bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 106: Hypothalamic PVN GαI2 Protein-mediated Renal Nerve Dependent Sympathoinhibition Facilitates Suppression of NCC Activity and a Salt-resistant Phenotype
Authors: Wainford Richard D
Published: March 2022

Aim:
We hypothesize hypothalamic paraventricular (PVN) specific Gαi2 proteins, which are up-regulated in the PVN during increased sodium intake, mediate global and renal sympathoinhibition to facilitate suppression of the activity of the sodium chloride cotransporter (NCC) and salt-resistance in the Sprague Dawley rat.

Methods:
Groups of intact or bilateral renal denervated (RDNX) salt-resistant Sprague-Dawley rats received a bilateral PVN infusion of a scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN-300ng/side/day) and a normal 0.4% (NS) or high 8% NaCl (HS) diet for 7-days. On day-7 24h Na+ balance was assessed - in sub-groups MAP, plasma norepinephrine (NE) content, kidney NE content and NCC activity (peak natriuresis to iv bolus hydrochlorothiazide (HCTZ)) was assessed (N=6/gp/study).

Results:
In SCR ODN infused rats HS-intake evoked a significant 3-fold site-specific increase in PVN Gαi2 proteins and suppressed plasma NE content (plasma NE [nmol/L] SCR NS 72±4 vs HS 40±5, P<0.05), endogenous kidney NE content (NE [pg/m] SCR NS 586±36 vs HS 496±41, P<0.05) and NCC activity (peak ΔUNaV to HCTZ [μeq/min] SCR NS 10.6±0.8 vs HS 7±1, P<0.05) without impacting sodium homeostasis or MAP. ODN-mediated PVN Gαi2 down-regulation caused renal nerve-dependent hypertension (MAP [mmHg] Gαi2 NS 126±2, Gαi2 HS 147±2, Gαi2 HS + RDNX 131±3 P<0.05) sodium retention (24h Na+ balance [meq] Gαi2 NS 0.6±0.3, Gαi2 HS 2.3±0.4, Gαi2 HS + RDNX 0.8±0.3 P<0.05), global and renal sympathoexcitation (plasma NE [nmol/L] Gαi2 NS 67±7, Gαi2 HS 105±11, Gαi2 HS + RDNX 74±6 P<0.05; kidney NE content [pg/mg] Gαi2 NS 614±48 vs HS 823±59, P<0.05) and a failure to suppress NCC activity (peak ΔUNaV to HCTZ [μeq/min] Gαi2 NS 10.2±2, Gαi2 HS 14.7±2, Gαi2 HS + RDNX 7.6±2, P<0.05).

Conclusion:
PVN Gαi2 protein-gated pathways represent a sodium sensitive CNS mechanism acting to regulate renal nerve-dependent sodium excretion via, in part, actions upon the activity of the NCC. Based on recent studies by other groups we speculate the regulation of NCC in this setting is mediated by the actions of NE released from efferent renal nerves on renal adrenoceptors to impact the signal transduction network that regulates NCC expression.
Abstract 108: Role of Primary Endothelial Cilia in Hypertension
Authors: Saternos Hannah , Hossain Md Zubayer , AbouAlaiwi Wissam
Published: March 2022

Primary cilia are mechanosensory organelles that are projected into the lumen of blood vessels. It has been demonstrated that vascular endothelia require primary cilia to sense and transmit external mechanical stimuli into internal biochemical reactions. One of these reactions includes the biosynthesis and release of nitric oxide, which is one of the most potent endogenous vasodilators. This idea has
only
been investigated in cultured endothelial cells
in vitro
. Based on this finding, however, a very bold hypothesis is formed to test that abnormal cilia function results in vascular hypertension. Our laboratory has recently generated and obtained several conditional mouse models to specifically study the function and structure of primary cilia in vascular endothelia. These models include
1)
mice without cilia function (
Pkd1
or
Pkd2
);
2)
mice without cilia structure (
Tg737
or
Kif3a
). Our data indicate that mice with abnormal cilia function (
Pkd1
) or structure (
Tg737
) show significantly higher systolic (150±19 for

Pdgfbcre:Pkd1
flox/flox

and 147±10 for

Tie2Cre:Tg737
flox/flox

vs. 128±9 for wild-type) and diastolic (120±21 for

Pdgfbcre:Pkd1
flox/flox

and 120±11 for

Tie2Cre:Tg737
flox/flox

vs. 102±7 for wild-type) blood pressure than the corresponding wild-type mice. Because there is a positive and continuous correlation between blood pressure and cardiovascular diseases, satellite hypotheses are developed to look at the pathophysiological roles of endothelial cilia in cardiac functions and focal vascular diseases
in vivo
. Our data clearly point towards deteriorating phenotypes in the cardiac muscle, including cardiac fibrosis due to an increased cardiac workload. As a result, a heart-to-body weight ratio was significantly increased by 17 weeks old (0.008

PdgfbCre;Pkd1
f/f

vs. 0.006

Pkd1
f/f

).The present study will very likely provide new insights for hypertension and offer advanced scientific understanding of vascular endothelial cilia in other cardiovascular diseases.
Abstract 110: Importance of the C-Terminal Transactivation Domain of STAT3 in Hypertension-Induced Cardiac Hypertrophy
Authors: Zouein Fouad A , Zgheib Carlos , do Carmo Jussara M , Vaka Ramana , Altara Raffaele , Kurdi Mazen , Booz George
Published: March 2022

Signal transducer and activator of transcription 3 (STAT3) is known to have protective effects in the heart in acute oxidative stress such as myocardial infarction; however, the role of STAT3 in the heart in response to chronic stress, such as hypertension, is not defined. Here, we assessed the importance of STAT3 on cardiac remodeling post abdominal aortic constriction (AAC) using mice expressing a STAT3 protein lacking the transactivation domain (TAD; aa701-732) selectively in cardiomyocytes (cm-STAT3Δ). Loss of the TAD region impairs both the mitochondrial and transcriptional actions of STAT3. Both WT and cm-STAT3Δ mice developed hypertension to a comparable extent. However, cm-STAT3Δ mice exhibited a significant (p < 0.01) decline in ejection fraction (58.3 ± 4.7 to 24.2 ± 3.6, n=4) and fractional shortening (30.8 ± 3.4 to 11.2 ± 1.7, n=4) over 28 days following AAC, while WT mice exhibited a compensatory response in EF (58.6 ± 3.2 to 59.4 ± 6.0) and FS (30.7 ± 2.1 to 33.0 ± 2.8). Notably, hearts of cm-STAT3Δ exhibited a marked increase in diastolic left ventricular internal diameter (LVIDd), symptomatic of eccentric hypertrophy and dilated cardiomyopathy. In contrast, the pattern of change in LVIDd for WT mice was consistent with concentric remodeling. Banding caused comparable increases in heart to body weight ratios in WT (4.1 ± 0.1 to 6.4 ± 1.0) and cm-STAT3Δ (4.0 ± 0.1 to 7.2 ± 0.6) mice, although on average the increase was larger in cm-STAT3Δ mice. Interestingly, we also found that miR-199a-5p levels were only moderately higher (36%) after AAC in the ventricles of cm-STAT3Δ mice vs. wild type hearts as compared to cardiomyocyte-targeted STAT3 KO mice in which the DNA binding domain of STAT3 is deleted. miR-199a-5p is a known inhibitor of peroxisome proliferator-activated receptor delta (PPARδ) expression and mitochondrial fatty acid oxidation in the heart. These new findings emphasize the distinctive and important role of the C-terminus of STAT3 in the hypertrophic response of the heart to hypertension and the progression to heart failure.
Abstract 112: Endothelial Mineralocorticoid Receptor Knock Out Protects the Endothelium From Aldosterone-Mediated Vascular Stiffness
Authors: Habibi Javad , DeMarco Vincent G , Jia Guanghong , Aroor Annayya , Garro Mona , Dodd Brittany , Jaffe Iris Z , Sowers James
Published: March 2022

Background:
There is accumulating evidence that increased levels of aldosterone (Aldo) and increased vascular mineralocorticoid receptor (MR) signaling increases vascular inflammation and oxidative stress leading to endothelial dysfunction and associated vascular stiffness. However, the specific role of endothelial cell (EC) MR activation in promotion of end stiffness in female mice has not been explored. Accordingly, we hypothesized that knocking out MR from ECs would attenuate the Aldo-induced endothelial dysfunction and vascular stiffness.

Methods and Results:
Twenty six week-old female ECMR knockout (ECMR-/-) and wild type (ECMR+/+) mice were infused with 250 μg/Kg/day Aldo for 3 weeks. To assess endothelial-dependent vasodilation, endothelial and aortic stiffness and blood pressure we utilized wire myography, atomic force microscopy (AFM), pulse wave velocity (PWV) (before and after Aldo perfusion) and tail cuff procedures. Aldo infusion did not increase BP or PWV and this was not affected by the presence or absence of ECMR. Aldo impaired endothelial-dependent vasodilation and increased EC stiffness 8.6 fold and these effects were mitigated in ECMR-/-. Aldo did not alter peri-aortic fibrosis by picrosirius red staining as measured by average gray scale intensities, nor did it cause medial thickening or aortic remodeling evaluated by the lumen to aortic wall ratio, in either Aldo-infused group. Moreover, levels of the oxidative marker, 3-nitrotyrosine (3-NT) did not differ in different compartments of the aortic wall in either Aldo treated group.

Conclusion:
ECMR protects the endothelium from aldo-mediated impaired vasodilation and endothelial cell stiffness, and this protection occurs without changes in BP, total aortic stiffness, or vascular remodeling.
Abstract 115: Hydrochlorothiazide Lowers Systemic Blood Pressure Predominantly Through Vasodilation in Conditions Associated with Vasoconstriction
Authors: Alshahrani Saeed , Rubinstein Jack , Jiang Min , Barone Sharon , Xu Jie , Zahedi Kamyar , Soleimani Manoocher
Published: March 2022

Thiazide derivatives are specific inhibitors of the Na+_Cl- Co-transporter (NCC) in the kidney distal tubules, and the most commonly used diuretic for the treatment of mild hypertension. The mechanism by which thiazides reduce the blood pressure (i.e. renal vs. extra renal) is not well understood. The blood pressure response to thiazides requires an initial volume loss (averaging about 1.5 kg) since it is not observed in individuals who are ingesting a high-salt diet. The objective of this study was to elucidate the renal and extra renal effects of thiazides. Using NCC deficient mice, we demonstrate that hydrochlorothiazide (HCTZ), a commonly used thiazide derivative, reduces blood pressure in the presence of volume-depletion and augmentation of circulating angiotensin levels but not in mice with normal vascular volume (NCC KO at baseline = 131.6 ± 4.4 vs. NCC KO with HCTZ = 125.8 ± 3.7 mm Hg) vs. (volume depleted NCC KO at baseline = 118.2 ± 4.1 vs. volume depleted NCC KO with HCTZ= 96.37± 4.5 mm Hg; p <0.05). The reduction in blood pressure by HCTZ occurs in the absence of increased salt excretion or urine output, indicating its extra-renal mechanism. Echocardiography demonstrated no significant changes in cardiac index in response to the drop in blood pressure by HCTZ (WT at baseline = 0.639 ± 0.05 vs. WT with HCTZ = 0.590 ± 0.01 ml/min/g) vs. (volume depleted NCC KO at baseline = 0.617± 0.07 vs. volume depleted NCC KO with HCTZ = 0.509 ± 0.04 ml/min/g). The antihypertensive effects of HCTZ were abrogated in the presence of paxilline, a specific blocker of large conductance calcium activated potassium channels (BK channels) in the vascular smooth muscle cell. Western blotting demonstrated enhanced expression of BK channels in vascular system of volume depleted NCC deficient mice vs. WT mice. Our results indicate that systemic vasoconstriction, as observed in volume depleted states, will amplify the extra-renal effects of HCTZ to lower blood pressure through vasodilation irrespective of the status of its renal target NCC.

Patients with congestive heart failure who are on a combination of a loop diuretic and HCTZ are at increased risk of significant hypotension subsequent to the activation of extra-renal effects of HCTZ, specifically when they become volume depleted.
Abstract 116: Characterization of a Sodium Responsive Human Sodium Bicarbonate Transporter NBCe2 in Human Proximal Tubule
Authors: Gildea John J , Xu Peng , Carlson M , Gaglione Robert T , Bigler Wang Dora , Kemp Brandon A , Reyes Camellia M , McGrath Helen E , Carey Robert M , Jose Pedro A , Felder Robin A
Published: March 2022

The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure (BP), which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT), another site of bicarbonate transport. The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by siRNA and Western blot, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and HEK293 cells. We immune-localized (peptide pre-absorbable) NBCe2 protein in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and in isolated RPTC apical membrane. NBCe2 was primarily found in the Golgi while NBCe1, another electrogenic member of the same family, was primarily found at the basolateral membrane, under basal conditions. Following a short-term increase in intracellular sodium, NBCe2 expression increased at the apical membrane in cultured slices of human kidney (1.75±0.15 fold increase over basal, N=3, p<0.05) and polarized, immortalized RPTCs (0.08±0.015 vs 0.03±0.006 control, NBCe2/CD13 RFU, N=3, p<0.05), while the basolateral localization of NBCe1 was decreased (30.7±5.4% over basal, N=3, p<0.05). DIDS (electrogenic sodium bicarbonate inhibitor, 500 μM, 10 min) inhibitable bicarbonate dependent pH recovery in the presence of EIPA (NHE inhibitor,100 nM, 10 min) after ammonium chloride prepulse (20 mM, 5 min) was higher in the cells cultured under high salt conditions (26.7±7.2% over basal, N=3, p<0.05). Thus, NBCe2 appears to be important in apical sodium and bicarbonate cotransport under high salt conditions. Future studies will examine the role of NBCe2 in mediating increased renal sodium transport in human salt sensitivity of BP.
Abstract 124: Luminal Flow Induces NADPH Oxidase 4 Translocation to the Nuclei of Thick Ascending Limbs
Authors: Saez Fara , Garvin Jeffrey L
Published: March 2022

Superoxide (O2-) exerts its physiological and pathophysiological actions in part by causing changes in gene transcription. In the thick ascending limb of the loop of Henle (TAL), luminal flow-induced O2- production is mediated by NADPH oxidase 4 (NOX4). Recently, polymerase (DNA-directed), delta interacting protein 2 (poldip2) has been shown to increase NOX4 activity. However, it is not known whether NOX4 translocates to the nucleus when activated or whether poldip2 participates in this process. We hypothesized that luminal flow causes translocation of NOX4 to the nucleus of the TAL facilitating its interaction with poldip2 in a protein kinase C (PKC)-dependent process. To test our hypothesis, we studied the subcellular localization of NOX4 and poldip2 using confocal microscopy and measured O2– production. Isolated TALs were studied in the absence and presence of luminal flow. To disrupt the cytoskeleton and inhibit PKC we used cytochalasin D (1uM) and staurosporine (10nM), respectively. Luminal flow increased the amount of NOX4 in the nucleus from 4±1 arbitrary units (AU) to 31±1 AU (p <0.0001; n=6). This redistribution was blocked by cytochalasin D (7±2, p < 0.04 vs. controls; n=6). Similarly, staurosporine decreased the intensity of NOX4 within the nuclear region of perfused TALs (3±2 AU vs controls, p < 0.01; n=5), Luminal flow increased poldip2 in the nucleus from 18±1 AU (n=3) to 29±1 AU (p<0.01; n=6). Net O2- production increased in response to luminal flow from 89±15 AU/S to 231±16 AU/S (p < 0.001; n=6). Inhibiting PKC decreased flow-induced O2– production in isolated TALs. However, disrupting the actin cytoskeleton with cytochalasin D did not block flow-induced O2– production (no flow + cytochalasin: 135± 19 vs. flow + cytochalasin: 285 ± 41 AU; p <0.04; n=7). We conclude that NOX4 translocates to the nucleus when activated by luminal flow, as does poldip2. Translocation is dependent on the actin cytoskeleton but O2– production is not. In contrast PKC mediates both processes. Translocation of NOX4 to the nucleus may facilitate changes in gene transcription caused by O2–.
Abstract 125: Overexpression of the Transcription Factor Dlx5 in the Placenta of Preeclamptic Patients Leads to Decreased Trophoblast Proliferation - A Novel Mechanism Involving Loss of Imprinting
Authors: Rugor Julianna , Singh Manvendra , Herse Florian , Haase Nadine , Golic Michaela , Staff Anne Cathrine , Müller Dominik , Schulz Herbert , Hurst Laurence D. , Dechend Ralf , Izsvak Zsuzsanna
Published: March 2022

Objectives:
Preeclampsia (PE) is one of the most-common, life-threatening pregnancy complication that affects maternal and offspring health, with short and long-term implications.

We hypothesize that epigenetic abnormalities and/or dysregulation of imprinted genes together with genes located in their proximity during placentation affect expression pattern of a broad spectrum of genes leading to preeclampsia. These genes can induce life-long epigenetic changes in the genome of the mother following preeclamptic pregnancy.

Methods and Results:
Using large-scale bioinformatical analysis of human placenta gene expression in 25 preeclamptic patients and 23 controls we identified characteristic pattern for PE. It consists of three clusters of patients, suggesting that preeclampsia is a complex, heterologous disease. Moreover, the analysis identified distal-less homeobox 5 (DLX5), a maternally expressed gene, which is significantly upregulated in preeclamptic placenta (1.5 fold). We confirmed upregulation of DLX5 on mRNA level in the early onset (0.8 (n=6), indicating that LOI is involved in overexpression of DLX5 in PE. DLX5 is primarily known as a developmental transcription factor and is crucial for bone development. Although DLX5 is highly expressed in human placenta, its function and the target genes in trophoblasts as well as its role during placentation still remains unknown. Using Sleeping Beauty (SB) transposon system, we stably overexpressed DLX5 in SGHPL-4 trophoblast cell line that significantly reduced trophoblast proliferation as indicated by high throughput sampler cell count (654.2 ± 395.5 vs. 1709 ± 1119) and MTT colorimetric assay (A570: 0.154 ± 0.097 vs. 0.29 ± 0.099).

Conclusions:
We show that the imprinted transcription factor DLX5 is upregulated in preeclampsia and propose loss of imprinting as one underlying mechanism. Overexpression of DLX5 in first trimester trophoblasts reduces cell proliferation. Our data support the importance of dysregulated epigenetic mechanism as an important feature in preeclampsia.
Abstract 137: Conditional Knockout of Sphingosine-1-Phosphate Receptor 1 in Renal Collecting Ducts Promotes Sodium Retention in Mice
Authors: Zhu Qing , Wang Weili , Wang Lei , Hu Junping , Li Ningjun
Published: March 2022

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite formed by phosphorylation of sphingosine. We have recently shown that renal medullary infusion of S1P agonist increases sodium excretion possibly through inhibition of epithelial sodium channel (ENaC) activity via S1P receptor 1 (S1P1), which is prominently localized in the collecting ducts. The present study was to test the hypothesis that the S1P1 in collecting duct plays a pivotal role in the regulation of renal sodium handling. We generated conditional knocked-out (CKO) mice with collecting duct-specific ablation of S1P1 using loxP-Cre system. Functionally, urinary sodium excretion in S1P1 CKO mice was significantly decreased by about 60% compared with that in control mice after acute IV sodium loading (0.84±0.16 vs. 2.22±0.62 μmole/min/g kwt). Moreover, the pressure natriuresis was severely impaired in S1P1 CKO mice compared with that in the control mice (4.32±1.04 vs. 8.73±0.19 μmole/min/g kwt). Furthermore, chronic high salt-induced sodium retention was remarkably enhanced in S1P1 CKO mice comparing to the control animals (5.27±0.39 vs. 2.38±1.04 mmole/24h/100g bw). Our results suggested that S1P1 in collecting duct plays an important role in the regulation of renal sodium excretion and that deficiency of S1P1 in collecting duct impairs renal sodium handling and promotes sodium retention. Modulation of S1P1 function in the renal medulla could be a therapeutic approach for salt-sensitive hypertension.
Abstract 140: Deficiency of Serum Glucocorticoid Kinase 1 (SGK1) in T Lymphocytes Blunts Hypertension and Abrogates Renal/Vascular Inflammation
Authors: Norlander Allison E , Saleh Mohamed A , Harrison David G , Madhur Meena S
Published: March 2022

We have previously shown that the T cell-derived pro-inflammatory cytokine, interleukin 17A (IL17A), is upregulated by and promotes angiotensin II-induced hypertension and contributes to renal and vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride enhances IL17A production from CD4+ T cells in an SGK1 dependent manner. We confirmed the effect of salt on CD4+ T cell differentiation and extended this finding to CD8+ T cells in which 40mM of excess salt increased the expression of IL17A (4.7 fold, p=0.0003), TonEBP (2.3 fold, p=0.002), and the salt-sensing kinase SGK1 (2.2 fold, p=0.001) in naive CD8+ cells cultured under Th17 polarizing conditions. As dietary salt intake is linked to hypertension, we hypothesized that T cell SGK1 is essential to the ability of T cells to mediate hypertension. To test this hypothesis, we crossed SGK1
fl/fl
mice with CD4cre mice to delete SGK1 in T lymphocytes. Interestingly, loss of T cell SGK1 resulted in a blunted blood pressure response to angiotensin II infusion (by 31.25 mmHg, p=0.01). Moreover, angiotensin II-induced renal and vascular inflammation was abolished in these mice compared to SGK1
fl/fl
control mice. Angiotensin II infusion increased total (CD45+) leukocytes in the kidney from 55.4 to 120.4x10
3
(p<0.01) in SGK1
fl/fl
mice while there was no increase in mice with T cell deletion of SGK1 (48.1 to 47.5x10
3
, p=ns). Similarly, angiotensin II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4x10
3
(p<0.01) in SGK1
fl/fl
mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1x10
3
, p=ns). Further characterization of these mice will lead to a more thorough understanding of how T cells sense salt and promote hypertension. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and the associated end-organ inflammation.
Abstract MP15: Afferent Arteriolar Endothelial-dependent Dilation is Impaired Prior to the Development of Radiation-induced Nephropathy and Hypertension
Authors: Imig John D , Sharma Amit , Khan Md. Abdul H , Fish Brian , Cohen Eric P
Published: March 2022

Chronic kidney disease (CKD) occurs in 15% of hematopoietic stem cell transplant (HSCT) patients, and has been clearly linked to irradiation at the time of the HSCT. CKD due to radiation also occurs after radionuclide therapy for cancer. Radiation nephropathy is expressed in rats and in humans as proteinuria, azotemia, and hypertension. There is a latent period of 6-8 weeks after irradiation to the development of proteinuria, azotemia, and hypertension in rats. This study tested the hypothesis that afferent arteriolar responses to the endothelial-dependent dilator acetylcholine are impaired prior to the development of proteinuria, azotemia, and hypertension in rats exposed to total body irradiation (TBI). Male WAG/RijCmcr rats were subjected to TBI (11 Gy) and afferent arteriolar responses to acetylcholine using the juxtamedullary nephron technique were determined at one, three, and six weeks. Systolic blood pressure (117 ± 6 vs. 119 ± 4 mmHg) and BUN (15.6 ± 1.4 vs. 15.8 ± 0.8 mg/dl) were not different between control and TBI groups at 6 weeks. Afferent arteriolar diameters averaged 22.5 ± 0.8 μm (n=30) in controls and 21.7 ± 0.7 μm (n=27) in TBI rats and were not different between control and TBI groups at 1, 3, or 6 weeks. Acetylcholine dilator responses were progressively attenuated from one to six weeks in TBI compared to control rats. During exposure to acetylcholine (0.01, 0.1, 1, and 10 μmol/L), afferent diameter increased by 8 ± 2%, 18 ± 3%, 27 ± 2% and 31 ± 3% in control rats (n=6), and 3 ± 2%, 9 ± 3%, 12 ± 3%, and 16 ± 3% in kidneys of 3 week TBI rats (n=8, p<0.05). However, TBI did not change the norepinephrine-mediated vasoconstrictor responses or the nitroprusside vasodilator responses at 1, 3, or 6 weeks. Renal microvessels were isolated from control and TBI groups at 3 and 6 weeks for protein expression assessment of endothelial enzymatic pathways Cyp2C, COX, and NOS. We found that epoxygenase Cyp2C23 and Cyp2C11 protein expressions were significantly lower 3 and 6 weeks following TBI compared to control rats. Taken together, these results indicate that the impaired afferent arteriolar endothelial-dependent acetylcholine responses and decreased epoxygenase enzymes precede the development of proteinuria, azotemia, and hypertension in irradiated rats.
Abstract MP16: ReninAAV Uninephrectomized db/db Mice as a Novel Diabetic Kidney Diesease Model for Testing New Therapeutics on Top of RAS Inhibition
Authors: Harlan Shannon M , Baker Hana E , Shrake Cynthia R , Breyer Matthew D , Heuer Josef G
Published: March 2022

ReninAAV comprises a novel approach to induce persistent hypertension in diverse murine models. ReninAAV induced hypertension is persistent and facilitates the development of animal models of diseases accelerated or driven by hypertension such as diabetic kidney disease (DKD). A single injection of ReninAAV (5x10
9
GC) in uninephrectomized (uNx)
db/db
mice results in progressive DKD. Impaired renal function was detected as early as one week post injection with an 8.9±3.1-fold increase in ACR (albumin:creatinine) as compared to baseline values, and at 11 weeks post injection significantly (p<0.01) increased serum creatinine (0.20±0.03mg/dL) and ACR were detected (68,890±7,209μg/mg) versus LacZAAV uNx
db/db
controls (0.08±0.01mg/dL and 1,364±83μg/mg, respectively). To determine if treatment with the ACEi Linsinopril halted progression of renal disease uNx
db/db
were given ReninAAV and followed for 4 weeks until ACR reached approximately 15,000μg/mg. Mice were randomized into 4 treatment groups: untreated, Lisinopril low (30mg/L), Lisinopril medium (100mg/L) and Lisinopril high (300mg/L) and followed for an additional 8 weeks. Untreated mice showed progressive increase in ACR developing a final ACR of 35,318±4,177μg/mg. Lisinopril dose dependently reduced ACR significantly compared to baseline (p<0.001, final ACR of 7,869±1632, 2,730±582 and 2,014±44μg/mg for the 30, 100 and 300 mg/L groups respectively), albeit still elevated compared to control LacZAAV uNx
db/db
mice (1,364±831μg/mg) that did not receive ReninAAV. Lisinopril treatment at 100 and 300mg/L also resulted in improvement in final serum creatinine values (p<0.05, 0.18±0.01 and 0.14±0.01mg/dL respectively vs untreated ReninAAV group 0.26±0.03mg/dL), yet residual increased serum creatinine were apparent compared to control LacZAAV uNx
db/db
mice (0.08±0.012mg/dL). This study establishes ReninAAV treated uNx
db/db
mice as a novel model of progressive DKD and demonstrates that suppression of RAS with Lisinopril improved, but only partially prevented progression of renal disease, thus enabling testing of new potential therapeutics on top of RAS inhibition.
Abstract P008: Impact of Hydrogen Sulfide on the Epigenetics of Diabetic Cardiomyopathy.
Authors: Veeranki Sudhakar , Kundu Sourav , Givvimnai Srikanth , Tyagi Suresh C
Published: March 2022

Although Diabetic cardiomyopathy results in enhanced risk for heart failure, epigenetic changes leading to diabetic heart failure are unclear. Hydrogen sulfide (H2S) has been implicated in the preservation of heart function owing to its anti-inflammatory and positive metabolic changes. In the current study, we investigated whether or not chronic H2S treatment (by giving NaHS) reverses diabetic cardiomyopathy using mouse model of type-1 diabetes: Akita mice. Regulators of mitochondrial biogenesis, calcium handling and molecules that regulate post-ischemic recovery were assayed by western blotting and Q-PCR. Further, we considered epigenetic modifications such as microRNA expression changes and DNA methylation alterations to understand the causes of diabetic heart failure with and without NaHS treatment for 30 days. Our data indicated that chronic H2S treatment significantly reduced the mitochondrial fission inducers: Drp-1 (24%) and Fis-1 (17%) in the Akita mouse hearts. Further, there was enhancement (10%) in the SERCA2a expression after NaHS treatment in the diabetic hearts. Also, there was significant decrease (16%) in TNF-1α protein expression in diabetic hearts after NaHS treatment. In addition, there was significantly increased expression of post-ischemic recovery regulators such as: Notch3 (157%), C-JUN (160%), PGC-1α (173%), HIF-2α (72%) , and NRF-1 (149%) after NaHS treatment. These results suggest that the chronic NaHS treatment ameliorates diabetic cardiomyopathy through decreasing mitochondrial fission and inflammation and enhancing Ca2+ handling; as well as mitigating epigenetic changes leading to enhanced post-ischemic recovery potential.
Abstract P015: Bilateral Common Carotid Artery Stenosis in Hypertensive Rats Impairs Dilation in Penetrating Arterioles and Posterior Communicating Arteries
Authors: Matin Nusrat , Fisher Courtney , Jackson William F , Dorrance Anne M
Published: March 2022

Hypertension and chronic cerebral hypoperfusion (CCH) are leading risk factors for cognitive impairment. We induced hypoperfusion in stroke prone spontaneously hypertensive rats (SHRSP) by bilateral carotid artery stenosis (BCAS) to create a novel, physiologically relevant model of cognitive impairment. We hypothesized that BCAS in SHRSP would impair endothelium dependent dilation and lead to outward remodeling of the penetrating arterioles (PAs) and posterior cerebral arteries (PCAs). The PAs are critical for maintaining parenchymal perfusion and controlling blood flow to the neurovascular unit, and the PCAs are integral in regulating the severity of CCH. Both artery types were studied by pressure myography and data are shown as mean±SEM, SHAM vs BCAS (an
n
=4 to 12 in each group) with p<0.05. Short-term memory, evaluated by novel object recognition testing, was impaired 8 weeks after BCAS (novel exploration quotient: 0.60±0.1 vs 0.45±0.1). Endothelium-dependent dilation was assessed with carbachol (data shown as % dilation at 10
-6
M). BCAS impaired endothelial function in PAs, as evidenced by the abolition of the dilation (14±3.3% vs -10±4.7%) but did not significantly alter sodium nitroprusside induced dilation (60 ± 9.7% vs. 49 ± 6.4%). Inhibition of nitric oxide and prostacyclin production with L-NAME (100 μM) and indomethacin (10 μM), respectively, did not change dilation in either group. This suggests that endothelium-derived hyperpolarizing factor plays a role in PA dilation in SHRSP. PAs from BCAS rats had a reduced wall thickness (at 60 mmHg: 7±0.5 vs 5±0.6 μm), and wall-to-lumen ratio (at 60 mmHg: 0.10±0.002 vs 0.07±0.0001). BCAS increased wall stress in PAs (at 60 mmHg: 306±30.2 vs 427±48.3 dynes/cm
2
). Endothelial dysfunction also was evident in the PCAs after BCAS (50 ± 13.0% vs 3 ± 14.9%) but the pattern of remodeling in these larger arteries was different. We did not observe a change in the wall thickness or wall-to-lumen ratio in PCAs after BCAS, but the lumen diameter was increased (at 80 mmHg: 105±7.7 vs 116±5.7 μm). These data suggest that endothelial dysfunction in the PAs and PCAs may worsen the severity of cerebral hypoperfusion and cognitive decline in SHRSP. The thinner walls in the PAs may also increase the risk of hemorrhage in the BCAS rats.
Abstract P017: Contribution of High Mobility Group Box 1 to Hyperhomocysteinemia-induced Podocyte Injury and Glomerular Sclerosis
Authors: Boini Krishna M , Xia Min , Conley Sabena M , Li Guangbi , Koka Saisudha , Gehr Todd W , Li Pin-Lan
Published: March 2022

High mobility group box 1 protein (HMGB1), a nuclear DNA binding protein is released under pathological conditions and locally act as one of potent damage-associated molecular patterns (DAMPs) to produce tissue injury and chronic inflammation. However, it remains unknown so far whether HMGB1 is implicated in homocysteine (Hcys)-induced podocyte injury and glomerular sclerosis during hyperhomocysteinemia (hHcys). In the present study, we found that homocysteine (Hcys) dose-dependently increased the production of HMGB1 in cultured podocytes, and that HMGB1 binder and inhibitor, glycyrrhizin (Gly) completely blocked its release induced by Hcys. Furthermore, inhibition of HMGB1 preserved podocyte function by restoring Hcys-induced suppression of VEGF secretion, decrease in expression of podocin and elevation of desmin level (podocyte damage markers). In
in vivo
studies, C57BL/6J wild type mice were fed a folate free (FF) diet or normal chow for 8 weeks to produce hHcys and administrated with vehicle or glycyrrhizin (1mg/kg/day) locally into the renal cortex. Western blot analysis of renal tissue showed that the FF diet significantly increased HMGB-1 (2 folds) and desmin expression (1.8 folds) compared to control mice, which was blocked by HMGB 1 inhibitor Glycyrrhizin. The urinary protein and albumin excretion were significantly higher in mice on the FF diet compared to ND fed mice. In glycerrhizin treated mice, however, the hHcys-induced albuminuria was significantly lowered (urinary albumin excretion in vehicle- and glycerrhizin-treated hHcys was 50 ± 3 and 32 ± 2.5 μg/24h/g BW, respectively). Morphological examinations showed that hHcys-induced more profound injury in glomeruli of vehicle treated mice than in glycerrhizin treated mice (the glomerular damage index in vehicle and glycerrhizin-treated hHcys mice was: 2.8 ± 0.4 vs. 1.3 ± 0.3). Based on these results, it is concluded that HMGB1 is one of important mediators of hHcys-induced podocyte injury and glomerular sclerosis. HMGB1 may be a therapeutic target for treatment or prevention of glomerulosclerosis associated with hHcys.
Abstract P021: The Combined Effects of Elevated Intrarenal Ang Ii and Blood Pressure Causes Greater Renal Injury in the Non-clipped Kidneys in 2k1c Rats
Authors: Miyata Kayoko , Shao Weijian , Prieto Minolfa , Satou Ryousuke , Katsurada Akemi , Seth Dale , Mitchell Kenneth D , Navar L. Gabriel
Published: March 2022

In 2-kidney 1-clip (2K1C) hypertension, intrarenal angiotensin II (Ang II) levels are increased in both kidneys, which are often implicated in the further augmentation of the intrarenal renin-angiotensin system and the development of hypertension and kidney injury. We recently reported that angiotensinogen (AGT) expression and secretion are increased only in the non-clipped kidneys (NCK). These findings provide the basis for the hypothesis that elevated Ang II levels, augmented AGT, and high arteriolar pressure in NCK synergistically cause greater kidney injury. Accordingly, we compared the degrees of kidney injury between clipped kidneys (CK) and NCK using a 2K1C model that maintains normal renal blood flow and GFR in the CK. Left kidneys of male rats were clipped for 21 days. Histological and immunohistological analyses were performed on both the CK and NCK. Twenty glomeruli and 20 microscope fields in the cortex and the medulla were examined for each group. The PAS-positive area in the glomeruli was higher in NCK compared with sham (33.9 ± 0.89 vs. 12.4 ± 0.51%) and CK (vs. 15.1 ± 0.58%). Similarly, the Masson's trichrome-positive area in the medullary region was greater in NCK compared with sham (2.21 ± 0.10 vs. 1.32 ± 0.05%) and CK (vs. 1.67 ± 0.10%), but the changes were not observed in the interstitial tissue of the cortex. Immunoreactivity for CD68, a marker of the macrophage and monocyte levels, was higher in NCK compared with sham (0.72 ± 0.07 vs. 0.36 ± 0.04%), but similar to that in CK. In contrast, accumulation of the immune cells in the glomeruli was greater in NCK compared with sham (8.99 ± 0.69 vs. 3.46 ± 0.46%) and CK (vs. 3.08 ± 0.24%). The proliferating cell nuclear antigen levels, a marker of cell proliferation, were greater in NCK (3.49 ± 0.09 %) but not in the CK. Levels of vimentin, a cell transformation and regeneration marker, were also higher in NCK compared with CK and sham. Increased vascular wall thickness (α-SMA) was observed in both kidneys. These results indicate that pathological factors associated with the high blood pressure are required for the development of renal injury in the 2K1C model including glomerular expansion, medullary fibrosis, immune cell infiltration in glomeruli and cell proliferation/transformation.
Abstract P032: High Glucose Increases Prorenin Receptor (PRR) at the Cell Plasma Membrane in the Collecting Duct Which Promotes the Induction of Downstream Fibrotic Factors
Authors: Gogulamudi Venkateswara R , Arita Danielle Y , Bourgeois Camille RT , Satou Ryosuke , Prieto Minolfa C
Published: March 2022

In streptozotocin (STZ)-induced Type-1 diabetic (T1D) rats, the collecting duct is the main source of intrarenal prorenin. The fact that binding of prorenin to the PRR triggers intracellular signals linked to tissue fibrosis has raised the expectation that prorenin might be directly responsible for diabetic nephropathy (DN). We showed that STZ-induced T1D rats exhibit greater apical distribution of PRR in the collecting duct. Therefore, we hypothesize that high glucose increases PRR abundance at the cell plasma membrane (PM) of collecting duct cells, which contributes to the stimulation of downstream fibrotic factors. To test this, we used male Sprague-Dawley rats (14w of age) with STZ-induced hyperglycemia for 7d (ip, single injection; 60 ng/kg), and cultures of collecting duct M-1 cells treated for 0, 5 min, and 1, 6, 12 and 24h with normal glucose (NG; 5mM glucose + 20mM mannitol) and high glucose (HG; 25mM). After 7d, STZ-induced rats (N=9) showed plasma levels of glucose as 428±13 vs.138±9 gr/dL and insulin as 0.07±0.02 vs. 2.4±0.01 ng/mL; compared to controls (N=7). Although PRR mRNA levels did not differ between groups; PRR protein levels and its downstream target TGF-β mRNA levels were augmented in the renal medulla of STZ-induced rats (0.55±0.03 vs.0.44±0.02 PRR/β-actin protein ratio; P<0.01; 1.22±0.06 vs.0.97±0.03 TGF-β/β-actin mRNA ratio; P<0.01). Interestingly, PRR protein levels were maximum elevated at 1h in PM extracts from M-1 cells treated with HG (0.95±0.04 vs. 0.33±0.2 PRR/E-cadherin protein ratio) but not in NG-treated cells. HG also unregulated downstream fibrotic factor fibronectin (1.9±0.2 vs.0.46±0.05 fold change) compared with NG-treated cells. Immunofluorescence studies of M-1 cells treated with NG for 1h showed that PRR is mainly localized in the perinuclear areas, whereas in HG-treated cells it was predominantly localized toward the cell surface, as it was more clearly observed in de-convoluted images. These data indicate that hyperglycemia leads to PRR trafficking alterations by inducing PRR translocation towards PM in the collecting duct cells and suggest that the activation of PRR during hyperglycemia conditions might be a novel mechanism underlying the development of DN, particularly tubulointerstitial fibrosis in DM.
Abstract P033: Acute Intravenous Glucose Injection Inhibits Tubuloglomerular Feedback and Increase Glomerular Filtration Rate
Authors: Zhang Jie , Lu Yan , Wei Jin , Yip Kay-Pong , Liu Ruisheng
Published: March 2022

Hyperfiltration is considered a risk factor for diabetic nephropathy, but the mechanism for alterations in glomerular filtration rate (GFR) in diabetes has not been clarified. Nitric oxide synthase 1 (NOS1) highly expresses in the macula densa (MD) and NO inhibits tubuloglomerular feedback (TGF). TGF is one of the major mechanisms for glomerular filtration rate (GFR) regulation. It is not clear whether glucose itself filtered into the tubules has any effect on GFR. We hypothesized the glucose enhances NOS1 activity in the MD, which blunts TGF and increases GFR.

First, we measured GRF in conscious mice by plasma FITC-inulin clearance. Three min after an intravenous infusion of 50μl of 4M glucose in wild type mice, GFR was increased by 19.1% from 236±9.7 to 281±13.4 μl/min. Intravenous saline infusion had no effect on GFR.

NO generation by the MD was then measured in isolated perfused juxtaglomerular apparatus with fluorescent probe DAF-2DA. In response to an increase in tubular glucose concentration from 0 to 300 mg/dl, NO production was increased by 78.4%. TGF, assessed in
vivo
by measuring the change in proximal tubular stop flow pressure (Δ) induced by an increase of perfusion rate in late proximal tubules from 0 to 40 nl/min, was significantly blunted from 6.5±0.9 to 5.0±1.6 mmHg when tubular perfusate glucose concentration was increased 0 to 300 mg/dl.

To determine whether the glucose-induced changes were mediated by NOS1 in MD, we repeated the experiments in MD specific NOS1 KO (MD-NOS1KO) mice. Intravenous glucose infusion did not significantly increase GRF (from 203±15.7 to 220±6.9 μl/min) in the KO mice. Elevating glucose content in tubular perfusate did not alter the TGF response (Δ was from 7.8±1.3 to 7.3±2.1 mmHg) in MD-NOS1KO mice. We concluded that increase of tubular glucose concentration increases GFR by inhibiting TGF response, which is mediated by NO production from NOS1 in the MD.
Abstract P044: Pleiotropic Effect of a <42.5 kb QTL Region on Rat Chromosome 10 Regulating Blood Pressure and Tumorigenesis
Authors: Cheng Xi , Waghulde Harshal , Mell Blair , Miller Shondra , Filipiak Wanda , Saunders Thomas , Joe Bina
Published: March 2022

This study is focused on a translationally significant, GWAS locus for cardiovascular disease (QT-interval) on human chromosome 17. We have already validated and high resolution mapped the homologous genomic segment of this human locus to <42.5 kb on rat chromosome 10. The locus in rats regulates both QT-interval and blood pressure and contains a single protein-coding gene, rififylin (Rffl). While there are no exonic variants, the expression of Rffl is differential between Dahl S and S.LEW congenic rats, which are strains used for mapping this locus. A previous study points to altered rate of endocytic recycling as the underlying mechanism, through which Rffl operates to control both cardiac QT-intervals and blood pressure. Rffl also contributes to tumorigenesis by negatively regulating caspases and tumor suppressor genes. Moreover, the expression of Methyl-CpG Binding Domain Protein 2 (Mbd2) is also differential between Dahl S and S.LEW congenic rats and Mbd2 can mediate the repression of methylated tumor suppressor genes. Therefore, we hypothesized that the higher expression of Rffl and Mbd2 render the S.LEW congenic strain more susceptible to tumor development. To compare the tumor susceptibility between S and S.LEW congenic strain, azoxymethane (AOM)-induced colon tumorigenesis was assessed. The number of colon tumors was significantly higher in the congenic strain compared with the S rat (p<0.01). Interestingly, the incidence of another phenotype, polyarteris nodosa (PAN), was also higher in the congenic strain. However, the identity of the quantitative trait nucleotides that regulates the expression of Rffl and Mbd2 is unknown. A novel long non-coding RNA (lncRNA), which we refer to as Rffl-lnc1, was identified within the rat Rffl 5’UTR intron locus. Compared to the S rat, the Lewis alleles of Rffl-lnc1 are polymorphic, with a large 19bp deletion. To further assess the role of Rffl-lnc1, the CRISPR/Cas9 system has been successfully applied to construct a Rffl-lnc1 knock-out model of the S rat and a 19bp knock-in rescue model of the S.LEW congenic strain. Functional evaluation of these targeted disruption and rescue models is underway to test the physiological role of Rffl-lnc1 and the 19bp polymorphism in regulating blood pressure and tumorigenesis.
Abstract P051: Renal Denervation Normalizes Arterial Pressure but Has No Effect on Renal Inflammation or Glucose Metabolism in Obese Hypertensive C57bl6j Mice
Authors: Asirvatham-Jeyaraj Ninitha , Han Ruijun , Fiege Jessica , Foss Jason , Razzoli Maria , Burbach Brandon J. , Bartolomucci Alessandro , Shimizu Yoji , Osborn John
Published: March 2022

Clinical studies have shown that renal denervation (RDNX) decreases arterial pressure (AP) and improves glucose metabolism in drug resistant obese hypertensive patients. In the present study, we used a murine model of obesity-induced hypertension (HTN) to test the hypothesis that RDNX lowers AP and improves glucose metabolism via an interaction of renal nerves with inflammatory mediators in the kidney. 8-week old C57Bl/6J mice were fed either a low fat diet (LFD; 10 KCal% from fat) or a high fat diet (HFD; 45 KCal% from fat) for 10 weeks. Two parallel protocols were conducted. In a metabolic protocol, body weight, food intake, body-composition and glucose metabolism were measured. In a cardiovascular protocol, radiotelemeters were implanted for measurement of AP. C57Bl6J mice on a HFD exhibited an inflammatory and metabolic syndrome phenotype including increase splenic and renal total T cells, increased fat mass, high blood pressure, hyperglycemia and glucose intolerance as compared to LFD mice. RDNX but not Sham surgery after 12 weeks of HFD normalized AP (116 ± 4 in sham vs. 97± 6 mmHg in RDX HFD mice). RDX had no effect on AP in LFD diet mice. RDX in obese hypertensive mice had no effect on renal T cells or cytokines. Finally, RDX had no effect on glucose metabolism in HFD mice as determined by the glucose tolerance test at 2 weeks after RDX. We conclude that the antihypertensive effect of RDX in obesity-induced hypertension is not associated with improvement in glucose metabolism or renal inflammation in the mouse.
Abstract P067: Novel Antagonists of Dopamine-β-hydroxylase Identified and Validated Through Structure Based Approach to Combat Hypertension
Authors: Dey Sanjay K , Joseph Toyanji , Kumar Santosh , Kamaladevi A , Sarkar Nabanita , Thelma B K , Sarkar Surajit , Balamurugan K , Kundu Suman
Published: March 2022

Human dopamine β-hydroxylase (hDBH), expressed in noradrenergic nerve terminals of nervous system and in chromaffin cells of adrenal medulla, is a key constituent of catecholamine biosynthetic pathway. DBH inhibition has been shown to help the treatment of hypertension, cardiac hypertrophy and cardiac heart failure, which are major causes of mortality and morbidity worldwide. Existing hDBH inhibitors are too few, often result in side effects and are frequently non-responsive to specific population. Since no three-dimensional structure existed for full-length hDBH, structure based rational drug design was elusive till date, an issue to which we provided solution lately by building an experimentally validated in silico model for hDBH. The model was used in Autodock, Glide SP and XP software for structure based virtual screening against small molecule databases from NCI, USA. The docked structures were scored using Autodock, X-Score and Prime MMGBSA. Thus, 69 compounds were identified as prospective inhibitors of DBH, which were then tested in vitro against human serum DBH and its nearly identical homologue, bovine DBH (586 of 617 amino acids homologous), with known inhibitors nepicastat and disulfiram as positive controls. Three lead molecules UDSC171, UDSC180 and UDSC142 were discovered in the process as potent inhibitors of DBH with IC50s of 1μM, 5.5μM and 18μM, respectively. The binding of the inhibitors to the enzyme were validated using fluorescence and CD spectroscopy as well as ITC, revealing KD values in the range of 100nm to 1μM. In silico pharmacokinetic analysis indicated the molecules to be latest generation of DBH inhibitors having very high cell permeability and inability to cross the blood brain barrier. High doses (up to 50μM) of the lead compounds showed acceptable cellular tolerance against HEK 293 cell line and insignificant hemo-toxicities against human RBCs. Hence, in vivo evaluation of the lead molecules were done in small model systems like C. elegans and D. melanogaster reconfirming their nontoxic properties up to 15μM doses. These three leads are now being tested in cardiac hypertrophy and hypertension rat models with exciting preliminary results.