bluuebunny/crossref_metadata_2025 huggingface dataset:
The Influence Of Obesity‐Associated Metabolic Disturbance On The Profile Of Angiogenic Regulators And Capillary Number Within Skeletal Muscle
Authors: Nwadozi Emmanuel , Liu Hsin‐yi , Roudier Emilie , Rullman Eric , Gustafsson Thomas , Haas Tara
Published: November 2023

Skeletal muscle capillaries are critical determinants of nutrient delivery and utilization and capillary network density is thought to correlate with insulin sensitivity. Conflicting data exist regarding the consequence of obesity‐associated metabolic disturbances on skeletal muscle capillarization, with positive, negative and no effect having been reported. The purpose of this investigation is to assess the influence of obesity induced by high fat feeding or by leptin receptor mutation, on the angiogenic profile within the skeletal muscle microenvironment, and ultimately, on muscle capillarization. In FVB/n mice fed a high‐fat (HF) or normal chow (NC) diet for 16‐weeks, mRNA levels of VEGF‐A did not change (NC: 1.06 vs. HF: 1.13; P>0.05) but VEGF protein levels were elevated (NC: 81 vs HF: 104 pg/mg; P =0.02). Relative FoxO1 protein levels increased in capillaries isolated from muscle of HF vs. NC mice (NC: 0.67 vs HF: 1.55; P =0.03). Capillary‐to‐fibre ratio (C:F) was not altered in HF‐ compared to NC‐ fed mice (2.22 and 2.04, P=0.34). In contrast, 13 week old mice with leptin receptor activity deficiency (Leprdb) had a significant reduction in VEGF‐A mRNA (1.85 vs. 1.09, P=0.03) and protein (WT: 98 vs Leprdb: 86 pg/mg; P<0.05) relative to age‐matched C57BL/6 (WT) mice. Both FoxO1 (0.58 vs. 0.86) and THBS1 (0.37 vs. 0.81) mRNA levels tended to increase in Leprdb vs. WT mice. Skeletal muscle C:F was substantially reduced in Leprdb vs. WT mice (1.92 vs. 1.39, P<0.001). Comprehensive profiling of angiogenic regulators will be conducted by microarray analysis. The skeletal muscle microenvironment differs substantially between HF and Leprdb models. Our data suggest that muscle capillarization is maintained during HF feeding due to a balance between pro and anti‐angiogenic factors, which may include VEGF‐A and FoxO1. This balance is disrupted in leptin receptor mutant mice, which results in a loss of capillaries and raises the intriguing possibility that leptin signaling itself modulates muscle capillarization. Thus, the Leprdb model may not be a suitable model to study obesity‐induced vascular alterations in skeletal muscle. Funded by CIHR.
Expression of Bim in Retinal Endothelial Cells or Pericytes is Essential for Normal Regression of the Fetal Vasculature
Authors: Sorenson Christine , Wang Shoujian , Zaitoun Ismail , Johnson Ryan , Jamali Nasim , Sheibani Nader
Published: November 2023

Apoptosis plays a central role in developmental and pathological vessel regression in the retina. Endothelial cells (EC) and pericytes (PC) each play unique roles during development, maintenance and regression of the retinal vasculature. Bim is a pro‐apoptotic Bcl‐2 family member that plays a prominent role in both normal and pathologic retinal vessel regression. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE‐cadherin‐cre (BimEC mice) or PDGR‐β‐cre (BimPC). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling, perhaps as a result of decreased apoptosis. Interestingly, even though conditional Bim mice displayed increased numbers of EC and/or PC their retinal vasculature still was susceptible to hyperoxia‐mediated vessel obliteration and subsequent neovascularization during oxygen‐induced ischemic retinopathy, unlike germline deleted Bim‐deficient mice. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to prevent pathological retinal vessel regression and preserve vision.

Support or Funding Information

RRF/Daniel M. Albert Chair and Lions Pediatric Ophthalmology Research Fund
VEGFR2 Signaling is Decreased During Necrotizing Enterocolitis
Authors: Yan Xiaocai , Liu Xueli , Managlia Elizabeth , De Plaen Isabelle
Published: November 2023

The pathogenesis of necrotizing enterocolitis (NEC), a common gastrointestinal disease affecting premature infants, remains poorly understood. We previously found that intestinal vascular endothelial growth factor A (VEGF‐A) expression is decreased in human NEC samples and in a neonatal mouse NEC model prior to detectable histological injury. Here we hypothesized that VEGF receptor VEGFR2 is developmentally regulated in the intestine during the perinatal period and that VEGFR2 expression and activation may be altered during NEC development. To test these hypotheses, fetal and neonatal mice of different ages (E16, 18, 20, D0, D1 and D2) were sacrificed. Also, neonatal mice were submitted to a NEC protocol consisting of adult commensal bacteria inoculation/hypoxia/cold stress/formula feeding, or allowed to be dam fed. Pups were sacrificed 24 hours later and their intestine processed for western blot analysis or immunofluorescence analysis of VEGFR2 and several cell‐type specific markers. We found that: 1) VEGFR2 protein expression significantly increases during late fetal life but decreases sharply after birth, remaining low until day of life 2, when it is significantly upregulated; 2) VEGFR2 is only detected in endothelial cells (CD31+ cells) but not in CX3CR1+ myeloid cells; 3) both VEGFR2 and pVEGFR2 are significantly decreased in the intestine of neonatal mice subjected to the NEC protocol. In conclusion, our data suggest that VEGFR2 is developmentally regulated in the perinatal intestine and VEGFR2 expression and activity are decreased during NEC development. We speculate that VEGFR2 signaling may play a critical role during NEC development.

Support or Funding Information

Northwestern Memorial Foundation (Friends of Prentice), NIH grants R01HD060876 (IDP)
Vitamin D Lowers Blood Pressure, ET‐1 and sFlt‐1 in AT1 Autoantibody Induced Hypertension During Pregnancy
Authors: Faulkner Jessica , Amaral Lorena M , Cornelius Denise , Cunningham Mark , Ibrahim Tarek , Thomas D'Andrea , Herse Florian , Dechend Ralf , LaMarca Babbette
Published: November 2023

Autoantibodies to the angiotensin II type I receptor (AT1‐AA) play a role in hypertension associated with preeclampsia (PE). We found that Vitamin D (VD) reduces AT1‐AA and blood pressure (MAP) in a rat model of PE. Here we tested the hypothesis that VD would improve MAP and endothelial dysfunction markers associated with AT1‐AA during PE. We infused AT1‐AA (1:40 in saline) via osmotic minipump on gestational day (GD) 12–19 in pregnant rats. Either VD2 (270 IU/day) or VD3 (15 IU/day) were given on GD14–18 by oral gavage. Uterine artery resistance index (UARI) was measured by Doppler sonography on GD18 and MAP measured on GD19 by indwelling catheter. MAP increased in AT1‐AA (121±4 mmHg) compared to normal pregnant (NP) (101±2) but was decreased in AT1‐AA+VD2 (105±2) and AT1‐AA+VD3 (109±2). UARI increased in AT1‐AA rats to 0.57±0.01 compared to NP (0.41±0.02). VD3 reduced UARI to 0.49±0.03. Placental preproendothelin‐1 (ET‐1) mRNA expression increased in AT1‐AA (18.1±2.9‐fold) from NP and was decreased with VD2 (4.3±1.4) and VD3 (1.3±0.3). Plasma 8‐isoprostanes (ROS) increased in AT1‐AA to 1633±179 pg/ml compared to NP (634±197) but was decreased with VD2 (436±81) and VD3 (752±94). sFlt‐1 increased in AT1‐AA (715±189 pg/ml) compared to NP (94±12) and was reduced with both VD2 (77±16) and VD3 (195±7). We have demonstrated that VD improved ET‐1, ROS and sFlt‐1 in response to AT1‐AA in pregnant rats. We believe reducing these factors led to reduced vascular resistance and MAP responses commonly seen in elevated AT1‐AA, as in the case of PE, thereby supporting a role for VD supplementation as a prevention for PE.

Support or Funding Information

Project funded by NIH RO1HD067541 and T32HL105324
Soluble guanylyl cyclase (sGC) stimulators and activators decrease blood pressure and proteinuria in a rat model of preeclampsia
Authors: Robinson Ta'Shariah , Gillis Ellen E , Garrett Michael R , George Eric , Granger Joey P , Sasser Jennifer M
Published: November 2023

Preeclampsia is a pregnancy disorder that adversely affects both the mother and the fetus and is characterized by the onset of hypertension and proteinuria after the twentieth week of gestation. While there are currently no effective treatments, agents that improve nitric oxide (NO) ‐ cyclic guanosine monophosphate (cGMP) signaling and endothelial function may be therapeutic targets to treat patients with preeclampsia. Recent studies suggest that soluble guanylyl cyclase (sGC) stimulators and sGC activators can enhance reduced sensitivity to NO and increase the enzymatic activity of sGC, respectively. The purpose of this study was to determine the efficacy of a sGC stimulator and a sGC activator on the maternal syndrome and fetal outcomes in the pregnant Dahl S rat. Baseline blood pressure (BP) was recorded (DSI telemetry), and rats were placed in metabolic cages for 24 hr urine collection before mating. On day 14 of pregnancy, rats were randomly placed on either the control diet (n=5), the BR5918 sGC stimulating diet (80mg/kg/day, n=7), or the BR5919 sGC activating diet (16mg/kg/day, n=4). Uterine artery resistance index (UARI) was measured by Doppler ultrasound (Vevo 770) on day 18, urine was collected again on day 19, and blood and tissues were harvested on day 20. Plasma and urinary cGMP concentrations were measured using a commercially available ELISA (Cayman Chemical). Only the sGC activator enhanced the sGC activity as evidenced by increased cGMP in the urine (p=0.06) and plasma (see Table, *P<0.05 vs Control); however, both treatments improved the maternal syndrome as measured by a decrease in blood pressure (from baseline to day 19) and proteinuria. These treatments did not affect UARI, litter size, or pup weight. These preliminary findings demonstrate reductions in blood pressure and proteinuria following treatment with either a sGC activator or stimulator with no adverse effects on fetal growth. Therefore, our findings support further investigation into the therapeutic effects of sGC stimulators and activators for the treatment of preeclampsia.

Urine cGMP (nmol/day)
Plasma cGMP (pmol/ml)
ΔBP (mmllg)
UPE (mg/d)
UARI
Pup weight
Litter size

Control
66.3±0.03
7.5±1.8
+7.9±1.1
264±49
0.67±0.05
2.3±0.1
10.0±0.6

BR5918 (stimulator)
63.8±5.9
5.6±0.9
−12.6±1.7*
128±39*
0.66±0.07
2.3±0.1
10.4±0.4

BR5919 (activator)
81.7±10.3
11.5±1.3*
−14.7±7.4*
106±17*
0.69±0.07
2.3±0.1
12.3±1.1
Homo‐ and Hetero‐dimerization of Uteroplacental and Uterine Arterial Matrix Metalloproteinase‐9 during Hypertension‐in‐Pregnancy
Authors: Chen Juanjuan , Ren Zongli , Zhu Minglin , Khalil Raouf A
Published: November 2023

Preeclampsia is a complication of pregnancy manifested as hypertension‐in‐pregnancy (HTN‐Preg) and often fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms leading to HTN‐Preg and growth restriction are unclear. To test the hypothesis that matrix metalloproteinases (MMPs) play a role in HTN‐Preg, the expression and activity of MMP‐2 and ‐9 and tissue inhibitor of MMP (TIMP‐1) were measured in uterus, placenta and uterine artery of Norm‐Preg rats and rat model of reduced uteroplacental perfusion pressure (RUPP). Consistent with our previous report, blood pressure (BP) was higher and gelatin zymography showed reduction in MMP‐2 (~60kDa), pro‐MMP‐2 (~70kDa), MMP‐9 (~80kDa) and pro‐MMP‐9 (~90kDa) in uterus, placenta and uterine artery of RUPP vs. Norm‐Preg rats. Careful examination of the zymographs showed additional bands at 220kDa, 200kDa and 135kDa. Western blots with MMP‐9 antibody suggested that the 200kDa band may represent MMP‐9 homodimer. Western blots with TIMP‐1 antibody as well as reverse zymography suggested that the 220kDa and 135kDa bands may represent MMP‐9 heterodimers (MMP‐9 homodimer/TIMP‐1 and MMP‐9/TIMP‐1, respectively). Importantly, the amount and gelatinase activity of MMP‐9 homodimer was decreased while MMP‐9 heterodimers were increased in uterus, placenta and uterine artery of RUPP vs. Norm‐Preg rats. Thus the increased BP in placental ischemia model of HTN‐Preg is associated with a decrease in MMP‐9 homodimer and an increase in MMP‐9/TIMP‐1 heterodimers in uterus, placenta and uterine artery, which together with the decrease in MMP‐2 and ‐9 could lead to a net decrease in collagenase activity, increased uteroplacental and uterine arteriolar collagen, and growth‐restrictive remodeling in the uteroplacental circulation in HTN‐Preg.

Support or Funding Information

This work was supported by grants from National Heart, Lung, and Blood Institute (HL‐65998,HL‐111775).
Perinatal taurine depletion depresses the renal tubular effect of estrogen in adult female rats independent of high sugar intake
Authors: Roysommuti Sanya , Lerdweeraphon Wichaporn , Wyss James Michael
Published: November 2023

Perinatal taurine exposure affects adult renal function. This study tests the hypothesis that the adverse effect of perinatal taurine depletion on renal excretory function in adult female rats is modified by estrogen status. Female Sprague‐Dawley rats were fed normal rat chow and given water alone (C) or water containing 3% beta‐alanine (taurine depletion, TD) from conception until weaning. After weaning, the rats received normal rat chow and tap water with (CG, TDG) or without (CW, TDW) 5 % glucose. At 7–8 weeks of age, renal function at rest and after acute saline load (5% body weight) was tested in conscious, restrained female rats treated with or without estrogen receptor blockade (tamoxifen; 10 mg/kg/day in drinking water for a week). Body, heart, and kidney weights were not significantly different among the eight groups. Compared to control, TD did not affect mean arterial pressure (MAP) or heart rate. Tamoxifen treatment significantly increased only resting MAP in TDG compared to TDW and heart rate at rest and after saline load in CG compared to CW groups. Effective renal blood flow, renal vascular resistance, glomerular filtration rate, and sodium and water excretion were not significantly different among the corresponding (tamoxifen treated and untreated) groups. Fractional sodium and water excretion were markedly and significantly depressed after a saline load in CG, TDW, and TDG compared to CW groups, but these differences were eliminated by tamoxifen treatment. These data indicate that in adult female rats, the adverse effects of perinatal taurine depletion on the renal tubule is modified by estrogen status, independent of high sugar intake.

Support or Funding Information

Khon Kaen University
Influence of Activity Before or During Pregnancy on Fetal and Infant Heart Outcomes
Authors: May Linda E , Reoyo Olga R , Moyer Carmen M , Newton Edward R , Strickland Diana
Published: November 2023

We hypothesized that exercise during pregnancy will have greater influence than exercise prior to pregnancy on improved offspring heart rate; this was tested by measuring fetal and infant heart rate. Women were classified into 4 groups: AA=active before and during pregnancy, AI=active before, inactive during pregnancy, IA= inactive before, active during pregnancy, and II= inactive before, inactive during pregnancy. Based on current guidelines, active was defined as ≥150min per week of moderate intensity exercise. Those not meeting criteria were classified as inactive. Fetal (36 wk) and infant (1 mo) heart rates were acquired via ECG and MCG recordings. Data from 83 uncomplicated pregnancies (AA=39, AI=13, IA=12, II=19) was analyzed. We found that fetuses from AA women had lowest heart rates of all groups, especially compared to fetuses of II women (p=0.002). In infant HR, we found significantly lower HR in infants exposed to exercise in utero relative to those not exposed with greatest differences in HR between AA and II groups (p=0.001). As we anticipated, we have found significantly lower fetal and infant heart rates in exercise‐exposed offspring regardless of women's activity prior to pregnancy. However, this dataset does not control for fetal/infant activity state, which may influence findings. These results further support the benefit of exercise as the primary mechanism for positive affects on fetal and infant heart rate. These data further support a prenatal programming effect on the fetal cardiac autonomic nervous system from maternal physiological events during gestation.

Support or Funding Information

East Carolina University StartUp funds
Chronic Flutamide Treatment Alters Intrarenal Renin Angiotensin System Expression in Intrauterine Growth Restricted Female Rats
Authors: Dasinger John Henry , Intapad Suttira , Rudsenske Benjamin R , Alexander Barbara T
Published: November 2023

Intrauterine growth restriction (IUGR) programs an increase in blood pressure associated with early reproductive senescence in female IUGR rats at 12 months of age. Serum testosterone (T) levels are increased in female IUGR rats at this age mimicking the hormonal milieu of postmenopausal women (PMW). Testosterone is positively associated with blood pressure in PMW. Thus, our laboratory previously reported that chronic blockade of the androgen receptor with flutamide abolished hypertension in female IUGR rats. However, the mechanism remained unclear. Activation the renin angiotensin system (RAS) may be important in the etiology of hypertension in PMW. Therefore, for this study we tested the hypothesis that activation of the vasoconstrictor arm of the RAS would be attenuated via chronic blockade of the androgen receptor in female IUGR rats. Following a two week treatment with flutamide (8mg/kg/day) or vehicle, kidneys were harvested and separated into cortex and medulla. Cortical RNA was isolated then converted into cDNA. Real‐time PCR was performed using Bio‐Rad SYBR Green Supermix and iCycler using primers for renin, androgen receptor, angiotensin converting enzyme (ACE) and angiotensin converting enzyme 2 (ACE2). Levels of mRNA expression are reported for cycle threshold method. Intrarenal renin mRNA expression was increased 2.5 fold (2.6±0.65 vs. 1±0.42, P<0.05) in renal cortex of IUGR vs. control kidneys. Following flutamide treatment, renin mRNA levels were normalized in IUGR vs. control (1.03±0.6 vs. 0.7±0.4). Androgen receptor and ACE expression were similar in vehicle control and IUGR rats; flutamide treatment had no further effect. There was a significant reduction in the ACE2 in vehicle treated IUGR relative to vehicle treated controls (0.84±0.12 vs. 1±0.11, P<0.05); however, flutamide treatment had no further effect on ACE2 expression. We previously reported that intrarenal ACE2 expression was increased in normotensive female IUGR offspring in young adulthood. Yet, we observe a reduction in intrarenal ACE2 expression with age in female IUGR rats. Thus, these data indicate that T may contribute to the etiology of hypertension in female IUGR rats via activation of the RAS. Since T causes activation of the RAS, targeting the androgen receptor could be a potential therapeutic intervention for postmenopausal hypertension.

Support or Funding Information

NIH: HL074927, HL51971, P20GM104357, AHA: GRNT19900004, 15PRE24700010
Effect of Aging on Kidney Function in Male Intrauterine Growth Restricted Rats
Authors: Newsome Ashley , Dasinger John Henry , Intapad Suttira , Davis Gwen , Alexander Barbara
Published: November 2023

Epidemiological studies show a correlation between low birth weight (LBW), reduced nephron number, and an increased risk of kidney disease later in life. We hypothesized that LBW males would have lower nephron numbers and diminished kidney function compared to their age‐matched controls. To test this hypothesis, our lab performed a reduced uterine perfusion or sham surgery on day 14 of gestation in timed pregnant rats to induce intrauterine growth restriction (IUGR). Their male offspring were aged to 18 months. Metabolic studies were performed to collect 24‐hr albuminuria data. Rats were then instrumented with jugular catheters and allowed to recover overnight. FITC‐sinistrin was infused intravenously in conscious rats to calculate glomerular filtration rate (GFR) by transcutaneous clearance measurements, and the right kidney was harvested to calculate nephron number through a semi‐quantitative acid maceration technique. Albuminuria was remarkably high in both groups, but there was no significant difference in IUGR (206.6±29.2 mg/day) compared to control (286.3±50.7 mg/day)(n=12/group). GFR examined in a subset was not significantly decreased in IUGR (0.48±0.07) compared to control (0.69±0.11)(p‐value=0.16, n=4/group). Nephron number was not statistically different in IUGR (75,000±4,074) compared to control (96,400±8,298) although there was a trend towards significance (p‐value=0.055, n=4/group). These data indicate that males have kidney injury and a reduction in kidney function that is independent of developmental programming effects by 18 months of age. However, this does not provide evidence about the time course of development of kidney injury or the susceptibility of IUGR offspring to secondary renal insults. Therefore, one future aim is to measure kidney function in younger males to determine whether there is a difference in the age of onset of GFR reduction and albuminuria in these animals. We will also study the effect of uni‐nephrectomy as a second hit in the progression of renal injury.

Support or Funding Information

NIH 074927, T32HL105324, AHA GRNT1990004
Reductions in βENaC and ASIC2 Induce Preeclampsia‐Like Symptoms in Pregnant Mice
Authors: Warrington Junie Paula , Granger Joey P , Ryan Michael J , Drummond Heather A
Published: November 2023

Preeclampsia is a complex, multi‐organ, pregnancy‐specific condition characterized by new onset hypertension with or without proteinuria past the 20th week of gestation. The underlying pathophysiological mechanisms have not been fully elucidated. Beta Epithelial Na+ Channels (βENaC) and Acid Sensing Ion Channel 2 (ASIC2), members of the degenerin protein family, play important roles in vascular function including smooth muscle cell migration. We and others have shown that βENaC is also important for trophoblast migration. While βENaC is reduced in placentas from preeclampsia patients and a rat model of placental ischemia‐induced hypertension, it is not known whether reductions in degenerin proteins lead to altered placental function and cardiovascular abnormalities during pregnancy. Therefore, the purpose of this study was to determine if reductions in ASIC2 and βENaC contribute to preeclampsia‐like symptoms during pregnancy using a novel genetic mouse model initially generated by crossing ASIC2 null mice with mice with reduced levels of βENaC (βENaCm/m) (ASIC2−/− × βENaCm/m). In this study, adult virgin female mice were mated with age‐ and genotype‐matched males overnight and separated the following morning (day of separation considered gestational day one). Mice were weighed every other day and on gestational day 18.5, tissues were harvested for analysis. Pregnant ASIC2−/− × βENaCm/m mice gained significantly less weight during pregnancy (8.3 ± 0.7 vs. 14.0 ± 1.4g, p = 0.003), had increased fetal resorptions (29.1 ± 6.7 vs. 2.1 ± 2.1%, p = 0.002), and decreased number of live pups (3 ± 1 vs. 9 ± 1, p < 0.001) compared to wild‐type (WT) pregnant mice (n = 5–6 per genotype). However, both fetal (1.0 ± 0.1 vs. 0.8 ± 0.1g, p = 0.005) and placental (0.14 ± 0.00 vs 0.10 ± 0.01g, p < 0.001) weights were significantly higher in the pregnant ASIC2−/− × βENaCm/m mice compared to WT pregnant group. Additionally, pregnant ASIC2−/−× βENaCm/m mice had decreased circulating levels of vascular endothelial growth factor (VEGF; 122 ± 11 vs. 206 ± 22 pg/mL, p = 0.009) and modest elevations in blood pressure (113 ± 2 vs. 102 ± 4 mmHg, p = 0.02) compared to WT pregnant mice. These data indicate that pregnancy, superimposed on the ASIC2−/−× βENaCm/m background, produces preeclampsia‐like symptoms. Future studies will assess the relative role of ASIC2 and βENaC in mediating these changes during pregnancy.

Support or Funding Information

NIH P20GM104357, P01HL051971, AHA 13POST16240000, and AHA20410022.
Modulation of Salt and Mineralocorticoid Sensitivity of Blood Pressure by the Circadian Clock Protein Per1
Authors: Solocinski Kristen , Wen Xuerong , Cheng Kit‐Yan , Lynch Jeanette , Wingo Charles S. , Cain Brian D. , Gumz Michelle L.
Published: November 2023

Increasing evidence demonstrates that circadian clock proteins are important regulators of physiological function. Consistent with this concept, we previously showed a role for the clock protein Per1 in the regulation of blood pressure. Global Per1 knockout mice on the salt‐sensitive 129/sv mouse strain exhibited dramatically lower mean arterial pressure (MAP) compared to wild type controls under standard conditions (Stow et al. Hypertension 2012). Interestingly, the normal circadian rhythm of blood pressure was not affected by loss of Per1 in this mouse model. The goal of the present study was to determine the effects of a high salt diet plus mineralocorticoid on Per1‐mediated blood pressure regulation in a salt‐resistant mouse model, C57BL6. As expected, MAP in wild type mice was not affected by a high salt diet (4%) plus injection with the long‐acting mineralocorticoid deoxycorticosterone pivalate (DOCP). In contrast, Per1 knockout mice exhibited significantly increased MAP (by 10 mm Hg, p<0.01) in response to a high salt diet plus DOCP. The day/night ratio of MAP in wild type mice was unchanged by high salt plus DOCP treatment. Conversely, this treatment caused Per1 knockout mice to completely lose the expected decrease or “dip” in blood pressure during the day compared to the night. Thus, loss of Per1 increased sensitivity to the high salt plus DOCP treatment and also resulted in a non‐dipper phenotype in this hypertensive model. Together these data support an important role for the circadian clock protein Per1 in the modulation of blood pressure in a high salt/mineralocorticoid model of hypertension.

Support or Funding Information

This work was supported by NIH DK085193 and DK098460, the ASN Foundation for Kidney Research to MLG, 2T32HL083810 to KS, and NIH DK082680 to BDC and CSW.
Loss of Protein Kinase C Alpha Disrupts Mean Arterial Pressure and Diurnal Rhythm
Authors: Wynne Brandi Michele , Molina Patrick , Klein Janet , Hoover Robert S
Published: November 2023

The sodium‐chloride cotransporter (NCC) is an essential regulator of sodium transport in the distal convoluted tubule (DCT). As such, functional aberrations will lead to dysregulation of sodium homeostasis and blood pressure. Although this role is well accepted, the mechanisms accounting for increased distal nephron sodium reabsorption is not completely understood. Previously published data from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) Study revealed that a single nucleotide polymorphism (SNP) in PKC‐α increases sensitivity to thiazide diuretics. To date, it is unknown whether this SNP is a gain or loss of function of PKC‐α. To investigate whether PKC‐α may be altering blood pressure via NCC, PKC‐α KO (PKC KO) and PKC Control (CTL) mice were used to measure blood pressure via radiotelemetry after normal and high salt (HS, 4%) dietary manipulations and Western blot analysis performed using kidney lysates. Using radiotelemetry (DSI) measurements obtained over an active and sleeping time periods, we report a significant decrease in mean arterial pressure (MAP) in the PKC KO mice as compared to control at baseline (111±1.3mmHg vs 120±2.4mmHg, n=12–16, p<0.001). When comparing active vs. sleeping MAP, PKC KO mice had depressed MAP during both time periods, yet blood pressure differences (day vs. night) double during active periods (5.4±1.3mmHg PKC KO vs. 10.9±3.9mmHg PKC CTL, n=6–7). These differences were abolished with HS chow feeding (17.3mmHg PKC KO vs. 16.1mmHg PKC CTRL, n=4). To determine a possible mechanism for this phenomenon, whole kidney lysate was used to assay for total NCC protein. Total NCC protein was significantly increased in PKC KO mice at baseline, and continued during HS chow feeding, as compared to PKC CTL. In addition, PKC KO mice exhibited a diuresis with both normal and HS feeding. Our data suggest that lack of PKC‐α leads to decreased MAPs and diuresis, which may lead to increased total NCC expression to compensate. In addition, PKC KO mice exhibited an aberrant diurnal rhythm, which was recovered with HS diet suggesting that PKC‐α may be necessary to maintain sodium homeostasis.

Support or Funding Information

NIDDK085097‐ RSH 2T32DK7656‐21 Emory Renal Division‐BMW
Blood pressure regulation under low K+ intake depends on the H,K‐ATPase type 2
Authors: Crambert Gilles , Salhi Amel , Lamouroux Christine
Published: November 2023

In industrialized countries, a large part of the population has a low daily K+ intake, which appears to be correlated with the development of salt‐sensitive hypertension. Among many processes, adaptation to K+ restriction involves the stimulation of H,K‐ATPase type 2 (HKA2) in the kidney and colon. In this study, we investigated whether HKA2 also contributes to the determination of blood pressure (BP). We compared wild‐type (WT) and HKA2‐null mice (HKA2 KO), and showed that after 4 days of K+ restriction (LK diet), WT remain normokalemic (3.7±0.1 mM) and normotensive (112 ±3 mm Hg) whereas HKA2 KO mice exhibit hypokalemia (3.2±0.1 mM) and hypotension (104±2 mm Hg). To elucidate the mechanism explaining the variations in BP between both strains, we measured, mRNA and protein expression as well as in vivo activity of NCC and ENaC. In WT mice under LK diet, we observed a switch between electrogenic (downregulation of ENaC activity and expression) and electroneutral (stimulation of NCC activity and expression) Na+ reabsorption. In these mice, the renal Na+ excretion remained similar to that measured under normal condition. Regarding HKA2 KO mice under LK diet, their NCC is not stimulated inducing a renal loss of Na+ and a decrease of the extracellular volume (by 20%). This increase of natriuresis in HKA2 KO mice under LK diet is correlated with 1/ the increase of PGE2 excretion (by 100% vs WT mice) and 2/ the observed renal resistance to vasopressin and the lower protein expression of AQP2 (by 50% vs WT).

Interestingly, the ability to stimulate NCC under LK diet renders the WT mice more sensitive to concomitant increased intake of Na+. Indeed, BP of WT mice increased by 15 mm Hg when given a salt load along with the K+ restriction. In contrast, the BP of HKA2 KO mice does not increase under LK diet and salt loading. We conclude that the presence of an activated HKA2 is required under K+ restriction not only to limit the decrease in plasma [K+] but also to maintain a normal BP level when salt intake is rather low rather low. However, when salt intake increases, the absence of the HKA2 appears to protect against the development of salt‐sensitive hypertension.
Role of Kir5.1 (Kcnj16) Channel in the Control of Potassium Homeostasis in the Kidney
Authors: Palygin Oleg , Levchenko Vladislav , Geurts Aron , Staruschenko Alexander
Published: November 2023

The inwardly‐rectifying potassium (K+) channels (Kir), Kir4.1/Kir5.1 (Kcnj10/Kcnj16) are critical channels responsible for the recycling of K+ in the distal nephron as well as regulation of basolateral membrane potential. Human mutations in KCNJ10 and KCNJ16 are associated with renal pathologies characterized by salt‐wasting, renal tubulopathy and hypokalemia (EAST/SeSAME and Gitelman syndromes) as well as cardiac phenotypes such as Brugada syndrome). The Kir4.1/Kir5.1 heteromeric channel is expressed on the basolateral side of distal convoluted tubule (DCT) cells and the principal cells of the cortical collecting duct (CCDs). Immunohistochemistry analysis of Dahl salt‐sensitive (SS) rats showed increased Kir5.1 abundance in the CCD compared to the DCT. Moreover, expression of Kir5.1 in both segments was significantly upregulated after 3 weeks of high salt diet (4% NaCl), showing a direct correlation between the development of salt sensitive hypertension and basolateral K+ channel function. To further explore the role of this channel in salt‐sensitive hypertension, we have created a loss‐of‐function mutation in Kir5.1 in the Dahl SS rat (SS‐Kcnj16em1Mcwi) using zinc‐finger nucleases. Overall, Kir5.1 loss‐of‐function mutation in the SS rat induced a renal phenotype characterized by hypokalemia (2.1±0.2 vs 4.3±0.1 mmol/L (mutant vs SS)), low aldosterone level (245.4±26.5 vs 1050±100 pg/ml), salt wasting (FEK+ 30.3±3.1 vs 5.8±0.6; FENa+ 0.42±0.06 vs 0.19±0.03; FEMg2+ 15.8±1.3 vs 6.1±0.9 %) and decreased mean arterial blood pressure (91.3±1.8 vs 104.7±5.5 mmHg). Furthermore, the absence of the Kir5.1 from distal nephron resulted in a compensatory increase of Kir4.1 protein abundance. IHC analysis of Kir4.1 localization revealed a rerouting from its expected location in the basolateral membrane of CCD and DCT tubules to the cytosol instead. We also found that loss‐of‐function mutation of Kir5.1 triggered upregulation of both total and phosphorylated forms of the NCC and NKCC2. In contrast, we were not able to identify any changes in the expression of ENaC subunits. Increasing the salt content of the diet from 0.4 to 4% triggered sodium and potassium excretion in Kir5.1 mutants, which resulted in a rapid reduction of blood K+ to critically low values (1.36±0.04 vs 2.1±0.1mmo/L (HS vs LS)). Na+ blood concentrations (141.6±0.5 vs 139.2±1.5 mmo/L) were unchanged between these two groups. This severe hypokalemia resulted in high mortality rate with most animals dying within 24 hours. These data demonstrate the critical role of renal basolateral Kir4.1/Kir5.1 channels in potassium handling, and identify them as a potential pharmacological target for the treatment of the imbalanced potassium homeostasis.

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Supported by Advancing a Healthier Wisconsin Research and Education Program #9520217 and HL122662
A novel in vivo renal ischemia reperfusion injury (IRI) model induced solely by cold ischemia
Authors: Zhang Jie , Wang Shaohui , Wei Jin , Wang Lei , Zhang Gensheng , Yip Kay‐Pong , Liu Ruisheng
Published: November 2023

Transplanted organs experience several episodes of IRI during procurement and transplantation in forms of both warm and cold ischemia. IRI‐induced kidney injury is crucial for the survival of transplanted kidney and considered as one of the most important nonspecific factor affecting graft function and is associated with decreased long‐term survival. We don't know whether they are the same or different of IRI induced between warm and cold ischemia, since no IRI in vivo model that is induced solely by cold ischemia available. In present study, we developed a novel IRI mouse model, which is induced exclusively by cold ischemia. C57BL/6 mice were anesthetized with pentobarbital (50 mg/kg i.p.) and body temperature was maintained at 37 °C during the surgery. Right renal artery, vein and ureter were ligated and right kidney was removed after a midline abdominal incision. Left kidney, aorta and inferior vena cava were exposed. The left kidney was placed in a kidney cup and incubated with cool saline, which temperature was maintained at 4±0.5°C by circulating sterilized saline solution from a reservoir on ice and driven with a pump. Four 5 mm microvascular mini clips were used to prevent bleeding and induce ischemia. First, the aorta between the left and right renal arteries was clamped, and distal aorta and inferior vena cava were clamped together. A small opening was cut on the left renal vein after clamping a short segment of left renal vein near vena cava. The aorta below left renal artery was infused with 1–1.5 mL of 4°C 0.9% saline using 30G needle until the solution from the renal vein became clear. The left renal pedicle between the opening and the left kidney was clamped to induce ischemia. The small opening was sutured with 10/0 suture. Then three clips on the aorta, inferior vena cava and left renal vein were removed. The clip on renal pedicle was removed 60 min after clamping and renal blood restored. The wounds were sutured and mice were recovered. The animals were anesthetized again 24 hours after IRI. Blood (500ul) was collected through retro‐orbital venous sinus before sacrifice and left kidneys were harvested and fixed in 10% formalin. Plasma creatinine level was significantly increased to 2.47±0.05 mg/dl compared with the control (0.08±0.03 mg/dl) and the plasma kidney injury molecule‐1 (KIM‐1) increased to 2917±492 pg/ml compared with the controls (2917±34 pg/ml). Histology with PAS staining showed that the most severe injury in corticomedullary region, with a percentage of tubular necrosis 31.4±8.1% and moderate damage in renal cortex, outer‐medulla and inner medulla with a percentage of tubular necrosis 1.6±1.3%, 8.9±4.7%, and 4.6±2.9%. In summary, we have developed a novel in vivo IRI mouse model that is exclusively induced by cold ischemia. This model could provide important information and mechanism for IRI that induced by cold ischemia in kidney transplantation.
Losartan May Reduce the Effect of Fluid Resuscitation Following Haemorrhage in Rats
Authors: Melville Jacqueline M , Hultström Michael
Published: November 2023

Acute kidney injury (AKI) affects 2% of hospital in‐patients. AKI is particularly a concern following surgery, as patients that require transfusions during surgery are at a greater risk for AKI, indicating haemorrhage as a cause. Angiotensin II AT1‐receptor blockers (ARB) are thought linked to postoperative AKI due to their hypotensive actions; as a result ARBs are often discontinued before surgery. We have previously found that there is no difference between losartan‐treated and control rats in Δ renal blood flow (RBF), Δ renal vascular resistance (RVR) and Δ kidney oxygenation in response to haemorrhage, indicating that ARBs may not be detrimental following haemorrhage. However, the impact of ARBs on fluid resuscitation and norepinephrine after haemorrhage is less well studied.

Wistar rats were treated with either control (n = 4) or the ARB losartan (60 mg/kg/day in drinking water, n = 6) for 7 days. Rats were anaesthetised with thiobutabarbital and surgery was performed for the insertion of catheters and a flow probe around the renal artery followed by a controlled haemorrhage of 20% blood volume. Five minutes following haemorrhage fluid resuscitation began (volume replacement with ringer's solution) over a period of 10 minutes. Mean arterial blood pressure (MAP) and RBF were measured before haemorrhage, and throughout fluid resuscitation.

The effect of fluid resuscitation on blood pressure (ΔMAP) tended lower in losartan (9.71±2.7 mmHg) compared to control (21.5±5.7 mmHg, p = 0.06). There was no statistical difference between ΔRBF or ΔRVR following fluid resuscitation between losartan (1.6±1.09 ml/min and −120.7±59.0 mmHg/ml/min, respectively) and control (1.2±0.16 ml/min and −142.2±116.3 mmHg/ml/min).

The response to a bolus dose of 0.08μg/kg norepinephrine after haemorrhage and resuscitation was not different between losartan and control rats for the area under curve (AUC) of blood pressure (15416±2404 vs. 12634±2472mmHg.s), RBF AUC (64.52±19.84 vs. 155.3±65.46 ml/min.s) or RVR AUC (75546±32617 vs. 88997±42130 mmHg/ml/min.s).

These preliminary data may demonstrate that losartan, an AT1‐receptor blocker, reduces MAP recovery following fluid resuscitation but does not affect the response to norepinephrine post‐haemorrhage.

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Swedish Society for Medical Research
Intrarenal Hypoxic Mesenchymal Stem Cells Transplantation Enforces Homing and Paracrine Effects to Ameliorate Anti‐Thy1.1‐Induced Nephritis in Rats
Authors: LAI YI‐AN , Chang Hao‐Hsian , Hsu Shih‐Ping , Chien Chiang‐Ting
Published: November 2023

In acute glomerlonephritis, activated resident cells or infiltrated inflammatory cells‐derived oxidative stress evoked GRP78‐mediated endoplasmic reticulum stress, Bax/Bcl‐2 family‐related apoptosis and Beclin‐1/LC3‐II‐mediated autophagy leading to glomerular injury. Mesenchymal stem cells (MSC) provide excellent self‐renewal and proliferation ability. In addition, MSC may release high levels of growth factors, cytokines and prostaglandins in the microenvironment by the paracrine mechanism to enhance cell proliferation, survival and angiogenesis to repair injuried tissue. Under hypoxic condition, MSC (HMSC) can not only prevent senescence but also increase differentiation efficiency. We aimed to evaluate the therapeutic effects and mechanisms of MSC or HMSC on anti‐thy1.1‐induced glomerulonephritis in the rats. Our results found that intrarenal arterial administration of MSC or HMSC (1×105, 2×105, 5×105 cells) can successfully implant in the glomeruli and proximal and distal tubules. Anti‐thy1.1 induced glomerulonephritis by the appearance of mesangial cell lysis, increase of collagen by Masson stain, collagen IV expression, ED‐1 infiltration, GRP78/endoplasmic reticulum (ER) stress, Bax/Bcl‐2/Caspase 3/PARP‐mediated apoptosis, Beclin‐1/LC3 II‐mediated autophagy and elevated urinary protein levels in nephritic rats. HMSC was more efficient than MSC significantly and dose‐dependently in reducing PAS‐injuried score, ED‐1 infiltration, GRP78/ER‐stress Bax/Bcl‐2/Caspase 3/PARP‐mediated apoptosis, Beclin‐1/LC3 II‐mediated autophagy, Masson stain, collagen IV expression and elevated urinary protein levels in nephritic rats. HMSC was more efficient than MSC in the reappearance of OX‐7 stain (Thy1.1 stain) in the nephritic glomeruli. Our results indicate that HMSC through higher efficiency in homing and paracrine effects attenuate autophagy, apoptosis, ER stress, inflammation and glomerular sclerosis in acute nephritis.

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Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, ROC
Suramin Protects From Cisplatin‐Induced Acute Kidney Injury
Authors: Dupre Tess , Doll Mark , Shah Parag , Sharp Cierra , Kiefer Alex , Scherzer Michael , Saurabh Kumar , Safaro Doug , Siow Deanna , Casson Lavona , Arteel Gavin , Jenson Alfred Bennett , Megyesi Judit , Schnellmann Rick , Beverly Levi , Siskind Leah
Published: November 2023

Cisplatin, a commonly used cancer chemotherapeutic, has a dose limiting side effect of nephrotoxicity. According to the Risk, Injury, Failure, Loss of Function, and End Stage Renal Disease (RIFLE) criteria of kidney injury, approximately 30% of patients administered cisplatin suffer from kidney injury and there is currently no treatment for cisplatin‐induced kidney injury other than palliative care. Suramin, FDA‐approved for the treatment of trypanosomiasis, improves kidney function following various forms of kidney injury in rodent models. We hypothesized that suramin would attenuate cisplatin‐induced kidney injury. Suramin treatment prior to cisplatin administration reduced cisplatin‐induced decreases in kidney function and injury. Furthermore, suramin attenuated cisplatin‐induced expression of inflammatory cytokines and chemokines, endoplasmic reticulum stress, and apoptosis in the kidney cortex. Treatment of mice with suramin 24 hours after cisplatin improved kidney function, suggesting that the mechanism of protection is neither via inhibiting tubular cisplatin uptake nor its metabolism to nephrotoxic species. If suramin is to be utilized in the context of cancer, then suramin cannot prevent cisplatin‐induced cytotoxicity within tumors. Mice harboring lung adenocarcinomas were pretreated with suramin and then administered cisplatin. Lung histology and markers of kidney function indicated that suramin protected mice from cisplatin‐induced acute kidney injury, but did not inhibit cisplatin induced cytotoxic effects within the tumor. These results provide evidence that suramin has great potential as a renoprotective agent for the treatment/prevention of cisplatin‐induced acute kidney injury.

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Support for these studies was provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01‐ DK093462, to L.J.S]. This work was also supported by the National Institutes of Health National Institute of General Medical Sciences [Grant R01‐GM084147 to R.G.S.]; the National Institutes of Health National Center for Research Resources [Grant UL1‐RR029882 to R.G.S.]; the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs [1BX000851 to R.G.S.]; the South Carolina Clinical and Translational Research Institute, with an academic home at the Medical University of South Carolina (to R.G.S.); and the National Institute of Environmental Health and Sciences (SBIR/STTR ES023767 to R.G.S.).
The Role of Par‐1a/b Polarity Proteins in Acute Kidney Injury
Authors: Kazi Ananna N , Pal Abhijeet , Du Zhongfang , Reidy Kimberly J
Published: November 2023

Acute Kidney Injury (AKI) is defined as the rapid loss of kidney function. Patients with acute kidney injury are unable to filter waste products from their body. The integrity of renal epithelial apico‐basal polarity is critical for many aspects of kidney function, including reabsorption of solutes. Loss of epithelial cell polarity occurs in several kidney disorders, including acute kidney injury. Partitioning defective 1 (Par‐1) is a member of the Par polarity protein family that localizes to the basolateral aspect of cells. Studies of Par‐1a+/−:Par‐1b−/− and Par‐1a−/−:Par‐1b+/− newborn mice determined that Par‐1 contributes to proximal tubular development. Complete loss of Par‐1a/b (Par‐1a−/−: Par‐1b−/−) leads to death early in fetal development of mice. Par‐1a and Par‐1b are expressed in developing nephrons and their expression decreases in adult nephrons. Tubular injury in mice correlates with increased expression of Par‐1a and Par‐1b. In our research, we hypothesized that Par‐1a/b polarity proteins are protective in the setting of acute kidney injury. In order to test this, cisplatin was injected intraperitoneal into Par‐1a/b global knockout mice at 5 weeks and they were sacrificed 3 days after injection. Cisplatin is one of the most widely used chemotherapy drugs that has been recently found to contribute to nephrotoxicity, especially on the signaling pathways leading to renal tubular cell death and inflammation. Hematoxylin and Eosin (H&E) staining was performed to assess tubular injury. Controls were non‐injected Par‐1a−/− and Par‐1b−/− mutants and injected wild type littermates. Ki‐67 staining was performed to identify cell proliferation. The level of tubular injury and cellular proliferation was examined by light microscopy. Our data showed that Par‐1b knockout mice were more susceptible to renal tubular injury than controls. Par‐1b knockout mice also had decreased cellular proliferation after cisplatin injury. Our ongoing experiment is testing the role of Par‐1a in AKI. In conclusion, Par‐1b polarity proteins contribute to renal tubular epithelial response to injury. Either decreased or delayed cellular proliferation may contribute to the increased tubular injury in Par‐1b−/− mice.

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This research was supported by American Physiological Society (APS) Short‐Term Research Experience for Underrepresented Persons (STEP‐UP) program, NIH NIDDK K08 5K08DK091507, R03 DK105242, and T32 DK007110. Thank you to Dr. Piwnica‐Worms for the Par‐1b+/− mice.