bluuebunny/crossref_metadata_2025 huggingface dataset:
Dopamine β‐hydroxylase Inhibition with BIA5‐1058 Normalizes the High Blood Pressure of Human GRK4g 142V Transgenic Mice
Authors: Asico Laureano D , Lin Hai , Jones John E , Soares‐da‐Silva Patricio , Jose Pedro A , Wang Xiaoyan
Published: November 2023

Overactivity of the sympathetic nervous system is one of the mechanisms involved in the pathogenesis of human essential hypertension. However, treating hypertension by inhibiting the sympathetic nervous system is associated with adverse side effects. Reducing the biosynthesis of norepinephrine (NE) by inhibiting the enzyme that catalyzes the conversion of dopamine to NE, dopamine β‐hydroxylase (DβH), in the peripheral but not the central sympathetic nervous system may be a good drug to treat hypertension; BIA5‐1058 is such a drug. Therefore, we used BIA5‐1058 to treat the hypertension of human GRK4γ 142V transgenic (142V) mice. 142V mice and non‐transgenic (NT) littermates (7 mo old, n=4/group) were gavaged (10:00 AM) with BIA5‐1058 (30 mg/kg) or vehicle, daily for 1 week. The mice were placed in metabolic cages for 24 hr urine collection before blood pressure (BP), under anesthesia, was measured, via the femoral artery on day 7. In additional studies, BP was also monitored by telemetry in conscious mice. Food and water intake, body weight, heart rate, urine flow and sodium and serum Na+, K+, and Cl− were similar in the 4 groups. Compared with NT mice, 142V mice had increased systolic (142v mice: 120±1.5 vs. NT mice: 98.8±2.7, mm Hg) and diastolic BPs (94.8±2.7 vs. 66.5±5.1) that were normalized by BIA5‐1058 (SBP: 97±2 vs. 97.3±0.6; DBP: 70.3±4.8 vs. 70.8±2.5). In conscious mice, 30 mg/kg but not 3 and 10 mg/kg BIA5‐1058, also normalized the BPs of 142V mice. Renal protein abundance of type 1 angiotensin II receptor (AT1R) was greater in 142V than NT mice (265±56 vs. 100±18%), similar to our previous report. BIA5‐1058 also decreased renal AT1R protein in 142V but not NT mice (27±6 vs. 100±14%). Our data suggest that inhibition of DβH normalizes the BP of 142V mice, not only by decreasing the synthesis of NE but also renal AT1R protein expression.

Support or Funding Information

P01HL074940

P01HL068686

R01HL092196

R37HL023081

R01DK039308
ETA Receptor Antagonism and Estrogen Replacement Prevent Ang II Hypertension in Menopausal Mice
Authors: Pollow Dennis P. , Romero‐Aleshire Melissa J. , Davies John , Nikolich‐Zugich Janko , Brooks Heddwen L.
Published: November 2023

Cardiovascular disease, including hypertension, increases dramatically in women after menopause, however the efficacy of hormone replacement therapy (HRT) in preventing such diseases has produced controversial results. Endothelin receptor antagonists have been tested as anti‐hypertensive agents in clinical trials and may represent a promising alternative to HRT in postmenopausal women. Therefore, we sought to directly compare the anti‐hypertensive capacity of estrogen replacement (17‐b estradiol pellet, 0.1mg) vs. the ETA receptor antagonist ABT‐627 (5mg/kg, i.p., daily) in the VCD mouse model of menopause (4‐vinylcyclohexene diepoxide, VCD). Ang II (800ng/kg/min, 14d) was infused into VCD‐treated menopausal female mice alone (Meno), or with either concurrent 17‐b estradiol replacement (E2) or ETA receptor antagonism (ETAA). Premenopausal mice received sesame oil vehicle with and without subsequent Ang II (Con, Con/Ang). Ang II infusion induced a significant increase in systolic blood pressure in VCD‐treated menopausal mice compared to Ang II infusion in premenopausal mice (Con Δ2 ± 2 mmHg, Con/Ang Δ15 ± 2* mmHg, Meno/Ang Δ37 ± 6*# mmHg, *P<0.05 vs Con, #P<0.05 vs Con/Ang). Both 17‐b estradiol replacement and ETA receptor antagonism prevented the increased blood pressure response in Ang II‐infused menopausal mice (E2 Δ10 ± 6* mmHg, ETAA Δ14 ± 3* mmHg, *P<0.05 vs Meno/Ang). Splenic and renal T cell profiles were assessed via flow cytometry. 17‐b estradiol replacement increased the percentage of Foxp3+ T regulatory cells in the spleen compared to all other groups (Con 17 ± 1%, Con/Ang 18 ± 1%, Meno/Ang 18 ± 1%, E2 23 ± 1%*, ETAA 18 ± 1%, *P<0.05 vs all groups). However, no changes in T cell profiles occurred with any treatment in the kidney. Quantitative real‐time PCR demonstrated that Ang II‐induced expression of whole kidney collagen type IV in VCD‐treated menopausal mice was significantly reduced by ETA receptor antagonism, but not 17‐b estradiol replacement (ETAA 0.68 ± 0.08 fold*, E2 1.25 ± 0.05 fold*, *P<0.05 vs Meno). These data suggest that ETA receptor antagonism may be an effective treatment strategy for preventing hypertension in postmenopausal women.

Support or Funding Information

American Heart Association Predoctoral Fellowship (DPP), NIH DK073611 (HLB), Sarver Heart Center (DPP), ARCS Foundation (DPP)
Altered Renal and Circulating Renin‐Angiotensin System Does Not Cause Spontaneous Hypertension in the African Green Monkey
Authors: Gutierrez Alex , Goleva Slavina B , Rhoads Megan K , Weaver Chelsea Christina , Beierwaltes William H , Osborn Jeffrey L
Published: November 2023

The renin‐angiotensin system (RAS) is important in the long‐term regulation of, sodium balance, body fluid volumes and blood pressure. The African Green Monkey, (AGM, Chlorocebus aethiops sabaeus) is a translational model of hypertension (HT) due to its close genetic similarity, behavioral activities and significant upright posture similar to humans. We hypothesized that upregulation of the RAS occurs in HT AGMs which may decrease Na+ excretion leading to Na+ retention and expansion of extracellular fluid volume. AGMs were phenotyped by systolic blood pressure (SBP) as HT (SBP>140 mmHg) or normotensive (NT, SBP<120 mmHg). Plasma and renal tissue samples were obtained from adult male AGMs with NT (n=26) having SBP of 98.2 ± 2.1 mmHg, HT AGMs (n=27) averaging 169.9 ± 6.1 mmHg. Plasma renin activity (PRA) and renal cortical renin content were determined by radioimmunoassay. Circulating PRA of NT (3.27 ± 0.36 ng AngI/ml/hr, n=15) and HT (3.34 ± 0.48 ng AngI/ml/hr, n=16) AGMs were not different (p>0.05). Renal cortex tissue was homogenized and incubated with excess renin substrate to measure renal cortical renin content (RCRC). Similar to PRA, RCRC of NT (10.73 ± 2.98 μg AngI/ml/hr/mg protein, n=11) and HT (8.94 ± 1.56 μg AngI/ml/hr/mg protein, n=13) AGMs were similar. Next, angiotensinogen (AGT) expression was assessed from renal cortex, outer medulla and liver using qRT‐PCR from extracted tissue RNA. Gene expression was normalized using RPS32 for kidney and RPS13A for liver. AGT expression was similar between NT and HT AGMs in the renal cortex (NT; 1 ± 0.55 vs HT; 1.74 ± 0.91), in the renal outer medulla (NT; 1 ± 0.16 vs HT; 0.59 ± 0.21) and liver (NT; 1 ± 0.14 vs HT; 1.19 ± 0.17, p > 0.05 for all tissues). To assess renal Na+ handling in HT (n=4) and NT (n=3) AGMs, animals were housed individually in pens and urine collected at 24 hour intervals for 5 consecutive days. Daily urinary sodium excretion of HT AGM's was greater than that of NT animals (HT; 2.86±0.40 mmol/day vs NT; 1.17±0.16 mmol/day, p<0.05) but urine osmolality was not different. The similar quantities of plasma and tissue renin, combined with unchanged gene expression of AGT, indicate that renin activity and renin substrate (angiotensinogen) in the RAS do not contribute to hypertension in the AGM. Thus, the long term altered blood pressure regulation and hypertension may result from elevated peripheral resistance due to other pressor mechanisms, as our data rules out any abnormal expression or activity of the renin‐angiotensin system in this nonhuman primate model of heritable, genetic hypertension.

Support or Funding Information

Supported by American Physiological Society, National Heart, Lung and Blood Institute (NHLBI; 1 R25 HL115473‐01), and Biomedical Science Research Group, LLC.
Salt Inducible Kinase (SIK): A potential intracellular signal involved in DOCA‐salt induced hypertension?
Authors: Gonsalez Sabrina Ribeiro , Ferrão Fernanda Magalhães , Gomes Dayane Santos , de Lima Dayvid Goncalves Bezerra , Lowe Jennifer , Prieto Minolfa C , Lara Lucienne S
Published: November 2023

Sodium sensitizes the cardiovascular‐renal axis resulting in salt‐sensitive hypertension, renal injury, and inappropriate activation of the renin‐angiotensin system (RAS). However, the molecular mechanisms and influences of sodium on blood pressure (BP) remain unclear. In the present study, we aimed to determine the unique effects of Na+ on renal function and the intracellular signaling involved in a rat model of DOCA‐salt induced hypertension. Adult Wistar male rats were subjected to left uninephrectomy, followed by SC administration of deoxycorticosterone acetate (DOCA; 8 mg/kg) or vehicle (mineral oil) and either normal or high salt diet (HS, 4% NaCl) in four groups, as follows: CTRL, CTRL/Salt, DOCA and DOCA/Salt (n=15, each group). CTRL and CTRL/Salt rats exhibited normal systolic BP. In CTRL/Salt rats, GFR was normal, despite BUN and proteinuria. Both, Na+ filtration and excretion loads increased from 0.23 ± 0.0074 in CTRL to 0.30 ± 0.011 (mEq) in CTRL/Salt and from 6.6 × 10−4 ± 1.5 × 10−4 in CTRL to 0.0095 ± 1.5 × 10−4 (mEq) in CTRL/Salt, (P<0.05), respectively. In addition, FENa increased 10 times, as Na+ reabsorption load (mEq) from 0.22 ± 0.064 in CTRL to 0.29 ± 0.011 in CTRL/Salt rats. Na+ reabsorption was increased two‐fold and in parallel with the Na,K‐ATPase activity in the renal cortex. In the hypertensive DOCA rats, these functions were preserved. DOCA/Salt rats showed exacerbated hypertension, increased FENa to a similar extent than CTRL/Salt rats along with augmentation of BUN, and proteinuria. In these rats, the high salt intake lead to 45% reduction in Na,K‐ATPase activity in renal cortex. The histological analyses revealed the presence of renal inflammation in DOCA and DOCA/Salt rats. High‐salt diet and DOCA increased sodium‐inducible kinase (SIK) immunoexpression in the glomeruli. These data suggest that in the absence of high BP, sodium contributes to increase Na,K‐ATPase activity and kidney injury without glomerular function alterations. However, in the DOCA/Salt rat model with established hypertension, increases in sodium excretion include alterations in Na,K‐ATPase activity and SIK, which are potential molecular intracellular signal involved in DOCA‐salt induced hypertension.

Support or Funding Information

CNPq (420584/2013‐7 Science Without Borders grant, Government of Brazil)and NIH‐NIDDK (RO1DK104375)
Effect of low‐dose of multi‐glycoside of Tripterygium wilfordii Hook. f. on renal injury in mice with salt‐sensitive hypertension
Authors: Wang Youping , Song Chundong , Zhu Mingjun , Xu Hui , Wang Xiaoxiao , Cui Lin , Liu Suxiao , Xie Shiyang , Shen Si , Zhang Songjiang , Wang Donna H
Published: November 2023

To clarify the effects of low‐dose of multi‐glycoside of Tripterygium wilfordii Hook. F. (GTW), an extract from a Chinese herb thunder god vine, on salt‐sensitive hypertension‐induced renal damage, we induced hypertension by uninephrectomy and deoxycorticosterone (DOCA)‐salt in mice with or without low‐dose of GTW treatment. DOCA‐salt treatment for 5 weeks increased mean arterial pressure (MAP) (P<0.01) and urinary albumin excretion (P<0.05), and also induced renal hypertrophy (P<0.01) compared with the control. Periodic acid‐Schiff and Masson's trichrome staining showed that DOCA‐salt treatment caused obvious glomerulosclrosis and tubulointerstitial injury in the renal cortex (P<0.05), and tubulointerstitial fibrosis compared with the control mice. Consistent with the results, renal collagen level determined using hydroxyproline assay was higher in DOCA‐salt‐treated mice compared with the control mice (P<0.05). Treatment with GTW at the low dose of 11.5 mg/kg/day by gavage had no effect on MAP and renal hypertrophy. However, it partially prevented renal morphological damage and reduced the increases in urinary albumin excretion and renal collagen (P<0.05). Our data showed that treatment with the low dose of GTW prevented renal damage induced by DOCA‐salt hypertension independently of their effects on blood pressure. The results indicate that low‐dose of GTW exhibits a protective effect on renal damage during salt‐sensitive hypertension. This work was supported in part by the National Natural Science Foundation of China (No. 81170243).

Support or Funding Information

National Natural Science Foundation of China (No. 81170243)
Zinc Deficiency Stimulates Sodium Chloride Cotransporter (NCC) Upregulation via Modulation of Calcineurin Activity in Mouse Distal Convoluted Tubular Cells
Authors: Williams Clintoria R. , Mistry Monisha , Li Mirandy S. , Ko Benjamin , Gooch Jennifer L. , Hoover Robert S.
Published: November 2023

Background

Zn2+ deficiency (ZnD) has been implicated in the development of hypertension. However, the mechanisms continue to be defined. The Sodium Chloride Cotransporter (NCC) plays a critical role in Na+ handling by the distal convoluted tubule and subsequent blood pressure homeostasis. Furthermore, experimental studies support a direct role for the Zn2+‐dependent enzyme calcineurin in NCC regulation.

Hypothesis

The objective of this study is to investigate the effects of ZnD on NCC. We hypothesize that Zn2+ regulates NCC via modulation of calcineurin activity.

Methods

To test this hypothesis, mouse distal convoluted tubular (mDCT) cells were exposed to the Zn2+ chelator N,N,N′,N′‐Tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN) or vehicle (DMSO) for 24 hours. To investigate the role of calcineurin in NCC regulation, mDCT cells were treated with the calcineurin inhibitor tacrolimus for 24 hours. NCC expression (qRT‐PCR, Western blot and immunocytochemistry) and activity (thiazide‐sensitive Na+ uptake) were examined as well as calcineurin activity (phosphatase assay).

Results

The data show that NCC expression is upregulated in mDCT cells following intracellular Zn2+‐depletion with TPEN compared to vehicle‐treated cells. Additionally, calcineurin phosphatase activity is blocked in Zn2+ depleted mDCT cells. Consistent with TPEN findings, inhibition of calcineurin with tacrolimus stimulates NCC expression and activity.

Conclusions

Taken together, data suggest that ZnD induces NCC upregulation by inhibiting calcineurin activity.

Significance

These novel findings identify NCC as a possible mediator of ZnD‐induced hypertension. Understanding the mechanisms by which ZnD contributes to blood pressure dysregulation may have an important impact on the treatment of hypertension.

Support or Funding Information

T32‐DK00756 (CRW), R25‐DK101390 (MSL), K08‐DK081728 (BK), VA‐MERIT (JLG), R01‐DK085097 (RSH)
Meprin β deficiency reduces leukocytes infiltration in mouse kidneys with unilateral ureteral obstruction (UUO)‐induced inflammation
Authors: Fernander Mizpha Courtney , Niyitegeka Jean‐Marie Vianney , Conklin Dawn , Yahaya Ismaila , Minor Radiah C , Ongeri Elimelda Moige
Published: November 2023

Meprins are metalloproteases that are most abundantly expressed in the proximal kidney tubules. Meprins are also expressed in leukocytes (monocytes, and macrophages) suggesting a role in the immune response. Meprins were previously shown to play a role in the movement of leukocytes to sites of inflammation in the small intestines. Furthermore, meprins have been shown to cleave pro‐inflammatory cytokines such as interleukin 1 β (IL‐1β), interleukin 6 (IL‐6), and pro‐interleukin 18 (IL‐18). Proteolytic processing by meprins inactivates IL‐6, but activates IL‐1β and pro IL‐18. The objective of the current study was to determine how meprin β deficiency impacts renal leukocyte infiltration in a unilateral ureteral obstruction (UUO)‐induced inflammation mouse model. Surgical procedures were used to ligate one ureter in 12 week‐old wild‐type (WT) and meprin β knockout male mice. The contralateral kidney served as a control for each mouse. The mice were sacrificed at 7 days post UUO. The kidneys were perfused with saline, subjected to collagenase digestion, and flow cytometry was used to determine the number of leukocytes [T‐cells (CD3+), macrophages (CD11b), and neutophils (LY6G+)] present in kidney tissues. Compared to wild‐type counterparts, we observed a significant decrease (P ≤ 0.05) in Ly6G+ and CD3+ cells in meprin β KO kidneys subjected to UUO. The data suggests that meprin β enhances leukocyte infiltration in the mouse kidneys subjected to UUO‐induced inflammation. Proteomic analysis will determine how the presence of meprins impacts the levels and/or fragmentation of pro‐inflammatory cytokines.

Support or Funding Information

NIH/NIGMS SC3GM102049
RORγt‐Dependent Preferential Induction of IL‐17F by TLR3 Activation in SHR Immune Cells
Authors: Singh Madhu V , Cicha Michael Z , Chapleau Mark W , Abboud François M
Published: November 2023

We have shown that spontaneously hypertensive rats (SHR) have an abnormally large population of CD161+ immune cells at birth that increases further with age compared with age matched normotensive Wistar Kyoto rats (WKY). We also found in SHR spleens a greater abundance of CD161+CD4+ cells that expressed IL‐17A and IL‐17F cytokines known to be synthesized predominantly by T helper 17 (Th17) lymphocytes that are prohypertensive and anti‐T regulatory cells. Here we further define the phenotype of the proinflammatory Th17 in SHR vs. WKY. When subjected to in vitro programming by T cells receptor activation in the presence of IL‐6 and TGF‐β, SHR splenocytes showed significantly greater induction of IL‐17F than WKY (1.4 fold in WKY vs 4.7 fold in SHR, p< 0.05). In contrast activation of the TLR3/TRIF‐mediated pathway with the TLR3 agonist poly‐IC resulted in a preferentially much greater expression of IL‐17F in SHR (2.9 fold in WKY vs. 27 fold in SHR) compared to the modest increase in IL‐I7A in both WKY and SHR (1.7 fold in WKY vs. 2.2 fold in SHR). We also found that this enhanced IL‐17F expression in SHR is dependent on a master regulator transcription factor (the orphan receptor RORγt) which polarizes immune cells into IL‐17 producing Th17 cells. RORγt RNA expression was significantly greater in SHR than in WKY splenocytes (5.7 fold more RORgt in SHR). Using flow cytometry, we confirmed the greater expression of RORγt in CD4+CD161+ cells of SHR. Furthermore, the pronounced induction of IL‐17F by TLR3 activation was suppressed by digoxin (5μM), a specific inhibitor of RORγt. We conclude that SHR has an abnormally large population of CD4+CD161+ Th17 cells with a markedly enhanced potential for expression of proinflammatory, prohypertensive IL‐17F in response to TLR3 activation, driven by the master regulator transcription factor orphan receptor RORγt.

Support or Funding Information

National Institutes of Health Program Project Grant to FMA (HL 14388)

VA Merit Review Award to MWC (1 I01 BX001414).
The cGMP/PKG Signaling Pathway Underlies the Inhibitory Effect of Sildenafil Citrate on NHE3 Activity in Rat Renal Proximal Tubules
Authors: dos Santos Priscilla Marys Costa , Pessoa Thaissa Dantas , Malnic Gerhard
Published: November 2023

Sildenafil (Sil) increases cGMP and inhibits RhoA, and this intracellular pathway has pronounced effects on NHE3 activity. The aim of this work was to determine the effects of Sildenafil on NHE3 and to study the intracellular signaling pathway involved. The studies were performed by the stationary microperfusion technique, in which we measured H+ secretion in rat renal proximal tubules (PT), through a H+‐sensitive microelectrode. PT of Wistar rats (WR) were perfused with 24mM HCO3− (CTRL) +/− 10μM Sil to determine bicarbonate reabsorption (JHCO3−). Luminal perfusion of Sil decreased JHCO3− by 20% compared to CTRL. To test if this effect was NHE3‐dependent, PT were perfused with CTRL, Sil plus 5μM S3226, a specific NHE3 inhibitor, which decreased JHCO3−(39,14% in CTRL and 45,41% in Sil perfused tubules). The remaining S3226‐insensitive JHCO3− was not different among groups, showing that only NHE3 was modulated by Sil. To determine if RhoA was involved in these effects, the PT were perfused with the Rho GTPase inhibitor Y‐27632 (1μM). Perfusion of Y‐27632 alone decreased JHCO3− by 20% compared to CTRL. The perfusion with Y‐27632 increased the inhibitory effect of Sil by 26.16%, suggesting that this effect on JHCO3− was additive and that the inhibitory effect of Sil was caused by another intracellular pathway. Moreover, the inhibitory effect of Sil was completely abolished by the protein kinase G (PKG) inhibitor KT5823 (1μM). These data showed that the inhibitory effect of Sil on NHE3 transport activity in proximal tubule is mediated by activation of cGMP/PKG and that this effect is RhoA independent.

Support or Funding Information

FAPESP; CNPq; CAPES
AMPK Stimulates Osmotic Water Permeability in Rat IMCDs
Authors: Wang Yanhua , Efe Orhan , LaRocque Lauren M. , Klein Janet D. , Sands Jeff M.
Published: November 2023

Our previous studies showed that adenosine monophosphate activated kinase (AMPK) is a possible alternate, vasopressin‐independent, regulatory pathway for urea and water transport. Mice lacking a vasopressin receptor (V2R‐KO) are polyuric. Intraperitoneal (IP) injection of metformin to stimulate AMPK significantly increased urine osmolality from 196 ± 32 (control) to 399 ± 52 mOsM at 1h, and 586 ± 41 mOsM at 5h post treatment in the V2R‐KO mice. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we showed that metformin and AICAR, two AMPK activators, significantly increased urea permeability (control:metformin 16 ± 2 to 22 ± 4 ×10−5 cm/s; control:AICAR 19 ± 2 to 23 ± 3 ×10−5 cm/s). In this study, we investigated the role of AMPK in the regulation of water transport in IMCDs. In perfused rat IMCDs, metformin (400 μM) significantly increased osmotic water permeability from 72 ± 42 to 104 ± 61 μm/s. Immunohistochemistry showed that injecting rats IP with metformin increased the membrane accumulation of total aquaporin‐2 (AQP2) and Serine 256 phosphorylated AQP2 (pSer256‐AQP2). Phosphorylation of Serine 256 is required for AQP2 membrane accumulation. In contrast, western blot analysis of biotinylated proteins showed that metformin did not increase urea transporter UT‐A1 membrane accumulation. We conclude that AMPK activation increases: 1) water permeability by increasing the membrane accumulation of total and pSer256‐AQP2; and 2) urea permeability by stimulating the UT‐A1 transporter that is already present in the membrane. These data show that AMPK activation increases water and urea permeability in rat IMCDs. Thus, activation of AMPK may be a novel therapeutic approach to treating nephrogenic diabetes insipidus due to defective vasopressin signaling.

Support or Funding Information

NIDDK
Impaired Urinary Concentration in Car2 Deficient Mice
Authors: Alexander R. Todd , Krishnan Devishree , Cordat Emmanuelle
Published: November 2023

Pharmacological inhibition of carbonic anhydrases and deficiency of carbonic anhydrase II (Car2) in mice cause polyuria. We therefore set out to determine in Car2 deficient mice by which mechanism(s) polyuria is mediated. Blood gas analysis revealed that Car2 deficient mice have a respiratory acidosis, as reported previously. Moreover, the acidotic mice failed to excrete an acidic urine and had significant bicarbonaturia, consistent with a type III renal tubular acidosis. Carbonic anhydrase inhibitors induce a natriuresis, however Car2 deficient mice did not have altered urinary sodium excretion (either total excretion or FENa). Deletion of NHE3 in mice results in calciuria, and inhibition of NHE3 in cell culture reduces transepithelial calcium flux. Carbonic anhydrase II enhances NHE3 activity and calciuria induces polyuria. However, Car2 deficient mice do not demonstrate altered urinary calcium excretion. Further examination revealed a dilute urine in Car2 deficient mice. Urinary concentration remained reduced in Car2 deficient mice relative to wild‐type animals even after water deprivation. Expression of aquaporin‐2 was not different between genotypes, however Car2‐deficient mice had increased renal aquaporin‐1 expression. Carbonic anhydrase II associates with and increases water flux through aquyaporin‐1. Water flux through aquaporin‐1 in the thin descending limb (TDL) of the loop of Henle is essential to the concentration of urine, by generating a concentrated medullary interstitium. Car2 is expressed in the thin descending limb. We therefore measured cortical and medullary interstitial concentration in wild‐type and Car2 deficient mice. Mice lacking Car2 had equivalent cortical interstitial osmolarity to WT mice however, they had a reduced medullary interstitial osmolarity. We propose that reduced water flux through aquaporin‐1 in the TDL in the absence of Car2 prevents the generation of a maximally concentrated medullary interstitium and thus the maximal concentration of urine in Car2 deficient mice resulting in polyuria.

Support or Funding Information

Canadian Institutes of Health Research
Evidence of Phospho‐degron regulating expression of urate secretory transporter ABCG2
Authors: Woodward Owen M. , Hofherr Alexis , Huang Qiong , Kottgen Michael
Published: November 2023

ABCG2 is a high capacity urate secretory transporter of the renal proximal tubule. Mutations in ABCG2 cause gout in humans. The ABCG2 patient mutation ABCG2Q141K leads to instability of the ABCG2 nucleotide‐binding domain, which can be rescued by enhancing nucleotide‐binding domain sandwich formation with phosphomimetic amino acid substitutions (G188E, S187D); raising the possibility that phosphorylation of residues in the NBD domain may regulate expression and trafficking of ABCG2. An in silico analysis of ABCG2 revealed a limited number of predicted phosphorylation sites including S195, a serine conserved in the mammalian lineage. The upstream RXRXS represents a target motif for both AKT1 and PKA, and we find both co‐immunoprecipitate with ABCG2. Specifically, endogenous AKT1 can pull down both over expressed ABCG2 in HEK293 cells as well as endogenous ABCG2 in mouse kidney lysate. AKT1 and ABCG2 transcript co‐localize in the proximal S2 segment of the mammalian nephron (Mark Knepper RNA‐seq database) and inhibiting the AKT1 kinase cascade with PI3K inhibitor LY294002 dramatically up‐regulated ABCG2 expression. Conversely, activating the AKT1 cascade with FBS down‐regulated ABCG2 expression. Replacement of the S195 residue with the phosphomimetic aspartic acid results in significant reduction in expression, localization of ABCG2 protein to peri‐nuclear compartments, and significant sensitivity to the proteasome inhibitor MG132; marking the S195 residue as a Phospho‐degron. Phosphorylation of the well conserved S195 residue may interfere with ATP binding and/or nucleotide‐binding domain sandwich formation, suggesting a novel regulatory mechanism for function and trafficking in ABC transporters.

Support or Funding Information

American Heart Association: 14SDG18060004 & Ardea BioSciences
Differential mechanisms of ammonia transport in rat duodenum and colon
Authors: Akiba Yasutada , Kaji Izumi , Said Hyder , Kaunitz Jonathan D
Published: November 2023

The major source of serum ammonia is colonic luminal bacteria, which contribute to ammonia production from amino acid metabolism. The ammonia transporters, rhesus (Rh) glycoprotein B (RhBG) and C (RhCG), expressed in the GI tract, are implicated in ammonia absorption. Since little is known about ammonia transport in the GI tract, including segmental differences, and since ammonia absorption is a major risk factor for hepatic encephalopathy, we investigated ammonia transport in the duodenum and colon.

We measured NH3/NH4+ transport in Ussing chambered mucosa‐submucosa preparation of rat duodenum, and proximal (PC) and distal colon (DC). Isc changes and serosal ammonia concentrations were measured before and after luminal application of NH4Cl (1 – 30 mM). We also perfused a proximal duodenal or distal colonic loop in vivo with an NH4Cl solution while measuring portal venous (PV) ammonia concentrations.

Luminal application of NH4Cl rapidly and concentration‐dependently increased Isc in the duodenum, whereas Isc gradually increased in response to NH4Cl in the colon. The peak response of NH4Cl‐induced Isc was duodenum = DC >> PC, while the time to peak was duodenum << PC = DC. NH4Cl‐induced Isc was greater when luminal pH was alkaline (pH 7.4 > 7.0 >> 6.4) in the colon, but pH‐independent in the duodenum. Serosal ammonia appearance was consistent with Isc changes in the duodenum, but inconsistent with Isc changes, and constantly increasing, in the colon. NH4Cl‐induced Isc in the duodenum was sensitive to ouabain, but insensitive to luminal Gd3+ and serosal bumetanide, most likely due to cation absorption. NH4Cl‐induced Isc in the colon was sensitive to luminal Gd3+ and serosal bumetanide, but insensitive to ouabain, suggesting that absorbed ammonia may induce Cl− secretion. Luminal perfusion of the duodenum and DC with NH4Cl similarly increased PV ammonia concentration. Quantitative PCR showed that RhBG was ubiquitously expressed in the GI mucosa, whereas RhCG was expressed in the duodenal mucosa, but not in the PC or DC mucosa.

Electrogenic, pH‐independent, ammonia absorption in the duodenum may be explained by apical RhCG‐mediated NH4+ transport, whereas luminal pH‐dependent ammonia absorption in the colon characterized by a gradual increase of Isc may be related to simple NH3 diffusion, followed by stimulated secretion. These results may explain why oral NH4Cl induces acute metabolic acidosis, whereas slow NH3 absorption in the distal colon may be implicated in hepatic encephalopathy.

Support or Funding Information

VA Merit Review, NIH R01 DK54221
Insights into the Role of Occluding Septate Junctions in the Maintenance of Salt and Water Balance in Mosquito (Aedes aegypti) Larvae
Authors: Jonusaite Sima , Kelly Scott Phillip , Donini Andrew
Published: November 2023

Septate junctions (SJs) are specialized occluding junctions that function as paracellular diffusion barriers in the epithelia of invertebrates. SJs are composed of transmembrane and cytosolic proteins which are reported to be essential for epithelium integrity. This is of particular importance to aquatic animals because their habitat is often compositionally different from their internal body fluids. Furthermore, aquatic habitats are subjected to alterations in their solute composition by anthropogenic and climatic factors. In this regard, the contribution of SJ proteins to the regulation of paracellular solute movement across epithelia during and/or following changes in an aquatic invertebrate's environmental conditions is not known. It can however be hypothesized that epithelial SJ protein abundance is altered in response to environmental changes, reflecting their involvement in the regulated movement of solutes across the paracellular pathway. To begin testing this hypothesis, a role for SJ proteins in the maintenance of salt and water balance in larval mosquito Aedes aegypti was considered. These animals reside in freshwater (FW), a hypoosmotic environment, but are capable of tolerating saline environments that reflect isosmotic conditions. Genes encoding A. aegypti SJ proteins Megatrachea, Sinuous, Kune‐kune, Coracle, Scribble, Discs large, Neurexin IV, Lethal giant larva, Gliotactin, Mesh and Snakeskin were identified and their expression was examined in midgut, Malpighian tubules, hindgut and anal papillae, which are tissues that participate in osmoregulation. SJ proteins exhibited tissue specific differences in transcript abundance and immunolocalized to SJ domains between epithelial cells and in the apical membrane domain of the syncytial anal papillae epithelium. Upon rearing larvae in brackish water (30% seawater) tissue‐specific alterations in SJ transcript and protein abundance were observed. Furthermore, in the midgut, salinity‐induced differences in SJ protein abundance occurred in conjunction with differences in the paracellular permeability of this tissue. Taken together, data support the hypothesis that SJ protein abundance is altered in response to environmental change and that SJ proteins play an important tissue‐specific role in the maintenance of salt and water balance in the aquatic larvae of A. aegypti. Since A. aegypti is a vector of re‐emerging viral diseases, Dengue and Chikungunya, and has been shown to successfully breed in both salinated and freshwater in regions where these diseases are active, a complete understanding of the role of SJ proteins in larval mosquito salt and water balance is important.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada Discovery Grants to S.P.K and A.D., an Ontario Graduate Scholarship to S.J
Urinary Excretion of Kidney Aquaporins as Possible Biomarker of Diabetic Nephropathy
Authors: Procino Giuseppe , Rossi Luigi , Nicoletti Maria Celeste , Carmosino Monica , Di Franco Antonella , Indrio Francesca , Lella Rosa , Laviola Luigi , Gesualdo Loreto , Svelto Maria
Published: November 2023

Background

Diabetic nephropathy (DN) is is classified into four hierarchical glomerular lesions on the basis of histological analysis of kidney biopsies. This is clearly a highly invasive diagnostic approach numerous studies have been conducted to identify a non‐invasive biomarker of the disease, but none of these is considered to be sufficiently specific and sensitive Experimental evidences showed that the water channel aquaporins (AQP), expressed at the plasma membrane of epithelial cells lining in the kidney tubule, are often dysregulated during DN. Interestingly, apical membrane protein are excreted in the urinary space as nano‐scale exosome vesicles, to an extent proportional to their amount expressed at the plasma membrane, thus allowing their quantitation in urine samples.

Methods

In this work we compared excretion of AQP5 and AQP2 (uAQP5 and uAQP2) in the urine of 35 diabetic patients: 12 with histological diagnosis of DN, 12 with with normal renal function and normoalbuminuric (DM) and 11 with non‐diabetic nephropathy (NDN). Both proteins were quantified by ELISA method on urine samples and the results were validated by Western blotting analysis on the exosome fraction isolated by ultracentrifugation.

Results

BothELISA and Western blotting analysis independently showed that uAQP5 was three‐ to fourfold higher in DN patients (7458 ± 2576, p <0.05) compared to the other two groups (D 847,2 ± 70,51; NDN1882 ± 215). uAQP5 was not statistically different between D and NDN patients. Strikingly, uAQP5 dramatically increased in parallel with the clinical severity of DN. The same analysis showed comparable results for uAQP2.

Conclusions

Our data showed, for the first time, that uAQP5 and uAQP2 dramatically and specifically increase in patients with DN and positively correlate with the histological class of DN. Taken together these data suggest a possible role of AQP5 and AQP2 as novel non‐invasive biomarkers to help diagnosing DN and classifying its histological stage.

Support or Funding Information

Non U.S.
Acute Pancreatitis Involves Alterations in the Transport of Amino Acids Necessary for Glutathione Synthesis
Authors: Kuster Evelyne , Graf Rolf , Verrey François , Camargo Simone Mafalda
Published: November 2023

Acute pancreatitis (AP) is a serious inflammatory disease of the exocrine pancreas. During its pathogenesis, reactive oxygen species (ROS) have deleterious effects and they can be neutralised by glutathione (GSH), an intracellular antioxidant. GSH is synthesised from glutamate, glycine and cysteine but amino acid transport into acinar cells remains poorly understood. It is the aim of this work to characterise the transport of glycine, glutamate and cysteine (as well as their precursors glutamine and cystine) for the production of GSH during AP in isolated pancreatic acinar cells. For this purpose, AP was induced in C57BL6/J mice using supraphysiological doses of cerulein (12 times hourly 50μg/kg intraperitoneally) and acinar cells were isolated 12, 24, 36 and 72 hours after the first injection.

Glutamate, glycine and cysteine concentrations are rapidly depleted in acinar cells during AP. Concurrently, gene expression levels of enzymes necessary for GSH synthesis are increased. Levels of free GSH (oxidised and reduced) are initially diminished but they are increased during the following regeneration phase. Gene expression levels of the acinar glutamine transporters (LAT1 (slc7a5), LAT2 (slc7a8), SNAT2 (slc38a2), SNAT3 (slc38a3) and SNAT5 (slc38a5) as well as the glycine transporter GlyT1 (slc6a9) are impaired during AP, while the cystine/glutamate exchanger xCT (slc7a11) is remarkably increased during the early phase. Immunofluorescence analysis revealed low xCT expression on pancreatic acinar cells during the healthy state, whereas the protein level increases significantly during AP, as was also shown via Western blotting. Uptake experiments using radiolabelled cystine confirmed an enhanced capacity of isolated acinar cells to import cystine during AP, presumably via the up‐regulated xCT protein levels.

Our data suggests that acinar cells adapt amino acid transport and enzymes necessary for GSH synthesis to match the cells’ needs in the oxidative stress situation occurring during AP. Specifically, xCT expression is up‐regulated in acinar cells to enhance cystine import, the rate‐limiting factor of GSH synthesis.
Smoking‐induced urothelial cell accumulation of platelet‐activating factor: Implications for interstitial cystitis/bladder pain syndrome
Authors: McHowat Jane , Marentette John , Isbell T. Scott , Campian E. Cristian
Published: November 2023

Interstitial cystitis/bladder pain syndrome (IC/BPS) may encompass many diseases with causes such as stress, irritants, previous bladder infections, a dysregulated immune response, inappropriate diet or cigarette smoking. We have determined previously that cigarette smoke extract (CSE) results in accumulation of platelet‐activating factor (PAF) via activation of calcium‐independent phospholipase A2 and inhibition of PAF acetylhydrolase (PAF‐AH). We hypothesized that cigarette smoking may predispose smokers to IC/PBS via PAF accumulation in the bladder wall. Incubation of human urothelial cells (HUC) with cigarette smoke extract (CSE) resulted in a significant inhibition of PAF‐AH activity and PAF accumulation. Inhibition of HUC with CSE resulted in a decrease in electrical resistance and rate of wound healing. Histological examination of bladders removed from mice exposed to cigarette smoke showed urothelial cell thinning and sloughing. Immunohistological staining indicated an increase in PAF accumulation in the bladder wall of mice exposed to cigarette smoke when compared to room air. PAF content in the urine obtained from IC/BPS patients was greater than that observed in controls (PAF:creatinine ratio 13.5 ± 1.5 compared with 5.4 ± 0.6, n=3, p<0.05). PAF content was greater in IC/BPS patients who were current smokers (PAF: creatinine ratio 34.3 ± 10.4, n=3) than in IC/BPS patients who did not smoke. We conclude that cigarette smoking is associated with PAF accumulation in the bladder wall that could contribute to impairment of urothelial function in IC/BPS patients.

Support or Funding Information

Saint Louis University President's Research Fund
Mutation in the PPARγ Ligand Binding Domain Impairs the Anti‐inflammatory Action of PPARγ
Authors: Mukohda Masashi , Ketsawatsomkron Pimonrat , Stump Madeliene , Liu Xuebo , Guo Deng F. , Rahmouni Kamal , Quelle Frederick W. , Sigmund Curt D.
Published: November 2023

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand‐activated transcription factor with anti‐inflammatory properties. One of the anti‐inflammatory mechanisms is antagonism of nuclear factor kappa B (NFκB) activity. Synthetic activators of PPARγ are known to inhibit the formation of atherosclerotic lesions in animal models. In contrast, transgenic mice expressing a dominant negative (DN) mutant of PPARγ specifically in vascular smooth muscle cells (SMC) exhibited exacerbated atherosclerosis with increased expression of NFκB target genes in aortic tissue. Based on this, we hypothesized that the exaggerated atherosclerosis in these mice is due to increased NFκB‐mediated inflammation. We tested the role of SMC PPARγ in controlling inflammatory gene expression. Mesenteric SMCs were cultured from transgenic mice designed to conditionally express wild‐type (WT) or DN PPARγ (P467L) in response to Cre‐recombinase (delivered by adenovirus). PPARγ expression remained silent in control SMC infected with AdGFP but was induced in SMC infected with AdCRE. The PPARγ target gene aP2 was induced in WT‐Cre SMC but not in P467L‐Cre SMC. TNF‐α, IL‐1β or LPS all induced NFκB target gene (VCAM1, MCP‐1, iNOS and MMP9) expression in WT SMC infected with AdGFP (WT‐GFP). Expression of these genes was reduced in WT SMC infected with AdCRE (WT‐Cre). For example, TNF‐α induced MMP9 expression by 6.0±0.3‐fold in WT‐GFP vs 0.7±1.2‐fold in WT‐Cre (p<0.01). In contrast, the ability of TNF‐α to induce NFκB target gene expression was increased in P467L cells infected with AdCRE (P467L‐Cre) compared to those infected with AdGFP (P467L‐GFP, MMP9: 6.3±0.2 vs 3.2±0.5, p<0.01). Similarly, TNF‐α or LPS‐induced NFκB activity assessed by an adenovirus‐NFκB‐Luciferase reporter was blunted in WT‐Cre SMC but increased in P467L‐Cre SMC. Although expression of the NFκB subunit, p65, was not altered in any of these cells, nuclear export of p65 was accelerated in WT‐Cre SMC, but prevented in P467L‐Cre SMC. WT PPARγ co‐precipitated with p65, but the interaction between p65 and P467L PPARγ was severely impaired in HEK293 cells. Finally, to examine mechanisms in vivo, we crossbred transgenic mice expressing WT PPARγ specifically in SMC (S‐WT) with mice expressing luciferase under control of a NFκB‐responsive promoter. TNF‐α (500 ng/ml, 24 hr)‐induced NFκB activity was decreased in aorta and carotid artery from S‐WT mice compared to control mice (aorta: 4.7±1.1 vs 6.7±0.7, p<0.05, carotid artery: 4.0±0.6 vs 8.7±1.2, p<0.01). We conclude that PPARγ in SMC exerts anti‐inflammatory effects by inhibition of NFκB activity through a mechanism promoting nuclear export of p65. This mechanism is abolished by P467L mutation in PPARγ.

Support or Funding Information

This work was supported by National Institutes of Health Grants HL048058, HL062984 and HL08307 (to Curt D. Sigmund).
Toll‐like Receptor 4 Mutation Moderates Hyperhomocysteinemia‐ Induced Hypertension
Authors: Familtseva Anastasia , Chaturvedi Pankaj , Kalani Anuradha , Metreveli Naira , Kunkel George H , Tyagi Suresh C
Published: November 2023

Hyperhomocysteinemia (HHcy) has been observed to promote hypertension, but the mechanisms are unclear. Toll‐like receptor 4 (TLR‐4) is a cellular membrane protein that is expressed on immune cells and non‐immune cells (endothelial cells, vascular smooth muscle cells). TLR‐4 activation has been known to promote innate immune system response with inflammatory cytokine up‐regulation. TLR‐4‐ driven inflammatory cascade has been shown to be involved in pathogenesis of hypertension and atherosclerosis. In this study, we hypothesize that HHcy‐ induced TLR‐4 activation promotes inflammatory cytokine up‐regulation (IL‐1β, TNF‐α) and initiation of mitochondrial‐ dependent apoptosis leading to cell death and chronic vascular inflammation that predispose to hypertension. To test this hypothesis, we used 12 week old C57BL/6J mice (WT); Cystathionine‐β‐synthase deficient mice (CBS+/−) with genetic HHcy; C3H/HeJ (C3H) mice with TLR‐4 mutation and CBS+/−/C3H mice. Blood pressure measurement, western blotting (Caspase‐9, Caspase‐3, HSP60, TLR‐4), q‐PCR (Bax, IL‐1β, TNF‐α, NF‐kB), immunohistochemistry (cleaved Caspase‐3) and TUNEL assay were used. Blood pressure was up‐regulated in CBS+/− mice (systolic: 156.54 ± 10.2 mmHg; mean: 132.4 ± 11.16 mmHg) compared to WT mice (systolic: 121.8 ± 3.6 mmHg; mean: 97.2 ± 6.5 mmHg). Interestingly, blood pressure values were significantly decreased in CBS+/−/C3H mice (systolic: 130.13 ± 9.3 mmHg; mean: 105.8 ± 12.6 mmHg) compared to CBS+/− mice. q‐PCR showed significant up‐regulation of Bax, IL‐1β, TNF‐α mRNA expression in the mesenteric artery of CBS+/− mice compared to control and C3H mice. In addition, Bax, IL‐1β, TNF‐α mRNA expression was decreased in the mesenteric artery of CBS+/−/C3H mice compared to CBS+/− mice. Western blotting validated increase of Caspase‐9 and Caspase‐3 protein expression in the mesenteric artery of CBS+/−mice compared to WT and C3H mice. Immunohistochemistry confirmed cleaved Caspase‐3 up‐regulation in the mesenteric artery of CBS+/− mice compared to control and C3H mice. TUNEL assay showed DNA fragmentation in the mesenteric artery of CBS+/− mice compared to control and C3H mice. In addition, active Caspase‐3 and DNA fragmentation were significantly decreased in the mesenteric artery of CBS+/−/C3H mice compared to CBS+/− mice. In conclusion, our data suggested that HHcy‐induced TLR‐4 activation promotes inflammatory cytokine (IL‐1β, TNF‐α) and Bax up‐regulation followed by mitochondrial apoptosis leading to cell loss and chronic vascular inflammation that contributes to hypertension. Therefore, TLR‐4 mutation abates vascular inflammation and mitochondrial‐dependent apoptosis that moderate hypertension.

Support or Funding Information

This work was supported by NIH grants: HL‐74185
Domain of hCLCA1 that is responsible for macrophage activation
Authors: Ching John C.H. , Loewen Matthew E
Published: November 2023

The CLCA gene family produces both secreted and membrane‐associated proteins that modulate ion‐channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The mechanism of how hCLCA1 regulates airway inflammation remains unclear. However, hCLCA1 is highly expressed in airway diseases in which alveolar macrophages’ are central to disease progression. Thus, it is possible that hCLCA1's role in airway inflammation is to regulate the immune response of macrophages. Previously, we showed that secreted hCLCA1 is able to activate macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In this study, we have purified the functional domains of hCLCA1, and we activated macrophages with these domains. There are three functional domains in hCLCA1 (hydrolase domain, von Willebrand type A domain, and fibronectin type III domain), and we found that one of these hCLCA1's domains significantly induced the expression of pro‐inflammatory cytokines in macrophages. To further investigate this activation, we have also found the cell signaling pathways that are involved in this activation

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC) 371364/2010 to MEL