENaC), Na/K/ATPase, Na/K/2Cl cotransporter and aquaporin 1 and 2 (AQP1 and AQP2) were analyzed by RT‐PCR. 2K‐1C animals showed a progressive hypertension and MSC prevented BP to increase further. Urinary flow rate and sodium excretion were reduced in the stenotic kidney, but it was significantly increased in the contralateral kidney of the 2K‐1C animals. A suppression in the AQP1 but not AQP2 expression in the contralateral kidney was responsible for the rise in the urinary volume. In contrast, there was no change in the gene expression of the sodium transporters in the contralateral kidney, suggesting that the natriuresis was not dependent of tubular transport but probably of pressure origin (pressure natriuresis). GFR was significantly reduced in both stenotic and contralateral kidneys whereas RPF was reduced only in the stenotic kidney indicating that the hypertensive contralateral kidney presented signs of deficit of renal function. MSC treatment slightly improved GFR and RPF of both kidneys and caused further increases on diuresis and natriuresis of the contralateral kidney, suggesting that MSC improved the capacity of the contralateral kidney to excrete sodium that in turn could contribute to improve hypertension. In conclusion, in spite of a slight deficit of GFR the contralateral kidney was still able to excrete sodium by a tubular independent mechanism and MSC treatment improved the sodium excretory capacity of the contralateral kidney.

Support or Funding Information

Supported by FAPESP and CNPQ.
Conscious Dahl Salt‐Sensitive Rats Excrete a Volume Load Slower than Sprague Dawley Rats Due to a Defect in Thick Ascending Limb Transport
Authors: Saez Fara , Garvin Jeffrey
Published: November 2023

Dahl salt‐sensitive rats have an impaired excretory ability when compared to the parent Sprague‐Dawley strain due to a renal defect. However, nearly all studies have been performed in anesthetized animals raising the possibility that differences between strains are the result of the anesthesia. Although in vitro data indicate that at least part of the defect is due to enhanced NaCl reabsorption by thick ascending limbs in the salt‐sensitive strain, this has not been demonstrated in conscious, whole‐animal experiments. We hypothesized that conscious Dahl salt‐sensitive rats have an impaired ability to excrete an acute volume load to abnormal thick ascending limb NaCl reabsorption. To test our hypothesis, Dahl salt‐sensitive and Sprague‐Dawley rats were placed in metabolic cages and trained to drink their daily water intake in just two hours creating a modest volume load. On the day of the experiment, rats were given a 1% sucrose solution for just one hour with and without furosemide, an inhibitor of thick ascending limb NaCl reabsorption. Urine was collected every 30 minutes for 3 hours. At 60, 90, 120 and 150 min, cumulative urinary volume was lower in salt‐sensitive compared to Sprague‐Dawley rats (60 min: 1±0.3 vs. 7±1, p<0.05; 90 min: 7±1 vs. 18±2, p<0.01; 120 min: 14±1 vs. 24±1, p<0.01; 18±1 vs. 26±2, p<0.05). By 180 min, cumulative urinary volumes were no longer different. Cumulative urinary Na excretion, expressed as percent of total urinary Na excretion, was also lower in Dahl salt‐sensitive rats (60 min: 43±6 vs. 65±6, p<0.05; 90 min: 74±4 vs. 89±2, p<0.01; 120 min: 87±4 vs. 97±1, p<0.01). Furosemide increased dramatically both cumulative urinary volume and urinary Na excretion, and eliminated the differences between strains. We conclude that conscious Dahl salt‐sensitive rats have a defect in thick ascending limb NaCl reabsorption that impairs their ability to excrete an acute volume load. This defect likely contributes to the salt‐sensitivity of blood pressure in this strain.

Support or Funding Information

This work was supported in part by grants to Jeffrey Garvin from the National Institutes of Health (HL 70985) and to Fara Saez from the American Heart Association (15POST22650010 ).
Vascular Endothelial Growth Factor Down Regulates the Renal Epithelial Sodium Channel
Authors: Koenen Anna , Rettig Rainer , Grisk Olaf
Published: November 2023

Vascular endothelial growth factor receptor (VEGFR) inhibitors, such as sunitinib, are widely used in tumor therapy. Their clinical use is limited by a rapidly developing arterial hypertension. We found that fractional sodium excretion was significantly reduced in sunitinib‐treated rats without alterations in fractional lithium excretion suggesting increased sodium reabsorption in the distal nephron and/or collecting ducts (CD). Therefore, we tested the hypotheses that sunitinib increases renal epithelial sodium channel (ENaC) expression and alters subcellular ENaC localization in the rat renal CD. Further, we tested if VEGF down regulates ENaC expression in murine cortical CD (M1) cells.

Sunitinib [15 mg/(kg*d), n = 7, for 4 days] had no significant effects on renal ENaC subunit mRNA abundances but significantly increased α‐ENaC protein abundance by 57% in the renal medulla. Subcellular localization studies with confocal laser microscopy revealed that sunitinib increased the α‐ENaC subunit within the apical plasma membrane of the principal cells in cortical and medullary CD. The subcellular localization of the β‐ and γ‐ENaC subunits was similar in sunitinib‐treated and sham‐treated animals. In aldosterone‐treated M1 cells, VEGF significantly reduced α‐, β‐ and γ‐ENaC mRNA abundances by 30%, 55% and 40%, respectively. Sunitinib diminished (α‐ and γ‐subunits) or prevented (β‐subunit) these effects. In addition, VEGF significantly decreased α‐ and β‐ENaC protein abundances in M1 cells and sunitinib antagonized these effects.

We conclude that VEGF down regulates renal ENaC and that increased ENaC activity may contribute to the pathogenesis of VEGFR inhibitor‐induced arterial hypertension.
Neddylation‐Deficient K712R CUL3 Δ403‐459 Double Mutant Attenuates Proteasome‐Dependent KLHL3 Degradation
Authors: Cornelius Ryan J. , Zhang Chong , Erspamer Kayla J. , Terker Andrew S. , Singer Jeffrey D. , Yang Chao‐Ling , Ellison David H.
Published: November 2023

Cullin 3 (CUL3) is a member of the cullin RING E3 ubiquitin ligase family that mediates ubiquitylation and degradation of multiple cellular proteins via the proteasome. CUL3 facilitates ubiquitylation‐dependent WNK kinase degradation through the substrate adaptor Kelch‐like 3 (KLHL3). Activation of CUL3 requires a covalent modification called neddylation, in which a NEDD8 molecule is attached to a specific lysine residue. A form of Familial hyperkalemic hypertension (FHHt, or pseudohypoaldosteronism type II) has been shown to result from deletion of CUL3 exon 9 (consisting of 57 amino acid residues; CUL3 Δ403‐459). We showed previously that CUL3 Δ403‐459 is more heavily neddylated; this led to enhanced KLHL3 degradation and increased abundance of WNKs. Here, we sought to determine the molecular mechanism for this effect. We transfected HEK293 cells with a FLAG‐tagged neddylation‐deficient CUL3 plasmid construct in which a point‐mutation (K712R) was introduced at the neddylation site. Immunoprecipitation demonstrated that neddylation was nearly absent in both WT K712R CUL3 and K712R CUL3 Δ403‐459. When compared to CUL3 Δ403‐459, K712R CUL3 Δ403‐459 exhibited less capacity to degrade KLHL3. As a result, WNK4 abundance was lower when KLHL3 was co‐expressed with the K712R CUL3 Δ403‐459, compared with CUL3 Δ403‐459. To examine the mechanism of KLHL3 degradation, we incubated cells with the proteasome inhibitor MG132 for 18 h. This almost completely inhibited KLHL3 degradation; in contrast, inhibition of autophagy with two separate drugs, chloroquine and 3‐methyladenine, had only a modest effect. The results indicate that, 1) neddylation at the 712 lysine residue is important for CUL3 Δ403‐459 function, and 2) KLHL3 degradation, mediated by CUL3 Δ403‐459, occurs predominately via the proteasomal pathway. In summary, the FHHt‐causing CUL3 Δ403‐459 mutation exhibits increased neddylation at lysine 712, which causes proteasomal‐dependent degradation of KLHL3. This likely leads to increased WNK kinase abundance and human disease.

Support or Funding Information

Work was supported by National Institutes of Health Grants 2R01DK051496‐15A1 and 5T32DK067864‐10.
NEDD4‐2 reduces electroneutral Cl−/HCO3− exchange in mouse cortical collecting duct
Authors: Pham Truyen Derek , Nanami Masayoshi , Staub Olivier , Sutliff Roy L , Klein Janet D , Eladari Dominique , Chambrey Regine , Wang Xiaonan , Verlander Jill W , Wall Susan M
Published: November 2023

NEDD4‐2 is a ubiquitin ligase that associates with transport proteins, such as ENaC, resulting in their ubiquitination and then endocytosis and degradation. With global NEDD4‐2 gene ablation, salt‐sensitive hypertension is observed, in part, from the increased renal NaCl transporter expression. While principal cell NEDD4‐2 function has been studied, little is known about NEDD4‐2 within intercalated cells (ICs). Thus, the purpose of this study was to determine the effect of IC NEDD4‐2 ablation on blood pressure and on Cl− absorption in the cortical collecting duct (CCD). As such, we generated an IC‐specific NEDD4‐2 null mouse using Cre‐loxP technology. Blood pressure was measured by radiotelemetry and ion transport was measured in mouse cortical collecting ducts (CCDs) perfused in vitro. Following a high NaCl diet, IC NEDD4‐2 gene ablation markedly stimulated electroneutral Cl−/HCO3− exchange in the mouse CCD and increased mean arterial pressure by ~10 mm Hg. To determine the transporter(s) responsible for this increase in transport, we examined the effect of IC NEDD4‐2 gene ablation on mouse type B IC transporter abundance by immunoblot, immunohistochemistry and immunogold cytochemistry. We observed that IC NEDD4‐2 gene ablation increased ClC‐5 and pendrin protein abundance, but not the H+‐ATPase, AE4 or the Na+‐dependent Cl−/HCO3− exchanger, NDCBE. To determine if pendrin associates with NEDD4‐2 we transfected a rat intercalated cell line with adenovirus expressing Xpress‐tagged NEDD4‐2 and then immunoprecipitated for the Xpress tag. By immunoblot, pendrin was expressed in the Xpress tagged precipitates taken from cells transfected with full length NEDD4‐2, but not in precipitates taken from cells transfected with NEDD4‐2 lacking the C terminus.

Conclusions

IC NEDD4‐2 gene ablation contributes to the pathophysiology of salt‐sensitive hypertension, at least in part, by increasing Cl− absorption by ICs, through transporters such as pendrin.

Support or Funding Information

Interaction of NEDD4‐2 and Aldosterone in Interacalated Cell Function. Grant Number: DK104125
Angiotensin II (Ang II) Increases Phosphorylation of Rat Kidney Na, K‐Pump at S938 in Proximal Tubule (PCT) Cells through a Phosphatidylinositol 3′‐Kinase (PI3K)/Akt Pathway Facilitated via Epac
Authors: Yingst Douglas R , Mattingly Raymond R , Keezer Susan M
Published: November 2023

To understand how Ang II increases sodium reabsorption in PCT cells and thereby increases blood pressure, we earlier demonstrated that Ang II directly stimulates rat kidney Na, K‐pump activity by increasing phosphorylation of the Na, K‐pump at S938. Phosphorylation at S938, a site conserved in human kidney Na, K‐pump, was initially discovered under in vitro conditions to be mediated by protein kinase A (PKA). Nevertheless, when rat Na, K‐pump is stably co‐expressed with the AT1a receptor in opossum kidney (OK) cells, a PCT cell line, Ang II‐dependent increases in S938 phosphorylation were not blocked by H‐89, an inhibitor of PKA. Ang II‐dependent increases in phosphorylation at S938, as measured by a new antibody to phosphorylated S938, were instead blocked by wortmannin, a selective inhibitor of PI3K. We also found that Ang II activates Akt, likely via PI3K. In addition, we discovered that phosphorylation at S938 was increased by 8‐CPT‐2Me‐cAMP, an analog of cAMP that selectively activates Epac, but not PKA. We also showed that Akt is activated by Epac. Thus, we propose that Ang II acutely regulates Na, K‐pump activity in PCT cells via S938 and a novel Akt signaling pathway.
Short Term Regulation of SGLT2 by Insulin in vivo
Authors: Ghezzi Chiara , Scafoglio Claudio , Liu Jie , Barrio Jorge R , Wright Ernest M
Published: November 2023

Glucose homeostasis is a complex mechanism that requires the interplay between different organs. The kidney plays a central role in this process by re‐absorbing 99.5% of the filtered glucose in the proximal tubules, where SGLT1 and SGLT2 use the energy provided by the electrochemical gradient of sodium to drive glucose transport.

In recent years SGLT2 has received much attention as the target of inhibition by a class of drugs (i.e. dapagliflozin, canagliflozin) that lower blood glucose levels in type II diabetes patients by blocking glucose re‐absorption in the kidney tubules. In the present study, we used [F‐18] fluoro‐dapagliflozin (F‐Dapa), a specific, high‐affinity SGLT2 ligand (KB 4 nM), to study the localization and activity of SGLT2 in the mouse kidney in vivo, by microPET on wild type (WT) and knock‐out mice.

Our results showed, as expected, that in WT mice SGLT2 was highly expressed in the kidney. Binding was fast and reached saturation already after 10 minutes. The specificity of F‐Dapa binding was determined using SGLT2−/− and SGLT1−/−‐SGLT2−/− mice, in which binding was reduced to background levels, and competition experiments with phlorizin and dapagliflozin, in which we observed the blockage of F‐Dapa binding and the displacement of the bound ligand.

We have previously shown that hSGLT2 expressed in HEK‐293T cells is up‐regulated by insulin due to either a fast insertion of an intracellular pool of transporters into the plasma membrane, or an increase in the transporter catalytic turnover. To determine the effect of insulin on SGLT2 in vivo, we measured the change in F‐Dapa binding to the kidney after insulin injection. Our results showed that immediately after insulin administration, F‐Dapa binding rapidly increased by 30% to a new steady state, and the total binding was displaced by dapagliflozin and phlorizin.

In conclusion, our results showed that F‐Dapa is a SGLT2‐specific tracer that can be used to study the protein localization to the brush border membrane of the proximal tubule in vivo. Moreover, using this tool we showed that insulin rapidly recruits an intracellular pool of SGLT2 to the brush border membrane.

Support or Funding Information

NIH/NIDDK

DK077133
AT2R is involved in mediating the effect of high dietary K (HK) intake on basolateral Kir4.1 in the distal convoluted tubule (DCT)
Authors: Wu Peng , Gao Zhongxiuzi , Wang Mingxiao , Su Xiao‐Tong , Wang Lijun , Wang Wen‐Hui
Published: November 2023

The basolateral 40–45 pS K channel in the DCT is composed of two inwardly‐rectifying K channels (Kir), Kir4.1 and Kir5.1. Previous study has demonstrated that Kir4.1 plays a predominant role in determining the basolateral K conductance in the DCT and that the inhibition of Kir4.1 leads to the suppression of NCC. In the present study, we used patch‐clamp experiments to examine the effect of HK intake on the basolateral Kir4.1/5.1 channels in the DCT of mouse kidney. Increased dietary K intake not only decreased the probability of finding the 40 pS K channel in the basolateral membrane of the DCT but also channel open probability. The notion that HK intake inhibits the basolateral Kir4.1 channels is also supported by measurement of Ba2+‐sensitive whole cell K currents in DCT1. We observed that HK intake decreased the Ba2+‐sensitive K currents in the DCT by more than 50% in comparison to that on control K diet. Because AT2R is expressed in the distal nephron and a HK intake stimulates AT2R expression, we examine whether AT2R is involved in mediating the effect of HK intake on basolateral K channels in the DCT with patch‐clamp technique. Under control conditions (normal K diet), neither angiotensin II (AngII) nor AngII + losartan has a significant effect on the 40 pS K channel in the DCT. In contrast, in animals kept on a HK diet for 7 days, inhibition of AT1R with losartan reduced basolateral K channel activity in the DCT (control NPo, 1.4 ± 0.3; losartan 0.85 ± 0.25). Moreover, application of 100 nM AngII caused an additional inhibition of the basolateral 40 pS K channel (NPo, 0.3 ± 0.1). To study the role of AT2R in mediating the effect of HK intake on the basolateral K channel in the DCT, we performed the patch‐clamp study in mice that were treated with PD123319 for 7 days through an osmotic micro pump. Inhibition of AT2R did not significantly affect the basolateral K currents in the mice on a normal K diet but it largely abolished the HK‐intake‐induced decrease in the basolateral K conductance of the DCT. This suggests a role for AT2R in mediating HK intake‐induced inhibition of Kir4.1/5.1 in the basolateral membrane of the DCT. We conclude that high K intake decreases the basolateral K conductance in the DCT and that AT2R is involved in mediating the effect of HK intake on the basolateral Kir4.1/5.1.
Role of IGF1R in Tubular Remodeling of the Mouse Kidney
Authors: Walczak Elisabeth , Krauson Aram , Iyer Sonali , Nizar Jonathan , Dong Wuxing , Bhalla Vivek
Published: November 2023

Loop diuretics, commonly prescribed to treat hypertension and edema, increase sodium excretion by inhibiting the sodium‐potassium 2 chloride cotransporter (NKCC2) in the thick ascending limb. Chronic furosemide use leads to diuretic resistance in humans, but the molecular mechanisms are largely unknown. We developed a mouse model of chronic furosemide use to explore its effects on sodium transport and tubular remodeling. Mice treated with furosemide for seven days had greater sensitivity for thiazide diuretics with significantly increased levels of sodium‐chloride cotransporter (NCC), demonstrating compensatory sodium reabsorption of the distal nephron. Furosemide‐treated mice also exhibited tubular hyperplasia. After 21 days, mice treated with furosemide vs. vehicle, had persistently‐elevated NCC expression increased kidney‐to‐body weight ratio, increased tubular hypertrophy of distal convoluted tubule and connecting tubular segments, and changes in IGF‐1 signaling. There was no increase in IGF‐1 mRNA expression, but specific increases in IGF binding proteins (IGFBPs) which are known to sequester IGF‐1 in kidney cells, and IGF‐1 receptor. To further explore the role of IGF‐1 in tubular remodeling, we developed an inducible, pan‐tubular IGF‐1 receptor knockout mouse. Interestingly, furosemide‐treated IGF‐1 receptor (IGF1R) knockout mice lacked the increased kidney‐to‐body mass increase ratio seen in wild‐type mice, but they developed comparable hypertrophy in distal nephron segments and higher expression of NCC compared with furosemide‐treated wild‐type mice. These results suggest IGF1R signaling is involved in renal tubular remodeling of more proximal nephron segments and distinct factors control the tubular hypertrophy of the distal nephron beyond an increase in NCC expression.

Support or Funding Information

NIH/NIDDK
Effect of Oleanolic acid on regulation of renal function and hormonal balance in hypertension model
Authors: Ahn You Mee , Lee Yun Jung , Kim Hye Yoom Kim , Tan Rui , Kho Min Chol , Han Byung Hyuk , Lee So Heun , Lee Han Sol , Cho Kyoung Woo , Lee Ho Sub , Kang Dae Gill
Published: November 2023

Oleanolic acid is known to possess beneficial effects on the regulation of the cardiovascular homeostasis. However, the exact nature of the role of oleanolic acid on the regulation of body fluid balance and blood pressure homeostasis and its mechanisms involved are not well defined. We hypothesized that oleanolic acid inhibits the renin‐angiotensin system and accentuates renal function. Experiments were performed to identify the effects of oleanolic acid on the renal function, blood pressure, and the renin‐angiotensin system in normotensive and hypertensive rats. Oleanolic acid (0, 20, and 30 mg/kg/day) was administered orally for 1 or 3 weeks. Here, we found that oleanolic acid suppressed the renin‐angiotensin system. Oleanolic acid increased urinary volume, and urinary excretion of Na+, K+, and Cl−, concomitantly with an increase in clearance for creatinine in normotensive rats. Also, oleanolic acid decreased urinary osmolality. Oleanolic acid significantly suppressed arterial blood pressure in renal hypertensive rats. Furthermore, oleanolic acid suppressed plasma levels of renin activity, angiotensin II, aldosterone, and renal renin contents, and gene expressions by real‐time PCR of renin, angiotensin converting enzyme and angiotensin II type 1 receptor in the kidney cortex from normotensive and hypertensive rats. Oleanoliac acid elicited dual effects on the angiotensin II type 2 receptor: suppression with low and accentuation with high dose. These findings suggest that oleanolic acid modulates body fluid and salt balance and blood pressure homeostasis by suppressing the systemic and intrarenal renin‐angiotensin system.
1,2,3,4,6‐penta–O‐galloyl–β–D–glucose ameliorates renal injury in ischemia‐reperfusion induced acute renal failure rats
Authors: Park Ji Hun , Lee Yun Jung , Kim Hye Yoom , Yoon Jung Joo , Kho Min Chol , Lee So Heun , Lee Han Sol , Jin Xian Jun , Lee Ho Sub , Kang Dae Gill
Published: November 2023

Renal ischemia‐reperfusion (I/R) injury, an important cause of acute renal failure, cause decreased glomerular filtration rate and kidney microvascular inflammation. 1,2,3,4,6‐penta–O‐galloyl–β–D–glucose (PGG) has anti‐cancer, anti‐oxidation and angiogenesis effects. We examined protective effects of PGG on I/R‐induced acute renal failure rats. Ischemia was induced by clamping the both renal arteries during 45 min followed by reperfusion. Peritoneal injection of 10, 50 mg/kg/day PGG to ischemia‐reperfusion rat and I/R injury was measured after 2 or 4 days respectively following PGG treatment. Treatment with PGG significantly ameliorates urine volume, urine osmolality, creatinine clearance (Ccr) and blood urea nitrogen (BUN). In addition, PGG increases aquaporine 1/2/3 and Na+‐K+‐ATPase and decreases ICAM‐1, MCP‐1, and HMGB‐1 expression. In histopathologic study, PGG improved glomerular and tubular damage. Immunohistochemistry results showed that PGG increased aquaporine 1/2, and Na+‐K+‐ATPase whereas decreased ICAM‐1 expression. These findings suggest that PGG ameliorates renal injury including renal function and microvascular inflammation in I/R‐ induced acute renal failure rats.
Vasopressin‐Escape Does Not Involve Marked Changes in the Ratio of Intercalated‐to‐Principal Cells in the Cortical Collecting Duct
Authors: Chou Chung‐Lin , Ecelbarger Carolyn M. , Lee Jae Wook , Knepper Mark A.
Published: November 2023

The kidney collecting duct plays an important role in vasopressin escape, a phenomenon which helps to lessen vasopressin‐induced hyponatremia. To understand transcriptomic changes during escape, we recently carried out RNA‐Seq on the cortical collecting duct (CCD). A key question to interpret these results, however, hinges on whether the observed changes were attributable to cell remodeling in the CCD. Male Sprague Dawley rats infused with a sustained dose of DDAVP received either a high‐water load (50 mL/day) or a normal water load (25 mL/day). CCDs were collected by manual microdissection from collagenase‐treated kidney cortex at day 4, fixed with 4% paraformaldehyde, followed by immunofluorescent staining with anti‐pendrin (Slc26a4), anti‐proton pump (Atp6v1b1) antibodies, and DAPI. After acquiring the confocal stack images, automatic cell counting was performed on three dimensional reconstructed tubule images generated by Imaris Image Processing & Analysis Software. For each tubule, we counted the number of pendrin‐positive intercalated cells (IC), the number of pendrin‐negative IC cells, and the number of total cells per mm length of tubule. The total number of IC cells was calculated as the sum of pendrin‐positive and pendrin–negative IC cells. The number of principal cells (PC) was calculated as the total cell number minus total IC cells. With a total of 31 CCD analyzed, i.e., 12 from control rats and 19 from escape rats, we found no significant differences in either the proportion of principal versus intercalated cells, or in proportion of α‐versus β‐intercalated cells between the treatment groups (unpaired students t‐test, n=4). These results suggest that the differences observed in the CCD transcriptome in the early time course of vasopressin escape likely do not involve significant remodeling of the CCD in terms of cell‐type identity.
Distinct regulation of the basolateral conductance in the collecting duct by Na+ and K+ intake
Authors: Zaika Oleg , Tomilin Viktor , Mamenko Mykola , Pochynyuk Oleh
Published: November 2023

Distal renal tubule segments, such as the collecting duct, underlie final regulation of electrolyte transport to maintain systemic homeostasis. The collecting duct comprises of principal and intercalated cells having primarily Kir4.1/5.1‐mediated potassium and ClC‐K2‐dependent chloride basolateral conductance, respectively. Both dietary potassium load and sodium restriction are known to stimulate apical electrogenic Na+ reabsorption via ENaC. However, little is known whether this also affects ion fluxes across the basolateral membrane. Using patch clamp electrophysiology at the single channel and whole cell levels in freshly isolated mouse collecting ducts, we found a marked upregulation of the basolateral K+‐selective current and hyperpolarization of basolateral membrane potential in principal cells of mice subjected to a high (5%) K+ diet for one week compared to the control. This stimulation stemmed from the significantly increased functional levels of Kir4.1/5.1 but not from channel open probability. Moreover, elevated dietary potassium intake drastically decreased basolateral Cl−‐selective current and the number of active ClC‐K2 channels in intercalated cells. On the contrary, dietary Na+ restriction (<0.01% Na+) for one week had a minor effect on the basolateral K+ current and single channel Kir4.1/5.1 activity in the principal cells but significantly stimulated Cl− current and increased ClC‐K2 levels in intercalated cells. We conclude that this different response of the basolateral K+ and Cl− conductance may in part underlie the preferential Na+/K+ exchange observed during dietary K+ load and net Na+ and Cl− reabsorption to protect the circulating volume upon reduced sodium intake.
Chronic Lithium Treatment Induces Novel Patterns of Pendrin Localization and Expression in the Renal Inner Medulla
Authors: Himmel Nathaniel J. , Wang Yirong , Rodriguez Daniel A. , Sun Michael A. , Blount Mitsi A.
Published: November 2023

Prolonged lithium treatment is associated with various renal side effects, including hyperchloremic acidosis, and is known to induce inner medullary collecting duct (IMCD) remodeling. In animals treated with lithium, the fraction of intercalated cells (ICs), which are responsible for acid‐base homeostasis, increases as compared to renal principal cells (PCs). In order to investigate the intricacies of lithium‐induced IMCD remodeling, male Sprague Dawley rats were fed a 0.3% LiCl‐enriched diet for 1, 2, 3, 6, 9, or 12 weeks. Urine osmolality began to decrease at 1 week, and from 2–12 weeks, animals were severely polyuric. Beginning at 6 weeks, approximately one‐quarter of cells counted in initial IMCD sections expressed vATPase, an IC marker. These cells were localized in portions of the IM where ICs are not normally found. Pendrin, a Cl−/ HCO3− exchanger, is normally only expressed in two IC subtypes found in the convoluted tubule, the cortical collecting duct, and the connecting tubule. Beginning at 6 weeks of lithium treatment, we observed novel and various pendrin localization and expression patterns in the IMCD, including co‐expression with AQP4, a PC marker. These data collectively suggest that renal IMCD cell plasticity may play an important role in lithium‐induced IMCD remodeling.

Support or Funding Information

Norman S. Coplon Extramural Grant ‐ Satellite Healthcare
Basolateral cholesterol depletion of mpkCCD cells results in ubiquitylation of aquaporin‐2
Authors: Fuglsang Cecilia H , Fenton Robert A , Moeller Hanne B
Published: November 2023

Apical expression of the water channel aquaporin‐2 (AQP2) is regulated by the antidiuretic hormone arginine‐vasopressin (AVP). Post‐translational modifications (PTMs) of the C‐terminal tail of AQP2 mediate AQP2 function by various mechanisms. Ubiquitylation of K270 can mediate apical membrane AQP2 internalization, whereas phosphorylation of AQP2 (at S256 and S269) promotes its apical membrane accumulation. Here, we examined the effect of the cholesterol depleting reagent methyl‐β‐cyclodextrin (MβCD) on AQP2 PTM. The aim was to understand if a specific distribution of AQP2 in the lipid bilayer is involved in its apical membrane accumulation. mpkCCD cells grown on filters and exposed to MβCD from both apical and basolateral sides increased apical expression of AQP2. These effects may be due to inhibition of AQP2 endocytosis. MβCD‐treatment decreased dDAVP induced S269 phosphorylation of AQP2 and dramatically increased ubiquitylation of AQP2. The effects of MβCD on AQP2 ubiquitylation occurred in a dose dependent manner, and only following basolateral and not apical application of MβCD. Basolateral MβCD also decreased AQP2 phosphorylation (S269). Treatment of mpkCCD cells with cAMP increased S269 phosphorylation, whereas this effect was decreased in the presence of basolateral MβCD. In conclusion our data suggest that basolateral cholesterol depletion affects proper regulation of AQP2 trafficking by disturbing PTMs of the protein.
SGLT1 and Osmotic Water Flow across the Small Intestine
Authors: Her Ka , Gorraitz Edurne , Loo Donald , Wright Ernest M
Published: November 2023

Passive water flow is important in the physiology of the small intestine in response to osmotic gradients across the epithelium following the ingestion of food and water; and malabsorption syndromes. However, the mechanism of osmotic water flow across the small intestine is poorly understood. Questions arise on the relative contributions of the cellular and paracellular pathways; and the identity of water channels in the apical and basolateral membrane of the epithelium. It is often assumed that the tight junctions are highly permeable to water simply because they are permeable to small ions; and orthodox aquaporins are not expressed in the small intestine. We have previously postulated that the intestinal Na+/glucose cotransporter SGLT1 plays a key role in fluid transport based on studies of SGLT1 expressed in Xenopus laevis oocytes (see Loo et al 1999. J. Physiology 518:195–202; and Loo et al 1996 PNAS 93:13367–13370). The osmotic water permeability of SGLT1 in oocytes (Pos) is independent of the magnitude and direction of the osmotic gradient, and the presence of ligands (Na+ and sugar), but is inhibited by phlorizin in the presence of Na+ (Ki 5 μM). Furthermore, osmotic water flow has a low temperature dependence (5 kcal/mole) relative to Na+/glucose cotransport (>20 kcal/mole).

To investigate the importance of SGLT1 in osmotic flow we measured the effect of phlorizin on osmotic flow. Our approach was to prepare everted sacs of mouse jejunum and measure water flow by recording the change in weight of the sacs with time in response to osmotic gradients. The osmotic gradients were generated by the addition of mannitol to the mucosal buffer, and the contribution of SGLT1 to the osmotic flow was determined by the addition of phlorizin. The experiments were conducted at 21–23°C to maintain the viability of the tissue, a long standing practice in studies of mammalian tissues in vitro. In the absence of an osmotic gradient between the mucosal and serosal solutions there was no significant change in the weight of the sac but the addition of 100 mM mannitol to the mucosal fluid produced a loss of weight equivalent to 16 ± 2 nl min−1 cm−1 and a Pos of 0.012 cm s−1. Phlorizin (250 μM) added to the mucosal fluid reduced the flow to 6.5 ± 2 nl min−1 cm−1.

These results indicate that 65% of the osmotic flow across the mouse small intestine occurs through SGLT1, and less than 35% of the total flow occurs through the paracellular pathway.

Support or Funding Information

NIH NIDDK DK19567
Ablation of the Renal Urea Transporters UT‐A1 and UT‐A3 Replicates the Benefits of a Low‐protein Diet in Attenuating Diabetic Nephropathy
Authors: Wang Yirong , Himmel Nathaniel J. , Mallow G. Michael , Sun Michael A. , Borchart Matthew R. , Blount Mitsi A.
Published: November 2023

Diabetic nephropathy (DN) is a leading cause of kidney failure worldwide. Progression of DN can be slowed with a low‐protein diet; however, Western diets exceed the amount of protein physiologically needed, thus excess urea is generated and excreted by the kidney. Urea is reabsorbed in the inner medulla by the urea transporters UT‐A1 and UT‐A3. Although regulation of urea handling by these transporters controls urine concentration, the impact of these transporters on DN has not been explored. We induced DN in wild type (WT) and UT‐A1/A3 null mice (KO) using low‐dose streptozotocin injections and fed the mice a low (LP; 14%)‐, normal (NP; 25%)‐ or high (HP; 40%)‐protein diet over 10 wk. All DN mice demonstrated glycosuria and polyuria regardless of diet or genotype. Creatinine clearance was dampened in DN WT mice on LP and NP diets and was significantly decreased in DN WT mice on a HP diet. Interestingly, DN KO mice did not have diminished creatinine clearance rates regardless of diet suggesting the inability to reabsorb urea preserves renal function in DN. Histological analysis showed advanced interstitial fibrosis in WT DN mice fed a NP and HP diet; however, diabetic KO mice on the same diets had minimal fibrosis. Both DN and high urea levels are associated oxidative stress, prompting us to investigate multiple markers of oxidative stress. We found that DN KO mice fed HP diet were protected from the oxidative stress observed in DN WT mice on the same diet. Our data indicate in the absence of UT‐A1 and UT‐A3, the collecting duct fails to reabsorb the excess urea from a high protein diet and in doing so, prevents the interstitial fibrosis that occurs as a consequence of increased oxidative stress in the diabetic kidney.

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HHMI Science Education Program Award #52006923
Tight junction protein abundance is altered in Metformin treated airway epithelial cells
Authors: Kalsi Kameljit K , Garnett James P , Baker Emma H , Baines Deborah L
Published: November 2023

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however during respiratory bacterial infection TJ abundance is altered. Metformin used for the treatment of diabetes is known to increase transepithelial resistance (TEER) of airway epithelial cells. We investigated the effect of metformin and Staphylococcus aureus inoculation on TJ abundance and the assembly/reassembly of the TJ protein in airway epithelial cells. Occludin protein was detected by immunoblot analysis with a major protein band at 60 kD with cleavage products at 46, 44 and 37 kD in human airway epithelial cells (H441), pre‐treatment with 1 mM metformin for 18h, resulted in an increase in the 60 kD protein band (13 ± 7%, P<0.05; n=4) and a decrease in the cleavage product at 44 kD (37 ± 4%, P<0.05; n=4). Inoculating H441 monolayers with S.aureus produced a significant reduction in abundance in both 60 and 44 kD protein bands (35 ± 11% and 41 ± 5% respectively, P<0.05; n=4) which was not reversed with metformin pre‐treatment. Similarly occludin and Z0‐1 abundance were both enhanced with metformin and reduced with S.aureus in immunostained epithelial monolayers. Disassembly and reassembly of TJs in the presence of a calcium chelating agent was not protected or enhanced by metformin pre‐treatment. These data provide evidence that metformin regulates TJ protein abundance through a mechanism that does not involve changes in assembly.

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MRC‐MICA MR/K012770/1
Dehydration Impairs Synaptic Plasticity by Interfering with Late‐Phase Long‐Term Potentiation (L‐LTP)
Authors: Vashisht Anirudh , Morykwas Michael , Hegde Ashok , Argenta Louis , McGee Maria
Published: November 2023

Dehydration impairs cognitive function. To explore the possible mechanisms, we dehydrated mouse brain tissue and measured LTP, the best understood cellular process of synaptic plasticity that underlies learning and memory in vertebrates.

We determined physiologic hydration by titrating water activity in mouse brain explants. Specifically, we immersed the 60–90‐mg explants in baths of artificial cerebrospinal fluid (ACSF) equilibrated at room temperature in which inert, non‐penetrating polymer polyethylene glycol 8000 was dissolved to attain colloidosmotic pressures of 0 mmHg (in control ACSF), 54 mmHg, 101 mmHg, and 196 mmHg. We then calculated the parameters reflecting brain hydration, including hydration potential and hydraulic conductance, from initial flow rates determined gravimetrically. To explore the effect of dehydration on synaptic plasticity, hippocampus slices were equilibrated for 30 min at the above pressures prior to recording field excitatory postsynaptic potentials (fEPSP). Parameters for basal synaptic transmission were derived from input‐output tracings following incremental stimuli. In a separate set of slices, L‐LTP was induced by spacing four 100‐Hz trains 5‐min apart and recording the results for 3 h. Water activity titrations were conducted at room temperature and LTP at 32 °C. All experiments were reproduced in at least 6 mice; results are presented as means and standard errors.

Fluid transferred either to or from the brain explants, depending on the bath pressure. Initial fluid transfer rates—3.486 ± 0.1919, 0.08950 ± 0.3907, −1.820 ± 1.258 and −4.248 ± 2.338 μl/min/g— increased linearly ( r2> 0.9, by linear regression analyses) with nominal bath pressures of 0, 54, 101, and 196 mmHg, respectively. The hydraulic conductance, calculated from the slope of the fitted regression lines, was 0.051 ± 0.0019 μl/min/g/mmHg, and the hydration potential, calculated from the pressure at initial rate = 0, was 56.44 ± 4.96 mmHg. In slices equilibrated at near this physiologic hydration potential, basal synaptic transmission resembled that of the slices in control ACSF. However, dehydrating the slices by increasing the colloid osmotic pressure increased basal synaptic transmission, indicating neuronal hyper‐excitability. Similarly, L‐LTP induced at the 54 mmHg level did not differ from that in controls, but progressive dehydration impaired its induction. Induction was completely abolished at a nominal pressure of 196 mmHg. The surprisingly high hydration potential measured in the explanted brain tissue indicates lower water activity in the brain interstitial matrix than in cerebrospinal fluid (CSF) and reflects competition for water in the crowded brain microenvironment. As a notable consequence, neuronal excitability is highly sensitive to changes in water activity. Our findings predict that local dehydration affects cognitive function and impairs synaptic plasticity by preventing L‐LTP induction.
The Anti‐Inflammatory Peptide Ac‐SDKP is Released from Thymosin β4 by Meprin α and Prolyl Oligopeptidase
Authors: KUMAR NITIN , Nakagawa Pablo , Janic Branislava , Romero Cesar A. , Worou Morel E. , Peterson Edward L. , Ongeri Elimelda E. , Niyitegeka Jean‐Marie V. , Rhaleb Nour‐Eddine , Carretero Oscar A.
Published: November 2023

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐SDKP) is a natural tetrapeptide with anti‐inflammatory and anti‐fibrotic properties. We have previously shown that prolyl oligopeptidase (POP) is involved in the Ac‐SDKP release from thymosin β4 (Tβ4). However, POP can only hydrolyze peptides shorter than 30 amino acids and Tβ4 is 43 amino acids long. This suggests that Tβ4 must be hydrolyzed by another peptidase that releases N‐terminal intermediate peptide(s) <30 amino acids before POP hydrolysis takes place. Our search in peptidase database for potential candidate(s) gave high score for meprin α metalloprotease. Therefore, we hypothesize that Tβ4 is hydrolyzed by meprin α prior to POP hydrolysis. To test this, in vitro and in vivo studies were performed. In vitro, we found that the incubation of Tβ4 with both, meprin α and POP releases Ac‐SDKP, whereas it failed when Tβ4 is incubated with either meprin α or POP alone. Incubation of Tβ4 with rat kidney homogenate increases Ac‐SDKP, which is blocked by actinonin (meprin α inhibitor). In addition, kidney from meprin α knockout mice showed a significantly lower Ac‐SDKP as compared to its wild type control. In vivo, we observed that rat treated with captopril (ACE inhibitor) increased plasma concentrations of Ac‐SDKP, which is inhibited by the co‐administration of actinonin (vehicle 3.1±0.22 nmol/L; captopril 15.1±0.7 nmol/L; captopril + actinonin 6.1±0.3 nmol/L; vehicle versus captopril, P<0.002; captopril versus captopril + actinonin, P<0.002). Similar results were obtained with urinary Ac‐SDKP excretion. We conclude that Ac‐SDKP is released from Tβ4 by the successive action of meprin α and POP.

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This work is supported by National Institute Health Grant HL028982
Involvement of the Cyclic AMP Pathway in Dendritic Cell Regulation of Th2 Immune Responses
Authors: Chinn Amy M , Lee Jihyung , Herdman Scott , Raz Eyal , Insel Paul A
Published: November 2023

Dendritic cells, the main antigen‐presenting cells, are involved in the induction of type II helper T cell (Th2) immune responses, but the mechanism by which this occurs is not fully elucidated. Mice that have a CD11c‐specific deletion of the Gnas gene (CD11cΔGnas), which encodes the Gαs protein of the heterotrimeric (αβγ) Gs signaling protein complex, display an allergic asthma phenotype; the corresponding decrease in cyclic AMP (cAMP) in dendritic cells biases them to provoke a Th2 response ex vivo and in vivo (Lee et al PNAS 112: 1529–34 [2015]). The molecular events involved in the Th2 bias that results from the increased Gαi:Gαs signaling ratio in this setting remain to be defined. Here, we compare the expression of G protein‐coupled receptors (GPCRs), including those that regulate cAMP formation, and the expression of other components that regulate cAMP levels of wild‐type (WT) and CD11cΔGnas mouse bone marrow‐derived dendritic cells (BMDCs). Quantitative PCR (qPCR)‐based Taqman GPCR arrays revealed that WT BMDCs express 120 non‐chemosensory GPCRs while CD11cΔGnas BMDCs express 145 GPCRs. Notably, GPCRs that couple to Gαi (rather than other Gα proteins) have the greatest increase in gene expression in the CD11cΔGnas BMDCs compared to BMDCs from WT mice. Moreover, CD11cΔGnas BMDCs have reduced expression (as assayed by qPCR; p<0.05) of cyclic nucleotide phosphodiesterase (PDE) isoforms that degrade cAMP and are known to be induced by increases in cellular cAMP levels (PDE's1B, 3B, 4A, 4B, 4D, and 7B). PDE4B is also the predominant PDE isoform in mouse BMDCs. In addition, we found a significant (p<0.05) change in the mRNA expression of certain adenylyl cyclase isoforms: an increase in ADCY3 and decrease in ADCY4. Together, these data indicate that the expression of GPCRs (in particular Gαi‐coupled GPCRs) and other components that regulate cAMP levels are altered in dendritic cells that promote Th2 bias. Such GPCRs and post‐G protein components may contribute to the greater Th2 responses and allergic asthma observed in CD11cΔGnas mice.

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Supported by NIH grants R56AI110505 and T32GM007752.
Role of calcium signaling for GDNF secretion, ureter branching and early nephron formation
Authors: Fontana Jacopo Maria , Jess David Unnersjö , Blom Hans , Brismar Hjalmar , Aperia Anita
Published: November 2023

Low nephron endowment has an adverse effect on the outcome of many kidney diseases and is a risk factor for chronic kidney disease. The formation of nephrons occurs as a result of the reciprocal interaction between metanephric mesenchymal (MM) cells and the ureteric bud (UB). MM cells secrete the trophic factor GDNF, which binds to the Ret receptors on the UB. Signaling via GDNF and the Ret receptor promotes UB branching and plays a central role for initiating nephron formation. The mechanisms controlling the release of GDNF from the MM cells has been elusive, despite its fundamental importance for kidney development. Most secretory processes are controlled by calcium signaling. Here we demonstrate, using embryonic E14 rat kidneys cultured for 1–2 days, the occurrence of spontaneous calcium transients in MM cells with a frequency of 2–29 min−1. Depletion of calcium stores in the endoplasmic reticulum (ER) with cyclopiazonic acid (CPA) caused drastic reduction of calcium activity, while acute exposure of kidneys to calcium‐free extracellular solution had no effect. To chronically reduce the ER calcium stores and downregulate the spontaneous calcium activity in MM cells, kidneys were exposed to low concentration of CPA from DIV1 to DIV2. This resulted in swelling and significant reduction of UB as well as of significant reduction of UB branching points and of newly formed pre‐glomeruli. In addition, GDNF concentration in the culture media was significantly lower for CPA treated than for control kidneys. To further assess the role of calcium signaling for GDNF secretion, we stained the kidneys for MM and UB markers (WT‐1 and E‐Cadherin respectively) and for GDNF, as well as synaptotagmin‐1 (SYT1), a calcium binding protein that is the major calcium sensor activating secretory processes in the brain. By applying a tissue clearing protocol and super‐resolution microscopy, we could resolve the localization of SYT1 and GDNF in the MM cells, both surrounding the UB tips. In summary we have identified a novel calcium dependent link in the complex process of nephron formation. In the light of this, our results indicate that the vesicle transport and calcium‐regulated fusion that control neurotransmitter release at the synapsis could also have fundamental importance for early embryonic development of methanephric kidney.

Support or Funding Information

The Swedish Research Council (AA and HB).