bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 76: Toll-Like Receptor 4 Deficiency Protects C3H/HeJ Mice from Angiotensin-II Induced Renovascular Fibrosis
Authors: Pushpakumar Sathnur , Kundu Sourav , Givvimani Srikanth , Tyagi Suresh C , Sen Utpal
Published: March 2022

Toll-like receptor 4 (TLR4) is a molecule involved in innate immune response. Current evidence suggests that activation of TLR4 is associated with vascular and cardiac remodeling by a process which involves production of reactive oxygen species and inflammatory cytokines. Oxidative stress is well established in the development of renal fibrosis in chronic hypertension. In this study, we hypothesized that TLR4 deficiency in C3H/HeJ (C3H) mice protects the kidney from angiotensin induced renal remodeling by reducing mitochondrial oxidative stress. Wildtype (WT) and C3H mice were implanted with osmotic pumps loaded without or with Angiotensin-II (Ang-II) for delivery at 250 ng/kg/min for 4 weeks. Animal groups were 1) WT + Saline, 2) WT + Ang-II, 3) C3H + Saline, 4)

C3H + Ang-II. Blood pressure was measured weekly and end-point laser Doppler flowmetry was done to assess renal cortical blood flow. Superoxide content was measured by dihydroethidium staining. Mitochondrial oxidative stress was measured by H
2
O
2
production, manganese superoxide dismutase (MnSOD) and catalase was measured by gel and assay method respectively. Ang-II administration induced higher blood pressure in WT mice than in C3H mice (135.8 ± 5.1 vs. 109.4 ± 5.6 mm of Hg). Ang-II decreased renal cortical flux to a greater degree in WT mice compared to C3H mice [1052.8 ± 67.8 vs. 1182.9 ± 24.7 perfusion units (PU)]. Superoxide content was increased in WT + Ang-II mice compared to C3H + Ang-II. The expression and activity of mitochondrial MnSOD was increased in C3H mice compared to WT mice receiving Ang-II. H
2
O
2
production was increased in C3H + Ang-II compared to WT mice receiving similar treatment. Collagen accumulation and wall-to-lumen ratio was increased in the cortical vessels of WT mice compared to C3H mice receiving Ang-II indicating fibrosis. C3H mice also exhibited lower levels of inflammatory cytokines TGF-β, TNF-α and IL-6 than WT mice receiving Ang-II. Our data suggests that in TLR4 deficiency (C3H mice), upregulation of mitochondrial MnSOD reduces reactive oxygen species production and inflammation and thus renovascular remodeling in Ang-II induced hypertension.
Abstract 77: MicroRNA-34a Accelerates Aging in Vascular Smooth Muscle Cells (SMCs) Isolated from Transgenic Mice Overexpressing Nox1 in SMCs
Authors: Luo Zaiming , Fan Lijuan , Wellstein Anton , Griendling Kathy K , Welch William J , Wilcox Christopher S
Published: March 2022

Background:
Aging increases cardiovascular morbidity. Aging in vascular smooth muscle cells (VSMC) induces cell senescence, leading to impaired vasodilation with decreased vascular compliance, increased inflammation and oxidative stress. MicroRNA-34a (miR-34a) has been reported as a biomarker of aging.

Methods and Results:
After screening for a group of miRs, there was a > 2 fold of miR-34a in aortic SMCs isolated from transgenic mice overexpressing Nox1 in SMCs (Nox-1 SMCs). Therefore, we hypothesized that miR34 contributes to VSMC aging. TaqMan miR assay confirmed an increased miR-34a by 2.1 fold in young NOX-1 SMCs (passage 3-7) with an increase by 5.25 fold in activity of senescence-associated β-galactosidase (SA-β-Gal ) compared to WT. miR-34a in old NOX-1 and WT SMCs (passage 14-16) was increased by 3.8 fold and 2.1 fold, respectively, compared to young SMCs (both P< 0.01 ). There was 1.8 fold more miR-34a detected in old NOX-1 vs. old WT SMCs. SA-β-Gal positive cells accounted for 0.8±1% and 4±1.3% in young WT and NOX-1 SMCs (P<0.01), and 5±1.5% and 32±5% in old WT and NOX-1 SMCs (P <0.005). After transfection with miR-34a inhibitor, mature miR-34a was decreased by 75% and SA-β-Gal activity by 58% in old NOX-1 cells with parallel changes in the protein expression for Gamma-H2AX( a DNA damage marker) and p21( an activator for cell senescence). Moreover, phosphatase nuclear targeting subunit (PNUTS, a recently identified anti-age protein and direct miR-34a target) was not changed in young NOX-1 SMCs but reduced by 78% in old NOX-1 SMCs. Interestingly, nuclear factor (erythroid-derived 2)-like 2 (Nrf2, a key transcription factor for antioxidant genes) mRNA and protein were decreased by 55% and 32% in old NOX-1 SMCs (P<0.01 and 0.05). Nrf2 activator tert-butylhydroquinone (tBHQ) reduced miR-34a from 2.9±0.4 to 1.8±0.2 fold in those cells.

Conclusion:
Increased NOX-1 in SMCs induces miR-34a that accelerates SMC senescence. The pathway may involve downregulation of Nrf2 and increased P21 through P53 pathway. This provides potential targets for protecting against vascular aging.
Abstract 81: Hypo-Osmolality Pressor Stimulus is Linked to Transient Receptor Potential Vanilloid 4 (TRPV4) in the Portal Region
Authors: Mai Tu , McHugh Julia , Diedrich Andre' , Garland Emily , Robertson David
Published: March 2022

Patients with baroreflex impairment have a robust and sustained increase in blood pressure (BP) after water consumption. Using a sino-aortic denervated mouse model (SAD), we identified hypo-osmolality as the stimulus for this osmopressor response (OPR). However, the afferent mechanism of how changes in osmolality are relayed from the gut area to the central nervous system is still unknown. We tested the hypothesis that decreased osmolality is sensed in the portal area after water consumption and that TRPV4 channels in this region are essential for the response. We infused 0.45% or 0.9% saline directly into the portal vein of SAD wild type (WT) and TRPV4-/- mice. An immediate pressor response occurred in WT mice after 0.45% saline but not after 0.9% saline infusion (0.45%: 16 ± 6 vs. 0.9%: -6 ± 2 mmHg, p=0.03). On the other hand, infusion of 0.45% saline into TRPV4-/- mice failed to stimulate a pressor response (-5 ±2 mmHg, p=0.03 for comparison with WT). There are two known nerves that convey afferent signals from the gut to the brain: the vagal and splanchnic nerves. We explored the possible contribution of the splanchnic nerve to OPR by using celiac ganglionectomized (CGX) and sham WT mice. Both CGX and sham mice had a robust increase in BP after water infusion into the duodenum (CGX: 10 ± 2 vs. SHAM: 12 ± 1 mmHg, p=0.5). However, the response was shortened by ganglionectomy. Ten minutes after water, BP had returned to normal in CGX, while it remaining elevated in sham mice (ΔMAP for CGX: 0.3 ± 2 vs. 9 ± 1.7 mmHg, p=0.01). In mice that underwent both CGX and vagotomy, we observed full OPR (ΔMAP for CGX+Vagotomy: 17 ± 6mmHg vs. Sham: 30 ± 8mmHg, p=0.26). Therefore, we conclude that TRPV4 channels in the portal region are essential for the OPR, and the celiac ganglia play an important role in maintaining BP elevation, while the vagus nerve is unlikely to contribute to the water response.
Abstract 86: Tumor Necrosis Factor-alpha Suppresses miR-29 and Up-regulates Fibrotic Genes in the Kidney
Authors: Huang Baorui , Liu Yong , Yang Chun , Cowley Allen W , Liang Mingyu
Published: March 2022

Tumor necrosis factor-alpha (TNF-a) is a pleiotropic inflammatory cytokine. Interstitial fibrosis is prominent in the Dahl salt-sensitive (SS) rat, including in the outer medullary region where thick ascending limbs (TALs) are abundant. We found previously that members of the miR-29 microRNA family, which have potent anti-fibrotic effects, were expressed at a lower level in the kidneys of SS rats. In the present study, we examined the role of TNF-a in the regulation of miR-29 and fibrotic genes in the kidney. RNA-seq analysis showed that SS rats fed a high salt diet had higher expression levels of TNF-a and TNFRSF1a which is one of its main receptors compared with a 0.4% salt diet group. Rat medullary thick ascending limb (mTAL) cells were treated with TNF-a at 25ng/ml for 24h or 48h. At 24h, TNF-a significantly decreased the abundance of miR-29b (to 57% ± 9% of control, n=14, p<0.05) and miR-29c (to 74% ± 6% of control, p<0.01) and significantly increased alpha-smooth muscle actin (a-SMA) by more than two fold and up-regulated extracellular matrix genes such as Col4a1 (to 161% ± 25% of control, n=10, p<0.05) and Col4a2 (to 172%± 18% of control, p<0.01). At 48h, TNF-a significantly suppressed miR-29a and again up-regulated a-SMA and Col4a2. Similarly, TNF-a suppressed miR-29 in primary cultures of human renal epithelial cells, an effect that was attenuated by inhibition of NF-kB. This study shows that TNF-a can suppress miR-29 and up-regulate fibrotic genes in renal epithelial cells including mTAL cells, which may play an important role in the development of renal tubulointerstitial injury in SS rats.
Abstract 89: Male SHR Have Higher HMGB1 Mediated Renal T cell Activation Compared to Females
Authors: Tipton Ashlee J , Babak Baban , Sullivan Jennifer C
Published: March 2022

We have previously reported that there are sex differences in the renal T cell profile of spontaneously hypertensive rats (SHR), with females having more anti-inflammatory regulatory T cells (Tregs) and males expressing more pro-inflammatory Th17 T cells. High mobility group box 1 protein (HMGB1) is an endogenous danger molecule and when released by necrotic cells it activates pro-inflammatory immune cells. We hypothesized that males have higher HMGB1 mediated renal T cell activation compared to females. Serum and kidneys were isolated from 13 week old male and female SHR and spleens from normotensive male Wistar Kyoto rats (WKY). HMGB1 expression was measured in sera using an ELISA. To quantitate renal T cell activation, we performed a mixed lymphocyte reaction in the presence and absence of HMGB1 neutralizing antibody. For the reaction, T cells were isolated from kidneys using magnetic activated cell sorting and spleens were homogenized to obtain a single cell suspension of antigen presenting cells (APCs). T cells and APCs were plated together, incubated for 96 hours, proliferation was confirmed and T cell activation was assessed by the magnitude of CD71 using flow cytometry. Male SHR had greater serum HMGB1 levels than female SHR (ng/ml: 5.3±1.1 vs. 2.5±0.3; p=0.0225; N=4-6). In male SHR, 92% of T cells were activated by incubation with APCs in contrast to 65% of T cells in females (CD71
+
T cells: 92±1% vs. 65±1%; p<0.0001; N=3). HMGB1 neutralization decreased T cell activation in both sexes (males to 74±1%; females to 54±1%; effect of treatment: p<0.0001) but neutralization resulted in a greater decrease in T cell activation in male SHR (interaction: p=0.0012). These data support our hypothesis that male SHR have higher HMGB1 mediated renal T cell activation compared to females. We propose that in male SHR, circulating HMGB1 release promotes the expression of renal Th17 cells to a greater extent than in female SHR.
Abstract 9: The Vasopressin Pro-Segment Copeptin: A Novel, First Trimester Predictor of Preeclampsia
Authors: Santillan Mark , Santillan Donna , Leslie Kimberly , Hunter Stephen , Grobe Justin
Published: March 2022

Preeclampsia (PreE) is a unique late-gestational hypertensive disorder that affects 5-7% of all pregnancies and causes a disproportionate percentage of all perinatal morbidity and mortality. The prediction, prevention, modeling, and subsequent management of PreE have been severely limited by the lack of a known central etiology. Although placental dysfunction is considered the major mediator of PreE, many upstream and downstream vascular, immunologic, and hormonal mechanisms have been implicated in its cause. Vasopressin plays an important role in the maintenance of vascular tone in populations affected by low-renin hypertension such as African-Americans, the elderly, and those with chronic heart and renal failure. PreE is also a relatively low-renin hypertension state (vs. normotensive pregnancy), leading us to hypothesize a role for vasopressin in PreE. Copeptin (CP) is a stable 4 kDa pro-segment of vasopressin and secreted in a 1:1 molar ratio, thereby making CP an ideal biomarker of vasopressin secretion. Utilizing maternal plasma from the prospectively collected, IRB-approved Maternal Fetal Tissue Bank at the University of Iowa, CP was measured throughout pregnancy using a commercially available ELISA. Maternal plasma CP was significantly elevated in all trimesters in pregnancies that eventually developed PreE (n=50) versus (n=31) controls (1
st
trimester: 903±138 vs. 2045±371 pg/mL, p=0.03; 2
nd
trimester: 672±95 vs. 1754±37, p=0.05; 3
rd
trimester: 829±72 vs. 1832±247, p=0.002). After controlling for covariates such as age, BMI, chronic hypertension, twin gestation, diabetes, and history of PreE, the association of an elevated CP and the development of preeclampsia remains significant. ROC analyses reveal that CP is significantly predictive of the development of preeclampsia in all trimesters. Notably, these data establish CP as a novel, potent, 1
st
trimester predictor of PreE (AUC=0.80, p=0.005, at a cutoff of 1258 pg/mL). The temporal order of changes in CP (1
st
trimester) versus other known mechanisms of PreE (late pregnancy) supports our working hypothesis that vasopressin secretion may precipitate or induce other known humoral, vascular, immune, and morphological mechanisms of PreE.
Abstract 004: Interleukin 17a Regulates Salt And Water Retention Through Enhanced Sgk1 And Nhe3 Expression In The Renal Proximal Tubule
Authors: Norlander Allison E , Kirabo Annet , Gnecco Juan , Kamat Nikhil , Saleh Mohamed A , McDonough Alicia , Harrison David G , Madhur Meena S
Published: March 2022

We have previously shown that the pro-inflammatory cytokine, interleukin 17A (IL17A), is upregulated by angiotensin II and plays a critical role in promoting angiotensin II-induced hypertension and vascular dysfunction. However, the mechanism by which IL17A promotes hypertension is not known. Our preliminary studies demonstrated that angiotensin II-induced hypertension decreased sodium hydrogen exchanger 3 (NHE3) in the kidneys of IL17A-/- mice to a greater extent than in wild type (WT) mice. This likely enhances the pressure natriuresis in these mice and could explain the lower hypertensive response to angiotensin II in these animals. Serum glucocorticoid kinase 1 (SGK1) potentially regulates NHE3 in the kidney. Therefore, we hypothesized that IL17A upregulates SGK1 and NHE3 in the renal proximal tubule and that loss of IL17A would enhance natriuresis and diuresis. Using cultured human proximal tubule cells (HK-2 cells), we found that IL17A upregulated SGK1 mRNA expression 3.9±0.4 fold (n=4, p<.0001). In vivo, angiotensin II infusion upregulated renal SGK1 mRNA expression in wild type (WT) mice 2.6±0.3 fold, and this effect was abolished in IL17A deficient mice (n=3-4, p=.02). Upregulation of renal SGK1 expression in WT mice was specific to the cortex. Further, we found that IL17A induced phosphorylation of SGK1 at Ser78 2.01±0.4 fold (p=0.08, n=4) in HK-2 cells after 15 minutes. This effect was prevented when the cells were pretreated with a p38 inhibitor. We also found that NHE3 protein was upregulated 4±0.6 fold (n=2) by IL17A in these cells, suggesting a potential connection between phosphorylated SGK1 and NHE3 upregulation. Finally, we demonstrated that WT mice, in response to an intraperitoneal saline challenge, retained an additional 28.6% of the injected volume (p<.01), 32.7% of sodium (p<.01) and 30.7% of chloride (p<.05) after 10 days of angiotensin II infusion, and this effect was abolished in IL17A-/- mice (n=5-6), indicative of preserved natriuresis and diuresis in these mice. These studies are the first to describe a link between IL17A and the induction of renal SGK1 and NHE3 and thus provide a potential mechanism by which IL17A modulates salt and water retention and ultimately blood pressure.
Abstract 017: Spironolactone Improves Insulin Secretion In Metabolic Syndrome Subjects
Authors: Luther James M , Byrne Loretta M , Yu Chang
Published: March 2022

Renin-angiotensin-aldosterone system blockade reduces the incidence of type 2 diabetes in clinical trials. We previously demonstrated that aldosterone impairs insulin secretion via a direct beta-cell effect in mice. To test the hypothesis that aldosterone and the mineralocorticoid receptor contribute to impaired insulin secretion in humans, we enrolled subjects with metabolic syndrome treated with hydrochlorothiazide (HCTZ) 12.5 mg daily for 4 weeks (baseline) followed by either Spironolactone 25 mg daily (SPL, n=16) versus Aliskiren 150 mg daily (ALI, n=19) for 4 weeks. We assessed insulin secretion and sensitivity using hyperglycemic clamps during sodium controlled diet (160 mmol Na).

During HCTZ alone, plasma aldosterone correlated inversely with the initial insulin response and disposition index (Figure A), but not with insulin sensitivity index. Plasma aldosterone decreased during aliskiren (-1.5±0.78 ng/dL) and increased during spironolactone treatment (+4.0±0.8 ng/dL; p<0.001 between treatment). Blood pressure, serum potassium, and insulin sensitivity index were unchanged. The acute insulin response increased compared to baseline during SPL compared to ALI (+11.6±7.9 vs -10.7±7.7 μU/mL; p=0.048), whereas insulin sensitivity index was unchanged.

We conclude that aldosterone is inversely associated with the initial phase insulin secretory response, the earliest detectable change in impaired glucose tolerance. Treatment with spironolactone could improve glucose homeostasis by restoring insulin secretion in subjects with metabolic syndrome.
Abstract 018: Sympathetic Contribution to Obesity Hypertension: Differential Hemodynamic Mechanisms between Prehypertension and Hypertension
Authors: Okamoto Luis E , Gamboa Alfredo , Shibao Cyndya , Arnold Amy C , Celedonio Jorge E , Diedrich Andre , Farley Ginnie , Paranjape Sachin Y , Biaggioni Italo
Published: March 2022

Sympathetic activation contributes to blood pressure (BP) elevation in obesity, but it is unknown whether this contribution differs between obese prehypertensives (PreHTN) and obese hypertensives (HTN). We, therefore, assessed hemodynamic parameters during autonomic withdrawal with the ganglionic blocker trimethaphan (TMT) in 15 lean normotensives (NTN; 34±3 yrs., BMI 23.4±0.4), 11 obese PreHTN (38±2 yrs., BMI 33.6±0.8) and 14 obese HTN (40±1 yrs., BMI 33.8±0.7). As expected, baseline supine systolic BP (SBP) was higher in PreHTN and HTN compared to NTN (P<0.01, Figure A). HTN, however, had lower supine SBP on the study day compared to their seated levels at screening (127±4 mmHg vs. 142±5; P=0.04) presumably due to lower sympathetic tone at supine rest. Autonomic blockade lowered SBP similarly in PreHTN (-23±4 mmHg) and HTN (-24±4 mmHg) compared to NTN (-8±3 mmHg; P<0.01), but the “intrinsic” SBP in the absence of autonomic influences remained slightly higher in HTN compared to the other groups. Baseline systemic vascular resistance (SVR) was similarly increased in PreHTN and HTN compared to NTN (P<0.01, Figure B), but it normalized after TMT only in PreHTN. Conversely, the BP drop in HTN was due to a decrease in cardiac output (CO; Figure C) and stroke volume (SV; -37±2 vs. PreHTN -25±2 and NTN -26±3%; P<0.01). In conclusion, the autonomic contribution to BP elevation differed between obese PreHTN and HTN. Obesity prehypertension was due to a sympathetically driven increase in SVR, whereas obesity hypertension was due to a non-autonomic increase in SVR associated with impaired sympathetic modulation of CO and SV presumably due to sympathetic contraction of splanchnic capacitance.
Abstract 026: Renal Medullary (Pro)Renin Receptor Mediates Angiotensin II-Induced Hypertension via Enhancement of Local Renin Response and Renal Medullary α-ENaC Expression
Authors: Wang Fei , Lu Xiaohan , Feng Yumei , Yang Tianxin
Published: March 2022

(Pro)renin receptor (PRR) is predominantly expressed in the collecting duct and is stimulated by angiotensin II (AngII) via the COX-2/PGE2/EP4 pathway but the function of PRR is unknown. We examined the effect of intramedullary delivery of a novel PRR decoy peptide PRO20 on AngII hypertension via an interstitial catheter chronically placed in the rat medulla. Telemetry showed that 1-wk AngII infusion (100 ng/kg/min) elevated MAP from 108 ± 5.8 to 164.7 ± 6.2 mmHg and this value was lowered to 110.2 ± 4.8 mmHg (p<0.05) by intramedullary PRO20 infusion (IMPRO) at 120μg/kg/d but was only modestly affected by intravenous PRO20 infusion (IVPRO) (day 7: 151.7 ± 4.4 mmHg). The indices of kidney injury including proteinuria (2.7-fold increase in urinary protein), glomerulosclerosis and interstitial fibrosis following AngII infusion were completely normalized by IMPRO so were the polyuria and downregulation of renal AQP2 protein expression. AngII infusion elevated urinary renin activity from 0.17 ± 0.013 to 9.3 ± 1.7 AngI ng/24h, which was reduced to 0.09 ± 0.04 AngI ng/24h by IMPRO. A similar pattern of changes in renin activity was observed in the inner medulla, contrasting to opposite changes in renal cortex and plasma. α-ENaC mRNA and protein were elevated 158% and 180%, respectively, in the inner medulla but not in the cortex or plasma, and the elevations were abolished by IMPRO. By chopstick electrodes, amiloride-sensitive sodium transport was increased from 13.06 ± 1.2 to 115.19 ± 7.5 Ieq μA/cm
2
by aldosterone, which was abolished by PRO20 (10.5 ± 3.75 Ieq μA/cm
2
). Together, these results suggest that renal medullary PRR determines the hypertension likely via mediating the local renin response and the direct activation of ENaC during AngII infusion.
Abstract 027: T cell-Dependent B cell Activation Plays a Role in Mediating Hypertension and Pathophysiology in Response to CD4+ T cells from Reduced Uterine Perfusion Pregnant Rats
Authors: Cornelius Denise C , Castillo Javier , Porter Justin , Campbell Nathan , Thomas Alexia , Amaral Lorena M , Wallace Kedra , Moseley Janae , LaMarca Babbette
Published: March 2022

Preeclampsia, (PE) newly developed hypertension during pregnancy, is associated with altered immune activation. We have previously shown that adoptive transfer of CD4+T cells from a rat model of PE, the RUPP Rat, increases blood pressure, endothelin (ET) expression, oxidative stress (ROS), inflammation, and agonist autoantibodies to the AT1 receptor in NP recipients, all of which have been shown to play a role in hypertension during pregnancy. The objective of this study was to determine if blockade of lymphocyte crosstalk via the CD40-CD40 ligand interaction between RUPP CD4+ T cells with endogenous B cells in NP recipient rats would improve pathophysiology observed previously with RUPP CD4+ T cells. To do so splenic CD4+ T lymphocytes were magnetically separated, incubated with 2.5 μg/mL anti-CD40 ligand (αCD40L) overnight and transferred into Normal Pregnant (NP) rats on day 12 of gestation (NP+ αCD40L RUPP T cells). On day 19 of gestation, blood pressure (MAP), blood, and tissues were collected from all groups of rats.

MAP was 99+/-1.2 in NP (n=18), 120+/-2.3 mmHg in control RUPP rats (n=19;P<0.001); 121+/-1.2 in NP+RUPP T cells(n=5;P<0.001); but only increased to 103 +/-1.7 in NP + αCD40L RUPP T cells (n=21) (P<0.001 vs NP+RUPP T cells). Placental ROS increased from 160.4 +/- 31 in NP to 380.5 +/- 60.7 in RUPP (p<0.05) and 318.6 +/-89 in NP+RUPP T cells. CD40 ligand binding reduced placental ROS to 118.7 +/-24 in NP + αCD40L RUPP T cells (p<0.05). Plasma IL-6 increased from 38.71+/-8 in NP to 92.94+/-15 in RUPP (p<0.05) and 107.4+/-26 in NP+RUPP T cells and only increased to 50.1+/-3.6 pg/ml in NP + αCD40L RUPP T cells. TNF-alpha increased from 10.9+/-5 in NP to 79+/-15 in RUPP (p<0.05) and 23+/-17 in NP+RUPP T cells and was only 8+/-3 pg/ml in NP + αCD40L RUPP T cells. Plasma sFLT-1 increased from 79+/-14 in NP to 172+/-34 in RUPP (p<0.05) and 107+/-9 in NP+RUPP T cells and was only 74+/-21 pg/ml in NP + αCD40L RUPP T cells.

These studies indicate that placental ischemic induced T cell communication with B cells is one important mechanism of PE that leads to much of the pathophysiology of the disease.
Abstract 028: BPH/5 Pre-eclamptic Mice Have Increased Uterine Interleukin (IL)-15 And This Is Associated with Decidual Natural Killer (dNK) Cell Loss At The Maternal-Fetal Interface Early In Pregnancy
Authors: Sones Jenny L , Lob Heinrich E , Isroff Catherine E , Song Jiunn , Williamson Emilie , Davisson Robin L
Published: March 2022

Preeclampsia (PE) is a hypertensive disease that affects 5-10% of pregnant women worldwide. Although a leading cause of maternal and perinatal morbidity/mortality, its origin is unknown. Abnormal placentation resulting in reduced placental perfusion is implicated. Early in pregnancy dNK cells are recruited to the maternal-fetal interface to promote uterine angiogenesis during decidualization. Uterine IL-15 activates dNK cells, which produce IFN-γ to orchestrate vessel remodeling. Using the BPH/5 mouse model that spontaneously develops the cardinal features of PE along with placental abnormalities, we tested the hypothesis that uterine inflammation is associated with dNK cell loss at the maternal-fetal interface prior to placenta formation. At the start of decidualization (e4.5-5.5), IL-15 mRNA was increased in BPH/5 implantation sites vs C57 controls (60-fold vs 20-fold respectively, p<0.05, n=4). mRNA expression of the dNK cell activation marker NKp46 was similar in BPH/5 implantation sites vs C57 at e4.5, but reduced by 10-fold at e5.5 (p<0.05, n=4). This finding was confirmed by flow cytometry (BPH/5: 0.7 x 10
3
cells vs C57: 9.7 x 10
3
cells, p<0.05, n=4) and immunohistochemistry. Moreover, there was a 3-fold reduction of IFNγ mRNA in BPH/5 implantation sites vs C57 at the time of maximal decidualization (e7.5, p<0.05). Since prolonged IL-15 stimulation may initiate loss of NK cell function, we tested if elevated IL-15 is responsible for dNK cell loss in C57. IL-15 was increased in the pregnant uterus of C57 by intrauterine injection of recombinant IL-15/IL-15 Receptor-α complex (rIL-15/IL-15Rα) at e2.5. This did not change the number of implantation sites at e5.5 (veh: 8.7 ± 0.8; rIL-15/IL15-Rα: 8 ± 1.2, p>0.05), but blunted NKp46 mRNA expression (30-fold vs veh, p<0.05) and decreased dNK cell numbers (veh: 9.6 x 10
3
vs rIL-15/IL-15Rα: 0.4 x 10
3
cells, n=3-5, p<0.05). This shows that BPH/5 females have increased uterine

inflammation and dNK cell loss, and that artificial increases in intrauterine IL-15 in control mice cause dNK cell loss. This suggests that increased uterine expression of IL-15 contributes to dNK cell loss at the maternal-fetal interface in BPH/5 mice, which could lead to impaired placental perfusion and development of PE in this model.
Abstract 033: Novel Neurohumoral Responses to Head Upright Tilt Testing in Children with Chronic Nausea and Orthostatic Intolerance
Authors: Wagoner Ashley L , Shaltout Hossam A , Diz Debra I , Fortunato John E
Published: March 2022

Children with chronic unexplained nausea have a high incidence of orthostatic intolerance (OI), with exaggerated suppression of baroreflexes upon standing. The renin-angiotensin system (RAS) is known to play a role in disorders related to OI likely as a compensatory mechanism for CV regulation. The objective of this study was to characterize the neurohumoral response to head upright tilt testing (HUT) in children with chronic nausea and OI. Forty-eight patients (mean age [range] of 15 [10-18] years)(36 females, 12 males) completed HUT. GI symptoms including nausea were reported during HUT. Subjects were maintained supine for 15 min before 45 min 70° HUT. Plasma hormones catecholamines (Cat), epinephrine (Epi), norepinephrine (NE), vasopressin (AVP), aldosterone (Aldo), renin, and angiotensins [Ang-(1-7) and AngII] were measured in blood sampled immediately before and after 15 min of upright tilt. Of the 48 HUTs, 35 subjects demonstrated OI. There were no differences in supine blood pressures (BP) or humoral measures between groups. OI subjects had lower systolic (105 ± 4 vs. 127 ± 4 mm Hg, p=0.001), diastolic (61 ± 2 vs. 77 ± 2, p<0.0001), and mean arterial (76 ± 2 vs. 93 ± 2, p=0.0002) BPs during HUT compared to normal subjects (mean ± SEM). They also had higher Cat (625 ±44 vs. 466 ± 61 pg/ml, p=0.02), AngII (48 ± 3 vs. 35 ± 4 pg/ml, p=0.04), and AVP (46 ± 12 vs. 18 ± 14 pg/ml, p=0.01) during HUT compared to non-OI subjects. There was a negative correlation between Ang-(1-7) and NE at baseline (r=-0.6, p<0.05) and HUT (r=-0.6, p=0.03) in Non-OI subjects that was not seen in OI subjects. Nausea was reproduced by HUT in 42% (20 of 48) of this cohort. Nausea subjects did not have a change in DBP (61 ± 2 vs. 61 ± 3 mm Hg) upon tilt, but had significantly higher AVP (72 ± 20 pg/ml, p=0.01) during HUT compared to subjects who did not experience nausea (15 ± 7, p=0.001). Children with chronic nausea testing positive for OI have elevated Cat, AngII, and AVP levels upon HUT. Elevated serum AVP may be a key trigger to nausea with orthostatic challenge independent of OI on HUT. In addition to this humoral response, the absence of change in DBP upon standing suggests a failure in both the sympathetic nervous system and RAS compensatory mechanisms necessary to sustain HUT in children with chronic nausea and OI.
Abstract 038: MicroRNA-181a Mimic Inhibits the Renin-Angiotensin System and Attenuates Hypertension in a Neurogenic Model of Hypertension
Authors: Jackson Kristy L , Marques Francine Z , Nguyen-Huu Thu-Phuc , Stevenson Emily R , Charchar Fadi J , Davern Pamela J , Head Geoffrey A
Published: March 2022

Background:
Schlager BPH/2J mice are a genetic model of hypertension driven by greater activity of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS). During the dark period of the 24-hour light cycle when hypertension is at its greatest, BPH/2J mice display enhanced renal renin mRNA possibly related to lower levels of microRNA-181a, which is a negative regulator of renin mRNA.

Aim:
To determine whether lower renal miR-181a abundance contributes to elevated RAS activity and hypertension in BPH/2J mice.

Methods:
BPH/2J mice (n=6) were administered a mirVana miR-181a mimic (1, 5, 10 and 25nmol i.v) using an
in vivo
kidney specific transfection reagent and compared with established values in untreated normotensive BPN/3J and BPH/2J mice (n=6-8). Blood pressure (BP) was measured before and for two days after mimic treatment via pre-implanted radiotelemetry probes. The BP response to ACE inhibition (enalaprilat) and ganglion blockade (pentolinium) was determined during the dark period ~26-hrs after the 25nmol dose and kidney tissue was collected at ~50 hours.

Results:
The 25nmol dose of the miR-181a mimic caused a 4.0±1.4mmHg reduction from baseline in diastolic BP during the dark period (
P
<0.01) whilst the 1, 5 and 10nmol doses had no detectable effect (
P
>0.27). Renal renin mRNA abundance in mice treated with the miR-181a mimic was 0.87±0.1 which is lower than untreated BPH/2J mice (1.47±0.2,
P
=0.02) and comparable with untreated normotensive BPN/3J control mice (0.95±0.1,
P
=0.93), suggesting that the mimic effectively inhibited renin mRNA
in vivo
. Furthermore the depressor response to enalaprilat in untreated BPH/2J mice was abolished in BPH/2J mice treated with the mimic (-11.2±2mmHg vs 1.5±3mmHg respectively,
P
<0.001), suggesting the mimic reduced the RAS contribution to BP maintenance. The peak depressor response to pentolinium following enalaprilat pre-treatment, was comparable between untreated and mimic treated BPH/2J mice (-58±3 vs -50±3mmHg,
P
=0.08), suggesting the mimic does not overtly affect the SNS contribution to BP in BPH/2J mice. Importantly these findings provide the first
in vivo
evidence that low miR-181a levels contribute to greater activity of the RAS and hypertension in the BPH/2J model of hypertension.
Abstract 040: Cells of the Renin Lineage in the Peritoneal Cavity
Authors: Belyea Brian , Mehalic Theodore C , Gomez R. Ariel , Sequeira-Lopez Maria Luisa S
Published: March 2022

Renin cells have been traditionally associated with the control of blood pressure and fluid/electrolyte homeostasis. We recently described the presence of renin-expressing cells in the bone marrow, spleen and blood of mice. These cells are B-lymphocyte progenitors and show remarkable developmental, biochemical/transcriptional and functional similarities with the seemingly more distant kidney renin cell. While the function of these B
renin
cells within hematopoietic tissues remains unknown, we found that these progenitors are a vulnerable population for leukemic transformation: deletion of the Notch effector
RBP-J
within renin-expressing cells in mice leads to development of B-cell leukemia. Given their medical relevance, we sought to fully characterize the identity of renin cell descendants within the hematopoietic system. We generated

Ren1
dcre/

+
;
mT/mG
mice which after
Cre
-mediated recombination express
GFP
in renin precursors and their descendants and then performed lineage tracing and transplantation studies. We found that cells from the renin lineage are enriched in the peritoneal fluid (12.3 +/- 1.7% at 4 months) when compared to bone marrow (1.2 +/- 0.02%), blood (2.6 +/- 0.72%) and spleen (5.3 +/- 0.4%). Further, while renin-expressing cells in the bone marrow are pro-B cells, cells from the renin lineage within the peritoneal fluid exhibit increased expression of CD11b and CD5, and overall have an immunophenotype consistent with B-1 lymphocytes. Whereas the proportion of cells from the renin lineage in bone marrow, blood, and spleen decreases with age, renin lineage B-1 cells within the peritoneal fluid persist into adulthood. Finally, we found that renin lineage cells from the bone marrow of donor mice can repopulate the B-1 cell peritoneal compartment in irradiated host mice. This preliminary work suggests an association between renin, the key regulatory enzyme of the renin angiotensin cascade and the innate immune system, two major systems in the control of homeostasis.
Abstract 045: Endogenous Flow-induced Superoxide Stimulates Na/H Exchange Activity Via PKC In Thick Ascending Limbs
Authors: Hong Nancy J , Garvin Jeffrey L
Published: March 2022

Luminal flow stimulates Na reabsorption along the nephron and endogenous superoxide (O
2
-
) production by thick ascending limbs (TALs). The latter is due to protein kinase C (PKC) activation. Exogenously added O
2
-
augments TAL Na reabsorption, a process also dependent on PKC. Luminal Na/H exchange (NHE) and Na/K/2Cl cotransport initiate TAL Na reabsorption. However, the role of endogenously-produced O
2
-
in the stimulation of luminal NHE activity by flow, and the signaling pathway involved are unclear. We hypothesized that flow-induced production of endogenous O
2
-
stimulates luminal NHE activity via PKC in TALs. Intracellular pH (pH
i
) recovery was measured as an indicator of NHE activity in isolated, perfused rat TALs. Addition of the O
2
-
scavenger tempol decreased total NHE activity by 39% (0.168 ± 0.035 vs 0.103 ± 0.026 pH
i
units/min;
P
< 0.01;
n
= 4) when luminal flow was 20 nl/min; whereas it had no effect when flow was 5 nl/min (0.131 ± 0.027 vs 0.128 ± 0.027 pH
i
units/min;
n
= 5). Using the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in the bath to block basolateral NHE so that pH
i
recovery only reflected flow-enhanced luminal NHE activity, tempol caused a 30% reduction in pH
i
recovery (0.097 ± 0.018 vs 0.068 ± 0.015 pH
i
units/min;
P
< 0.04;
n
= 6). When these experiments were repeated with the general PKC inhibitor staurosporine, tempol had no effect (0.136 ± 0.018 vs 0.133 ± 0.007 pH
i
units/min;
n
= 4). Because PKC could mediate both induction of O
2
-
by flow and the effect of O
2
-
on luminal NHE activity, we used hypoxanthine/xanthine oxidase (HX/XO) to elevate O
2
-
and examined the role of PKC. HX/XO increased luminal NHE activity by 118% (0.140 ± 0.047 vs 0.305 ± 0.053 pH
i
units/min;
P
< 0.02;
n
= 5); whereas staurosporine blocked this effect (0.134 ± 0.043 vs. 0.125 ± 0.030 pH
i
units/min;
n
= 3). The PKCα/β1-specific inhibitor Gö 6976 also blunted HX/XO’s effect (HX/XO: 0.072 ± 0.023 vs 0.170 ± 0.035 pH
i
units/min;
P
< 0.008;
n
= 5; HX/XO in presence of Gö 6976: 0.116 ± 0.029 vs 0.137 ± 0.025 pH
i
units/min;
n
= 5). We conclude that flow-induced O
2
-
stimulates luminal NHE activity in TALs via PKCα/β1. This process may account for part of flow-stimulated bicarbonate reabsorption in this nephron segment.
Abstract 048: The Sphingosine-1-phosphate Receptor S1P1 Regulates Kidney Vascular Development and Heart Development
Authors: Hu Yan , Sequeira Lopez Maria Luisa
Published: March 2022

Sphingosine 1-phosphate receptor 1 (S1P1) is one of the five G-protein coupled receptors activated by Sphingosine 1-phosphate, which is a bioactive sphingolipid metabolite crucial in many biological processes, including vascular development. It has been reported that deletion of S1P1 in mice results in embryonic lethality at E12.5-14.5 due to failure of migration and/or differentiation of vascular smooth muscle cells (vSMCs) and pericytes and that S1P1 functions autonomously in endothelial cells (ECs) as an inhibitor of angiogenesis. However, due to the early lethality of the aforementioned S1P1-/- mice, the role of S1P1 in kidney vascular development has not been determined. This study was designed to test the hypothesis that the S1P1 is crucial for the differentiation, assembly and maturation of the renal vasculature.

Using EC-SCL-CreERT mice that specifically express tamoxifen inducible Cre in EC progenitors crossed with S1P1fl/fl and Rosa-LacZ reporter mice, we generated mice with timed deletion of S1P1 in ECs during kidney development (EC-S1P1KO). The knockout embryos with tamoxifen treatment develop severe edema, hemorrhages (36%, 8/22) or die (50%, 11/22) at E14.5 to E16.5, which may be caused by heart failure. Using histological methods including X-gal staining and immunohistochemistry for markers of ECs and mural cells, we found that the EC-S1P1KO embryos develop abnormal glomerular capillaries, dilated renal arteries and veins, disrupted vSMC coating of renal arteries and arterioles and absence of lymphatic endothelium. Further, thinner inter-ventricular septum, thinner ventricular myocardial compact layer and dilated myocardial capillaries are found in EC-S1P1KO embryos. Cross-transplantation studies of E12.5 kidneys from EC-S1P1KO and control mice under the kidney capsule of WT adult mice revealed that the kidney vascular abnormalities are intrinsic, due to the lack of S1P1 in renal EC precursors, and not secondary to the extra-renal anomalies.

These studies suggest that endothelial S1P1 is an essential regulator of both kidney vascular development and heart development.
Abstract 051: The Myogenic Tone Of Resistance Arteries Is Controlled By A Molecular Pathway Driven By Tgfβ Through Emilin1 And Converging On The Regulation Of Trp Channels.
Authors: Carnevale Daniela , Casaburo Manuel , De Lucia Massimiliano , Pallante Fabio , Bressan Giorgio , Lembo Giuseppe
Published: March 2022

Emilin1 (E1) is a protein of the extracellular matrix that regulates TGFβ bioavailability through proteolysis of the proTGFβ. E1 KO mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1 KO phenotype results from a developmental defect or lack of a homeostatic role in the adult. To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (VSMCs) of adult mice, by the use of floxed E1 and CreERT2 [tamoxifen (TAM) inducible Cre recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. When E1fl/fl mice carrying the Smmhc-CreERT2 were given TAM blood pressure significantly increased (124±1 vs basal condition 106±1 mmHg) as well as myogenic response in resistance arteries (16.3±0.7 vs basal condition 11.4±0.1 % at 125 mmHg).

In order to understand how E1 ablation in VSMCs could functionally determine the increase in myogenic tone, we used an ex vivo approach in resistance arteries, to target the molecular pathway that could regulate intracellular calcium fluxes, the main regulator of myogenic contraction. Experiments performed in isolated VSMCs from E1 KO as compared to WT arteries, suggested the involvement of a ROCE (receptor operated calcium entry) mechanism, whose main final molecular determinant is represented by the TRP channels, known to be regulated by a canonical PLC-DAG pathway, but also by a parallel one controlled by the growth factors signaling cascade. By exploring this pathway, we found that E1 regulates the activity of TRPC3 channels and thus of the myogenic response. The molecular pathway delineated by our studies started from the transactivation of EGFR through metalloproteinases and converged on the Src kinases that increased TRPC3 function and thus calcium entry. In particular, with an in vivo targeting of TRPC3 in E1 KO with an adenovirus carrying a dominant negative mutation, we demonstrated a rescue of the increased myogenic response of E1 KO resistance arteries.

Overall our results suggest that this pathway in VSMC, driven by TGFβ and regulated by E1, is crucial for the myogenic tone of resistance arteries and is linked to the regulation of blood pressure.
Abstract 063: Phosphodiesterase 5 Inhibition Improves Insulin Sensitivity and Disposition Index in Prediabetes without Altering Glucose-Stimulated Insulin Secretion
Authors: Ramirez Claudia E , Shibao Cyndya , Nian Hui , Yu Chang , Luther James M , Brown Nancy J
Published: March 2022

Chronic administration of the PDE5 inhibitor sildenafil citrate improves insulin sensitivity in high fat-fed mice. A small study in patients with metabolic syndrome found that one-month treatment with tadalafil with or without ACE inhibition improves disposition index (DI) and β-cell function, as measured by IVGTT. We report the results of a randomized, double-blind, placebo-controlled study of the effect of 3-month treatment with sildenafil (25mg t.i.d.) on insulin sensitivity and secretion assessed using hyperglycemic clamps in individuals with impaired fasting glucose and/or impaired glucose tolerance. Among 51 subjects randomized, 21 subjects completed sildenafil treatment [8 men, 2 black Americans, body mass index (BMI) 35.7±6.8 Kg/m
2
], and 21 completed placebo treatment (8 men, 6 black Americans, BMI 36.4±6.6 Kg/m
2
). There was no significant difference in acute- or late-phase glucose-stimulated insulin secretion (GSIS) between groups. In contrast, insulin sensitivity index (ISI) and DI were significantly higher in the sildenafil group (
Figure
, adjusted means and 95% CI after controlling for baseline measurements and BMI, *P<0.05). Sildenafil did not significantly affect blood pressure or heart rate compared to placebo. These data suggest that pharmacological strategies to increase cGMP enhance insulin sensitivity. Additional studies are needed to determine whether PDE5 inhibition prevents incident diabetes in high risk populations.
Abstract 087: Intrarenal Ghrelin Receptor Inhibition Attenuates Angiotensin II-Dependent Hypertension In Normal Rats
Authors: Kemp Brandon A , Howell Nancy L , Gildea John J , Padia Shetal H
Published: March 2022

Recent studies have shown that the intrarenal ghrelin receptor (GHSR) mediates distal nephron-dependent sodium (Na+) reabsorption in rats. Renal GHSR expression is increased in response to chronic high fat diet (HFD) and chronic intrarenal GHSR antagonism prevents HFD-induced hypertension. However, the role of the antinatriuretic renal GHSR in the development of other forms of hypertension is unknown. Normal female Sprague-Dawley rats (N=27) underwent uninephrectomy and telemetric blood pressure probe implantation. Following a 72h recovery, a subcutaneous osmotic minipump was inserted in the rat for the chronic systemic delivery of angiotensin II (Ang II; 200 ng/kg/min) or 5% dextrose in water (D5W) and received either bolus infusions of GHSR siRNA (15 μg x 3 over 5 days) or a second osmotic minipump for chronic intrarenal delivery of D5W (0.25 μL/h x 5 days) or GHSR antagonist [D-Lys-3]-GHRP-6 (D-LYS, 0.9 mg over 5 days). Four groups were studied: Group 1 (N=10); systemic D5W + intrarenal D5W; Group 2 (N=8): systemic Ang II + intrarenal D5W; Group 3 (N=4): systemic Ang II + GHSR siRNA (day 1, 3, 5); Group 4 (N=5): systemic Ang II + intrarenal D-LYS. While there was no change in mean systolic blood pressure (SBP) in the control group, systemic Ang II infusion significantly increased SBP from 126±5 to 192±2 mm Hg over 5 days (ANOVA F=47.9; P<0.0001). Intrarenal GHSR siRNA infusion markedly inhibited the pressor effect of systemic Ang II (136±2 mm Hg day 5; ANOVA; F=26.2; P<0.0001) and resulted in 65% knock down of collecting duct GHSR protein expression (P<0.05). Intrarenal GHSR antagonism with D-LYS also significantly reduced Ang II-induced hypertension, (151±13 mm Hg day 5; ANOVA F=10.7; P<0.001), albeit to a lesser extent. Collectively, these data demonstrate that chronic inhibition of intrarenal GHSR activity significantly reduces Ang II-induced hypertension in normal rats. Thus, renal GHSRs represent a novel therapeutic target for the prevention of hypertension.
Abstract 089: A Genetic Variant of Human Aldosterone Synthase Gene Causes Salt-Dependent High Blood Pressure in Transgenic Mice
Authors: Mopidevi Brahmaraju , Puri Nitin , Ponnala Madhusudan Reddy , Kaw Meenakshi , Keetha Narsimha Rao , Jain Sudhir , Rana Anita , Kumar Ashok
Published: March 2022

Aldosterone, synthesized by the enzyme CYP11B2, induces positive sodium-balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the hCYP11B2 gene to human hypertension. We have identified three SNPs, in linkage disequilibrium, in the hCYP11B2 gene: T/A at -663, T/C at -470 and C/T at -344. Variants ACT occur together and form the haplotype I (Hap I) while variants TTC constitute haplotype II (Hap II). We hypothesize that these SNPs, when present together, will lead to haplotype-dependent transcription of the hCYP11B2 gene, differentially increase aldosterone and affect blood pressure. To this end, novel transgenic (TG) mice with the hCYP11B2 gene, targeted to the mHPRT locus, with either haplotype II or I variant are used in the study. ChIP assay, using anti-RNA pol II antibody, shows increased Pol II binding to the chromatin from Hap I TG mice in adrenal (2.8 fold higher, p<0.05) and renal tissues (1.3 fold higher, p<0.05) as compared to chromatin extracts from Hap II TG mice. Immunoblot analysis shows upregulation of the hCYP11B2 in adrenal (2.7 fold higher, p<0.05) and renal tissues (1.35 fold higher, p<0.05) of Hap I vs. Hap II-TG mice; no significant difference was observed in mCYP11B2 between the two haplotypes. Complementary ELISA shows higher circulating levels (p<0.05) of aldosterone in Hap I mice (1504±48.7 pg/mL) as compared to both, Hap II (778±142.8 pg/mL) and C57 mice (740±28.9 pg/mL). Importantly, we observed increased baseline blood pressure in Hap I TG mice (Hap I- 117±2.5 vs. Hap II- 109±1.9 mm Hg, p<0.05), an effect accentuated by high-salt diet (Hap I- 135±2.6 vs. Hap II- 122±2.2 mm Hg, p<0.05). Elevated aldosterone was accompanied by up-regulation (p<0.05) of proinflammatory markers in renal tissues from Hap I TG mice (IL1β, MCP1, ICAM). Thus, this study identifies -344T as a reporter polymorphism for Hap I of the hCYP11B2 gene. SNPs in Hap I promote increased transcription and expression of the gene, in multiple tissues, with resultant elevation of plasma aldosterone levels. Pathophysiological impact of this haplotype-dependent transcriptional regulation of the hCYP11B2 is highlighted by increased inflammation and blood pressure in TG mice with the Hap I of this transgene.