bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 452: The Degree of Nephron Deficit is a Determinant of the Cardiovascular and Renal Risks Associated With a Reduced Nephron Endowment
Authors: Kett Michelle M , Makuei Leek , Cai Xiaochu , Brown Russell D
Published: July 2021

A reduced nephron endowment is associated with an increased sensitivity to hypertensive stimuli. We examined whether nephron endowment, and the degree of nephron deficit, impacts on the blood pressure and renal response to chronic angiotensin II (AngII) infusion. To address this aim we used the GDNF heterozygous mouse that, despite the same genotype, produces two distinct levels of nephron deficit due to the role of GDNF in both the initiation of kidney development and induction of nephron formation. GDNF het mice are born with either 2 small kidneys and a 30% nephron deficit (Het2K), or a single small kidney (Het1K) and a 65% nephron deficit compared to wild-type littermates (WT).

Sixteen week old male GDNF WT (n=9), Het2K (n=5) and Het1K (n=7) mice were implanted with radiotelemetry probes and baseline pressures recorded. Mice were administered enalapril (10mg/kg/day, drinking water) alone for 3 days, then concomitant with an AngII infusion (600ng/kg/min; s.c. osmotic minipump) for a further 21 days. 24hr albumin and urine excretion were assessed at baseline and at the end of the 21 days of blood pressure recording.

Baseline MAP was not different between WT, Het2K and Het1K groups (98.3±1.3, 97.1±1.8, 100.9±1.9mmHg respectively). Enalapril treatment led to a similar fall in MAP of 23mmHg in the 3 groups before the commencement of AngII infusion. AngII led to an immediate rise in MAP in all groups. At the end of the 21day infusion period, MAP of WT and Het2K mice were similar (122±3.3, 120.5±6.1mmHg), but MAP of Het1K mice (139.7±3.6mmHg; P<0.01) was significantly elevated over WT mice. Albumin excretion was not different between the groups at baseline, and AngII infusion did not significantly increase albumin excretion in WT or Het2K mice. However albumin excretion in Het1K mice increased 15 fold (4.86±1.56 to 76.05±30.3ug/24hr; P<0.001). Urine excretion also significantly increased in the Het1K (1.59±1.5 to 3.7±0.59ml/24h; P<0.001) in response to Angiotensin II, though WT and Het2k mice were not affected.

AngII infusion in Het1K, but not Het2k mice, led to significant exacerbation of hypertension and albuminuria. Our data suggests that the degree of nephron deficit is an important determinant of the risks associated with a reduced nephron endowment.
Abstract 455: Assessment of Pharmacokinetic Drug-drug Interaction between LCZ696 and Hydrochlorothiazide
Authors: Hsiao Hsiu-Ling , Greeley Michael , Pal Parasar , Langenickel Thomas , Sunkara Gangadhar , Chandra Priyamvada
Published: July 2021

Objective:
LCZ696 is a first-in-class angiotensin receptor neprilysin inhibitor (ARNI) being developed for the treatment of cardiovascular diseases, including hypertension and heart failure. Ingestion of LCZ696 results in systemic exposure to AHU377 (inactive prodrug of LBQ657, a neprilysin inhibitor) and valsartan (angiotensin receptor blocker). Hydrochlorothiazide (HCTZ) is indicated as first line treatment of hypertension. Since LCZ696 and HCTZ may be co-administered for optimal blood pressure control, this study was conducted to evaluate the pharmacokinetic (PK) drug-drug interaction potential between LCZ696 and HCTZ.

Methods:
An open-label, three-period, single sequence study in 27 healthy subjects was conducted. In Period 1, subjects received oral HCTZ 25 mg qd x 4 days and were discharged for a 4-10 day washout. In Period 2, subjects received LCZ696 400 mg qd x 5 days, and in Period 3, HCTZ 25 mg qd + LCZ696 400 mg qd x 4 days. Serial PK samples were collected and analyzed by a validated LC-MS/MS method. PK parameters (AUCtau,ss,Cmax,ss) of LCZ696 analytes (LBQ657, valsartan) and HCTZ in plasma were determined using non-compartmental analysis, and the results were statisticallyevaluated.

Results:
The 90% CIs confidence intervals (CIs) for the geometric mean ratio for AUCtau,ss of HCTZ fell within the ( 0.8 - 1.25) range, while those of Cmax,ss (0.74, 0.70-0.78) fell outside the range, indicating Cmax,ss of HCTZ decreased by 26% when co-administered with LCZ696. Those for AUCtau,ss of LBQ657 fell within the range but the upper bound for Cmax,ss (1.19, 1.10-1.28) was outside the range, indicating Cmax of LBQ657 increased by 19%; the upper bound for valsartan exposures(AUCtau,ss: 1.14, 1.00-1.29; Cmax,ss: 1.16, 0.98-1.37) were above the range, indicating AUCtau,ss and Cmax,ss of valsartan increased by 14%and 16%, respectively.

Conclusion:
When LCZ696 400mg qd and HCTZ 25mg qd were co- administered, AUCtau,ss of HCTZ was unchanged but Cmax,ss decreased by 26%; AUCtau,ss of LBQ657 was unchanged but Cmax,ss increased by 19%; and lastly, AUCtau,ss and Cmax,ss of valsartan increased by 14%and 16%, respectively. LCZ696 400 mg qd was safe and well tolerated in healthy subjects when administered alone and in combination with HCTZ 25 mg qd.
Abstract 463: Transregulation and Plasma Membrane Recruitment of the Dopamine-1 (D1R) and Angiotensin Type 2 (AT2R) Receptors in Human Renal Proximal Tubule Cells (RPTC)
Authors: Opper Phuong B , Gildea John J , Felder Robin A
Published: July 2021

Both the D1R and AT2R are key natriuretic receptors in that stimulation by dopamine agonists or AT2R agonist angiotensin III (AngIII) increases cAMP and cGMP, respectively, causing inhibition of sodium reabsorption. The interdependence of D1R and AT2R stimulation on receptor translocation has not been studied. We utilized normally coupled RPTC (nRPTC) and RPTC with defective D1R coupling to adenylyl cyclase (uRPTC). A 1 hr addition of the cell permeable cyclic nucleotide analogs 8Br-cGMP (1 mM) or 8Br-cAMP (1 mM) caused a similar recruitment of both D1R and AT2R, as measured by cell surface biotinylation and western blotting (see table below).

Flow cytometry confirmed these results using fluorescently labeled extracellular epitope-specific D1R and AT2R antibodies. When 8Br-cAMP and 8Br-cGMP were added together, no added effects were seen. AngIII-alone increased plasma membrane recruitment of D1R (1.23±0.04 fold, n=6, p<0.05, VEH=0.63) and AT2R (1.65±0.10 fold, n=6, p<0.05, VEH=0.69) in nRPTC but not in uRPTC. The nRPTC response was completely blocked by the AT2R specific antagonist PD123319 or the D1R antagonist LE300. Inhibition of either PKA with adenosine-3’, 5’-cyclic monophosphorothioate Rp-Isomer (100 μM) or PKG with 8-(4-chlorophenylthio) guanosine-3’, 5’ -cyclic monophosphorothioate Rp-isomer (10 μM) completely inhibited the cell surface recruitment of both D1R and AT2R when stimulated with 8Br-cAMP or 8Br-cGMP, respectively.

In conclusion, we found a novel AngIII-interdependent AT2R and D1R cell surface recruitment in human nRPTC cells. Furthermore, defects in this pathway in uRPTC can be bypassed by incubating the cells with either 8Br-cAMP or 8Br-cGMP.
Abstract 475: The Transcription Factor ETS-1: A Critical Mediator of Renal Injury in Salt Sensitive Hypertension
Authors: Feng Wenguang , Chumley Phillip , Fatima Huma , Rezonzew Gabriel , Hua Ping , Jaimes Edgar A
Published: July 2021

The transcription factor ETS-1 regulates the expression of several growth factors, chemokines and cytokines. We have recently shown that ETS-1 is a mediator of the pro-inflammatory and pro-fibrotic effects of Angiotensin II in the kidney (HTN ’12). Herein, we tested the hypothesis that the renal expression of ETS-1 is increased in the Dahl salt-sensitive (DS) rat, a paradigm of salt-sensitive hypertension in humans, and that ETS-1 mediates renal injury in this model of hypertension. DS rats (n=6 per group) were fed a normal salt diet (0.5% NaCl, NS) or a high salt diet (4% NaCl, HS) for 4 weeks. Four additional groups (n=6 per group) on HS diet received: ETS-1 dominant negative peptide (HS/DN, 10mg/kg/day) to block ETS-1, a control ETS-1 mutant peptide (HS/MU, 10mg /kg/day), the AT1 receptor blocker Candesartan (HS/ARB 10 mg/kg/day) or a combination of DN and ARB (HS/DN/ARB). HS rats had a ~ 3 fold increase in the cortical expression of ETS-1 as assessed by western blot (WB). Treatment with DN, MU, ARB or DN/ARB resulted in small and non-significant reductions in BP as compared to HS. (Table). Compared with LS, HS rats had increased proteinuria (Bio-Rad), higher glomerular injury score (GIS), increased fibronectin expression (WB) and urinary TGF-β (ELISA) that were improved by DN but not by MU (table). ARB reduced proteinuria and GIS (table). Treatment with DN/ARB resulted in further improvements in renal injury as compared to DN and ARB (table).

In conclusion we have demonstrated that hypertensive DS rats have increased cortical expression of ETS-1 and that blockade of ETS-1 improves renal injury in this model of hypertension.
Abstract 490: Redox-Sensitive Osteogenic Factors as Novel Mediators of Vascular Injury in Hypertension
Authors: Montezano Augusto C , Yusuf Hiba , Graham Delyth , Touyz Rhian M
Published: July 2021

Vascular calcification is associated with cardiovascular disease, including hypertension. Pro-osteogenic factors, such as BMP-2 and osteocalcin (OC) and anti-osteogenic factors such as osteoproteregin (OPG) (which is also pro-inflammatory through activation of RANKL and TRAIL), have been implicated in this process. In addition, reactive oxygen species (ROS) play a role in vascular calcification and hypertension-associated vascular remodeling. Here we postulated that in hypertension, vascular calcification leads to increased levels of osteogenic factors which may further promote vascular injury. Aorta and VSMCs from WKY and SHRSP (16-18 weeks) rats were studied. Vascular calcification was assessed by Von Kossa staining. VSMCs were exposed to control and calcification medium (CaM - Ca2+ 1.8 mmol/L, PO4 2.0 mmol/L) for 10 days. Osteocalcin (OC), BMP-2, BMP-7, OPG, TRAIL and fibronectin expression were assessed by immmunoblotting. ROS generation was studied by chemiluminescence. Aorta from SHRSP, but not WKY, exhibited positive Von Kossa staining. At basal levels, expression of osterix (86%), OPG (20%), TRAIL (28%) and fibronectin (51%) were increased in VSMCs from SHRSP rats. CaM, as predicted, induced an increase in OC (Ctl: 5 AU; CaM: 13 AU) and BMP-2 (Ctl: 0.60 AU; CaM: 0.75AU) (p<0.05), followed by a decrease in BMP-7 (Ctl: 1.12 AU; CaM: 0.7 AU) (p<0.05), expression in VSMCs from WKY, an effect that was inhibited by tempol (antioxidant). ROS production (Ctl: 30 AU/ug protein; CaM: 60 AU/ug protein, p<0.05) was increased by the CaM in VSMCs from WKY. Responses to CaM were augmented in VSMCs from SHRSP with respect to BMP-2 expression (p<0.05). Osteogenic regulation in SHRSP-derived VSMCs were sensitive to tempol . OPG stimulation of VSMCs from WKY and SHRSP induced ROS generation in a concentration-dependent manner (OPG 100ng/5 min: WKY - 40% increase; SHRSP - 75% increase, p<0.05 vs vehicle). In conclusion, the OPG system is upregulated in hypertension and may be related to activation of redox-sensitive pathways. Identification of vascular calcification-derived osteogenic factors as positive modulators of VSMC damage provides new insights into molecular mechanisms of vascular injury in hypertension.
Abstract 500: Reactive Oxygen Species and Insufficiency of Nitric Oxide Mediate Enhanced Microvascular Contractility and Impaired Endothelium Dependent Relaxation in Mice With Renal Failure
Authors: Wang Dan , Wang Cheng , Wilcox Christopher S
Published: July 2021

Background:
Cardiovascular disease (CVD) is common and major cause of death and disability in chronic kidney disease (CKD). Since CVD starts with endothelial dysfunction, we tested the hypothesis that reactive oxygen species (ROS) and insufficient nitric oxide (NO) contributed to microvascular contractility and endothelial dysfunction in C57BL/6 mice with normotensive reduced renal mass (RRM) 5/6 nephrectomy, a model of progressive CKD.

Methods and Results:
Mesenteric arterioles (MAs) were isolated from mice 3 months after sham-operation (Sham) or RRM (n=6/group) and were mounted on Mulvany-Halperin wire myograph, preconstricted with norepinephrine and relaxed with acetylcholine (ACh) for: endothelium-dependent relaxation (EDR); endothelium-dependent relaxation factor (EDRF; NOS-dependent relaxation); endothelium-dependent hyperpolarizing factor (EDHF; K
+
-channel dependent relaxation) and endothelium-independent relaxation (EIR; sodium nitroprusside). Contractions were tested to endothelium-dependent contracting factor (EDCF; ACh-induced contraction with blocked relaxation pathways); phenylephrine (PE); U-46,619 (thromboxane-prostanoid receptor agonist) and endothelin-1 (ET-1). NO activity (DAF-FM fluorescence) and ROS generation (tempo-9-AC fluorescence) were measured by fluorescence microscopy. Data are mean ±SEM. The MAs from RRM mice had diminished EDR (54 ±5 vs. 77±3%; P<0.01) and EDRF (13±5 vs. 27±4%; P<0.01) with reduced NO activity (0.18 ± 0.05 vs. 0.36± 0.04 ΔUnits; P<0.05), but unchanged EDHF (30±4 vs. 38±4%; NS). These vessels from RRM mice developed an EDCF (14±1 vs. 8±1%; P<0.05) and ACh-induced increased in ROS (0.17±0.03 vs. 0.06±0.02 ΔUnits; P<0.05). Contractile responses were enhanced to U-46,619 (107±4 vs. 87±6, P<0.05) and ET-1 (108±7 vs. 89±4, P<0.05), but not to PE (87±6 vs. 77±8%, NS).

Conclusion:
mice with RRM developed defective microvascular EDR, EDRF with reduced NO activity and enhanced new ACh-induced EDCF, contractilities to thromboxane and endothelin with increased ROS generation. These microvascular disturbances may contribute to the later development of thrombosis, vascular remodeling and dysfunction in patients with CKD.

*D. Wang and C.Wang: Equal contribution
Abstract 503: Dipeptidyl Peptidase-4 Regulates Proliferation of and Collagen Synthesis by Cardiac Fibroblasts
Authors: Jackson Edwin K , Gillespie Delbert G
Published: July 2021

Dipeptidyl peptidase-4 (DPP4) inhibitors, for example sitagliptin, are a new class of drugs for treatment of type 2 diabetes. Because there are multiple peptide substrates for DPP4, inhibition of DPP4 may entail risks due to augmented levels of biologically active peptides. The purpose of this study was to investigate the role of DPP4 in regulating the effects of two of its substrates, neuropeptide Y
1-36
(NPY
1-36
) and peptide YY
1-36
(PYY
1-36
) (naturally occurring Y
1
receptor agonists) on proliferation of and collagen production by cardiac fibroblasts (CFs) from spontaneously hypertensive (SHR) and Wistar-Kyoto normotensive (WKY) rats. For proliferation experiments, subconfluent cells were treated every 24 hours for 4 days with platelet-derived growth factor-BB (PDGF) and various treatments, and cells were dislodged and counted. For collagen synthesis experiments, confluent cells were treated with PDGF, tritiated-L-proline and various treatments, and after 48 hours radioactivity in cells was quantified. In both SHR and WKY CFs, both NPY
1-36
and PYY
1-36
(1 to 10 nmol/L) increased cell number and proline incorporation (index of collagen synthesis). Sitagliptin (1 μmol/L) significantly enhanced these effects. For example, in SHR CFs 10 nmol/L of NPY
1-36
increased cell number from 37,588 ± 503 to 51,990 ± 649; yet in the presence of sitagliptin, NPY
1-36
increased cell number from 37,964 ± 508 to 62,047 ± 939 (mean ± SEM, n=6; p<0.000001, p-value for interaction between sitagliptin and NPY
1-36
). In both SHR and WKY CFs and in both the absence and presence of sitagliptin, BIBP3226 (1 μmol/L; Y
1
receptor antagonist) blocked all effects of NPY
1-36
and PYY
1-36
. Because DPP4 metabolizes NPY
1-36
to NPY
3-36
and PYY
1-36
to PYY
3-36
, we also determined the effects of NPY
3-36
and PYY
3-36
on proliferation and collagen production. In both SHR and WKY CFs, these metabolites did not affect proliferation or collagen production, either in the absence or presence of sitagliptin.

Conclusion:
In cultured CFs, DPP4 inhibitors increase NPY
1-36
and PYY
1-36
induced activation of Y
1
receptors leading to increased proliferation and collagen production. Therefore, DPP4 inhibitors might increase cardiac fibrosis in patients with chronically elevated levels of NPY
1-36
or PYY
1-36
.
Abstract 507: Combined Exercise Training Induces Additional Cardiovascular Autonomic Benefits in Comparison to Dynamic Resistance Training in Ovariectomized Diabetic Rats
Authors: Sanches Iris C , Conti Filipe F , Quinteiro Hugo R , Buzin Morgana , Irigoyen Maria-Cláudia , De Angelis Kátia
Published: July 2021

The purpose of this study was to compare the cardiovascular autonomic effects between dynamic aerobic, resistance and combined exercise training in ovariectomized diabetic female rats. Female Wistar rats (200-220g) were divided into 4 ovariectomized (bilateral ovaries removal) diabetic (streptozotocin, 50mg/kg, iv) groups: sedentary (DOS) and trained by an aerobic protocol on a treadmill (DOTA), by a resistance protocol in ladder (DOTR), or by a combined protocol in ladder+treadmill on alternate days (DOTC). After 8 weeks of training, the animals were cannulated to arterial pressure (AP) recording and baroreflex sensitivity (BS) evaluation. Heart rate (HR) and systolic AP variabilities were analyzed in the time and frequency domains. Exercise training induced an increase of 77% in the run capacity in the DOTA group (DOS: 8.6±0.6; DOTA: 15.3±0.4; DOTR: 11.5±1.6; DOTC: 15.0±0.2 min), 60% in the maximum load in DOTR group (DOS: 179.7±11.0; DOTA: 167.3±1.5; DOTR: 288.4±7.5; DOTC: 284.6±10.2 % of body weight), and increases in both exercise tests in DOTC (75% and 58%, respectively). All trained animals showed normalization of diabetic induced-resting hypotension and bradycardia, associated with increased HR variance (DOS: 39.6±2.6; DOTA: 71.2±7.2; DOTR: 70.9±12.1; DOTC: 65.7±6.1 ms2) and normalization of the LF band of pulse interval (DOS: 2.7±0.3; DOTA: 5.3±0.4; DOTR: 7.2±1.5; DOTC: 6.8±0.8 ms2) (vs. DOS). The DOTA and DOTC groups (but not DOTR) had higher BS (tachycardic and bradycardic responses), and attenuation in SAP variability (DOS: 7.4±0.8; DOTA: 12.7±1.1; DOTR: 8.9±1.0; DOTC: 13.3±1.0 mmHg2) in relation to DOS. Mortality was higher in DOS group when compared to trained group. In conclusion, the results showed that dynamic aerobic, resistance or combined exercise training induced benefits in physical capacity, hemodynamic and autonomic parameters after ovarian hormone deprivation in diabetic rats. However, the combined exercise training promoted additional effects than aerobic or resistance training in this condition.
Abstract 540: Increased Intrarenal CGRP Release From Afferent Renal Nerves Is Accompanied By Decreased Afferent Electric Activity
Authors: Ditting Tilmann , Rodionova Kristina , Heinlein Sonja , Fischer Michael , Reeh Peter , Tiegs Gisa , Ott Christian , Schmieder Roland E , Freisinger Wolfgang , Veelken Roland
Published: July 2021

Afferent peptidergic renal nerves exhibit a dual function. They influence intrarenal immunological processes by releasing peptides like CGRP and control central sympathetic outflow via afferent electrical activity. The former seems to be important in renal inflammation whereas the modulation of sympathetic outflow by afferent electrical activity is not fully understood. Hence, we wanted to test the hypothesis that augmented effects of CGRP in renal inflammation occur with increased afferent renal nerve activity.

As inflammatory model, normotensive renal inflammation (RI) was induced by i.v. injection of 1.75 mg/kg BW OX-7 antibody to rats. Animals were investigated neurophysiologically and pathomorphologically using standard techniques at day 6 after induction of RI.

There was no increase in blood pressure (BP) in RI animals, hence confounding effects of BP were unlikely. RI rats exhibited albuminuria (61±6 μg/24h), infiltration of macrophages in the interstitium (26 ± 4 cells/high-power field) and glomeruli (3.7±0.6 cells/glomerular cross-section). Pretreatment with the CGRP antagonist rCGRP 8-37 (Tocris Bioscience 15nmol/kg) significantly reduced proteinuria and macrophage infiltration suggesting increased activity of CGRP in RI. In an in-vitro assay, renal tissue of rats with RI exhibited increased release of CGRP (14 ± 3 pg/ml vs. controls 6.8 ± 2.8 pg/ml; p<0.05, n=8). RI tissue from renally denervated rats showed no detectable levels of CGRP. These results suggested CGRP release from renal nerves at a higher rate in RI. Directly recorded afferent renal nerve activity (ARNA; spikes/sec) was unexpectedly lower in RI compared to controls (8.0 ± 1.8 vs. 27.4 ± 4.1 Hz*,n=6, p<0.05). On the other hand, the burst-frequency of directly recorded renal sympathetic nerve activity (RSNA) in RI was significantly higher than in controls (14.7 ± 0.9 vs. 11.5 ± 0.9 bursts/sec*; n=6, *p<0.05).

In contrast to our assumption increased release of neurogenic CGRP aggravating renal inflammation occurred with decreased ARNA. Increased RSNA pointed to a reduced tonic inhibition by ARNA. In hypertension, the later mechanism might worsen sympathetic dysregulation and renal damage.
Abstract 563: Enhanced Renal Angiotensinogen Upregulation Under High Glucose Condition is Mediated by TRPA1
Authors: Satou Ryousuke , Zsombok Andrea , Miyata Kayoko , Katsurada Akemi , Derbenev Andrei V
Published: July 2021

Clinical and basic studies have revealed that the renin-angiotensin system (RAS) plays crucial roles in the development of kidney injury during progression of type 1 diabetes mellitus (T1DM) whereas the activity of systemic RAS is suppressed in T1DM. Thus, intrarenal RAS is increasingly being recognized as an important factor in the development of diabetic nephropathy. We recently demonstrated expression of TRPA1, an oxidative stress-sensitive calcium channel, in renal proximal tubular cells (PTC) and TRPA1 mediates angiotensinogen (AGT) augmentation in the cells. However, regulation and function of the intrarenal TRPA1 in T1DM have not been elucidated. Therefore, this study was performed to evaluate intrarenal TRPA1 expression and its contribution to AGT augmentation under high glucose condition. Streptozotocin (STZ, 200 mg/kg) was intraperitoneally injected to male C57BL/6 mice. Intrarenal TRPA1 expression, kidney AGT, urinary AGT, urinary 8-isoprostane, a maker of oxidative stress, and blood glucose levels were determined one week after STZ injection. Furthermore, contribution of TRPA1 to AGT regulation was tested by using mouse PTC which was cultured with normal (1.0 g/L) or high (4.5 g/L) glucose media. Under these conditions, PTC was treated with 100 μM hydrogen peroxide. Blood glucose and urinary 8-isoprostane levels were higher in the STZ-treated mice compared with control mice. TRPA1 mRNA levels were augmented in the kidney of STZ-treated mice (2.26 ± 0.2, ratio to control mice) accompanied by increases in kidney AGT and urinary AGT levels. TPRA1 upregulation was also observed in PTC which was cultured with the high glucose medium (1.55 ± 0.11, ratio to the normal glucose). Hydrogen peroxide stimulated AGT expression in PTC. The AGT elevation was enhanced in the cultured cells with the high glucose (1.52 ± 0.13-fold in the normal glucose medium vs. 1.97 ± 0.29-fold in the high glucose medium). Treatment with a TRPA1 antagonist attenuated the enhanced AGT augmentation in PTC. These results demonstrate that intrarenal TRPA1 mediates enhanced AGT upregulation under high glucose condition and suggest that intrarenal TRPA1 is implicated in intrarenal RAS activation leading to the development of T1DM-associated diabetic nephropathy.
Abstract 57: Angiotensin-converting Enzyme 2(ACE2) Activator, Diminazene Aceturate (DIZE), Improves Endothelial Progenitor Cell (EPC) Function and Attenuates Ischemia-induced Cardiac Pathophysiology
Authors: Qi Yanfei , Cole-Jeffrey Colleen , Zhang Juan , Shenoy Vinayak , Espejo Andrew , Song Chunjuan , Hanna Mina , Katovich Michael , Raizada Mohan
Published: July 2021

Background:
ACE2 [vasoprotective axis of the renin angiotensin system (RAS)], plays a protective role in cardiovascular diseases. In addition, the number and function of EPCs is impaired in heart failure. These observations led us to hypothesize that DIZE would protect against myocardial infarction (MI) and improve EPC function.

Methods:
DIZE treatment (15 mg/kg/day, s.c.) was initiated in 8-week-old SD rats two days prior to MI surgery and continued throughout the study-period. Cardiac function was measured four weeks post-MI. The number of circulating rat EPCs (CD4
-
/CD5
-
/CD8
-
/CD90
+
) was quantified via flow cytometry. EPCs were isolated from spleen mononuclear cells and function was assessed by ability to migrate towards SDF-1.

Results:
MI rats showed a 58% decrease in fractional shortening (FS,%) [control (C): 54.6 ± 3.0; DIZE alone (D) : 52.5± 4.8; MI: 22.5± 3.7], a 50% decrease in contractility (dP/dt
max
mmHg/s) (C: 9480 ± 425.3; D: 9585 ± 597.4; MI: 4716 ± 724.9), and a 27% increase in ventricular hypertrophy (VH, mg/mm) (C: 26.5 ± 1.5; D: 26.9 ± 1.4; MI: 33.4± 1.1). This was associated with 40% decrease in cardiac ACE2 activity (C: 246.2 ± 25.1; D: 254.2 ± 20.6; MI: 148.9 ± 29.2, RFU/min). There was a 23% decrease in the number of circulating EPCs (C: 4.3 ± 0.1%; D: 4.2 ± 0.2%; MI: 3.3 ± 0.1%) and 12% decrease in their migratory ability (MI: 20.0%). DIZE resulted in a 2.5 fold increase in ACE2 activity in MI heart, attenuated the MI-induced decrease in FS by 49%, improved dP/dt
max
by 49%, and reversed VH by 21%. In addition, DIZE increased the number of EPCs by 21% and improved their migratory ability by 9% in the MI rats. Furthermore, immunostaining for Islet-1, a marker for cardiac progenitor cells (CPCs) demonstrated that DIZE increased CPC number and decreased infiltration of macrophages in the peri-infarct area of the ischemic heart. All these beneficial effects were abolished by C-16, an ACE2 inhibitor.

Conclusions:
Collectively, our observations demonstrate that activation of endogenous ACE2 improves cardiac function and attenuates LV remodeling post-MI, increases EPC number and function, increases engraftment of CPCs and decreases inflammatory cells in peri-infarct cardiac regions. Thus, DIZE may represent a promising therapeutic strategy for treating MI.
Abstract 58: AT2 Receptor Agonism Regulates TIMP1/MMP9 Axis in the Heart Preventing Cardiac Fibrosis and Improving Heart Function After Experimental Myocardial Infarction
Authors: Lauer Dilyara , Slavic Svetlana , Sommerfeld Manuela , Thöne-Reineke Christa , Sharkovska Yuliya , Hallberg Anders , Dahlöf Bjorn , Kintscher Ulrich , Unger Thomas , Steckelings Ulrike Muscha , Kaschina Elena , Kaschina Elena
Published: July 2021

Aims:
A selective nonpeptide agonist for the angiotensin AT2 receptor compound 21 (C21) improved cardiac functions 7 days after myocardial infarction (MI). Here, we aimed to investigate what are the cellular mechanisms underlying cardiac protection in the late stage after MI.

Methods and Results:
MI was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with C21 (0.03mg/kg i.p. daily) started 6h after MI and continued for 6 weeks. Hemodynamic parameters were measured via transthoracic Doppler echocardiography and intracardiac Samba catheter. The expression of MMP9, TIMP1, TGF-β1 and collagen content were determined in left ventricle. Anti-proteolytic effects were additionally studied in primary cardiac fibroblasts.

C21 significantly improved systolic and diastolic function 6 weeks after MI in comparison with the vehicle group as shown by ejection fraction (71.2±4.7 % vs. 53.4±7.0%; p<0.001), fractional shortening (40.8±2.3% vs. 30.9±3.1%; p<0.05), LVIDs (4.4±0.5mm vs. 5.2±0.8mm; p<0.05), LV EDP (16.9±1.2mmHg vs. 22.1±1.4mmHg; p<0.05), E/A ratio, dP/dt
max
and dP/dt
min
(p<0.05). Moreover, C21 improved arterial stiffness parameter (AIx) (18±1.2% vs. 25%±1.8, p<0.05) and reduced collagen content (15%; p<0.05) in postinfarcted myocardium. TIMP1 protein expression in the left ventricle was strongly up-regulated (17.7-fold; p<0.05) whereas MMP9 and TGF-β1 were significantly down-regulated (1.5-fold, p<0.05; 3.4-fold p<0.001, respectively) in the treated group. In cardiac fibroblasts, C21 primarily induced TIMP1 expression followed by attenuated MMP9 secretion and TGF-β1 down-regulation.

Conclusion:
C21 improves heart function in the late stage after MI and prevents cardiac remodeling. Activation of TIMP1 and subsequent inhibition of MMP9-mediated proteolysis as well as down-regulation of TGF-β1 followed by decreased collagen accumulation may attenuate disintegration of the extracellular matrix and reduce fibrosis.
Abstract 60: Renal Denervation Prevents Dendritic Cell Activation and Renal T Cell Activation in Mice During Angiotensin Ii-induced Hypertension
Authors: Xiao Liang , Kirabo Annet , Harrison David G
Published: July 2021

Increased sympathetic outflow has been implicated in the pathogenesis of hypertension. Recent evidence has also suggested a role of T lymphocytes in hypertension. Renal sympathectomy is an effective approach to lower blood pressure in hypertensive patients and animal models. We hypothesized that renal sympathetics link the central nervous system to immune activation in hypertension. To test this hypothesis, we performed either unilateral or bilateral renal denervation (DNX) in C57BL/6 mice by applying phenol to the renal artery and cutting large visible nerves. On the same day mice received an osmotic minipump for angiotensin II (ANG) infusion (490 ng/kg/min, for 14 days). DNX decreased renal norepinephrine content from 162±13 to 42±11 ng/g (p=0.005) as measured by HPLC. Bilateral DNX lowered systolic blood pressure in mice with ANG infusion (137±4 vs. 159±5 mmHg, p=0.003) measured with tail-cuff. Analysis of T cells was performed using flow cytometry in single cell suspensions from kidneys. Angiotensin II significantly increased total leukocyte (CD45+) and T cell (CD3+) infiltration in the kidney, and this was prevented by DNX. Dendritic cells (DCs), which are the major antigen presenting cells that activate T cells, were isolated from the spleen and cultured in RPMI medium for 24 hours for cytokine measurements via Luminex100 system. Angiotensin II infusion increased DC production in IL-1α, IL-1β, and IL-6 production by 4 to 6-fold, and these increases were prevented by DNX. These results suggest that renal sympathetic nerves participate in T cell activation, and this is associated with dendritic cell activation in the spleen.
Abstract 616: The Population Impact and Cost-Effectiveness of Blood Pressure Treatment in U.S. Adults Age 75 and Older
Authors: Odden Michelle C , Coxson Pamela , Moran Andrew , Guzman David , Goldman Lee , Bibbins-Domingo Kirsten
Published: July 2021

Introduction:
In the next 30 years, the population of adults 75 years and older will more than double in the U.S., and high blood pressure is the most prevalent cardiovascular risk factor in this population. The impact of strategies for blood pressure control has not been evaluated in this population with special consideration of geriatric conditions that may alter the cost-effectiveness.

Methods:
Based on the Cardiovascular Disease Policy Model, a Markov model of the U.S. population, we forecasted the population impact of blood pressure treatment over 10 years in adults aged 75-94 years, using the health care system perspective, and assuming an annual discount rate of 3%. Based on epidemiologic and trial data, we projected the impact of four potential negative events on cost-effectiveness in this geriatric population: 1) mild side effects, 2) polypharmacy (use of 5+ medications) associated cognitive impairment, 3) increased risk of falls/fracture, and 4) attenuated effectiveness of therapy in frail older adults

Results:
We project that treatment to a systolic blood pressure target of 160 mmHg would result in the prevention of 65,000 cases of incident coronary artery disease, and 54,000 cases of incident ischemic stroke in adults 74-95 years. Based on a systolic blood pressure target of 140 mmHg, 191,000 cases of incident coronary artery disease, and 141,000 cases of incident ischemic stroke would be avoided. This would result in 729,000 additional years of life and 992,000 quality-adjusted life years (QALYs). The total health care costs of treating 75-94 year olds to a systolic blood pressure target of 140 mmHg would be $40.5 billion in the U.S., and the cost per QALY would be $40,800. The cost-effectiveness of treatment to a target of 140 mmHg would be worse in the presence of side effects ($ 51,000/QALY), polypharmacy ($ 58,400), fractures ($ 48,400), or frailty ($134,300).

Conclusions:
Treatment of systolic blood pressure to a target of 140 mmHg would prevent the most cardiovascular events and result in the greatest QALYs gained. The presence of side effects, polypharmacy-related cognitive impairment, fractures, or frailty could substantially offset this benefit. Treatment strategies that are tailored to the health status of older adults are warranted.
Abstract 620: Angiotensin AT2 Receptor Exerts an Anti-inflammatory Response in Lipopolysaccharide-activated THP-1 Macrophages
Authors: Dhande Isha S , Hussain Tahir
Published: July 2021

Macrophages have been shown to be an important contributor to the pathogenesis of hypertension and stroke. The angiotensin AT2 receptor (AT2R), which is expressed in macrophages, is known to promote vasodialation, natriuresis and lower inflammation. The goal of the present study was to explore the anti-inflammatory role of AT2R stimulation in human macrophage-like THP-1 cells activated by lipopolysaccharide (LPS). Phorbol 12-myristate 13-acetate (PMA) differentiated macrophage-like THP-1 cells were treated with AT2R agonist C21 (1 μmol/L) for 30 minutes prior to activation with LPS (1 μg/ml). Media and cells were collected after 24 hours and were analyzed for levels of pro- and anti-inflammatory cytokines and proteins. Pre-treatment with C21 resulted in a 4-fold increase (104.8±6.1 vs 406.7±52.3) in anti-inflammatory interleukin-10 (IL-10) production and a 5-fold decrease (3560±237 vs 588.8±15.94) in pro-inflammatory tumor necrosis factor-α (TNF-α) levels in the media in response to LPS. Predictably, LPS resulted in a 6-fold up-regulation of iNOS expression which was prevented with C21 pre-treatment. A modest decrease in the anti-inflammatory macrophage mannose receptor C type 2 (MRC2) expression was detected with LPS treatment. AT2R agonist pre-treatment, however, increased this receptor expression by ~70% after LPS activation. C21 alone also resulted in a 20% increase in MRC2 expression compared to untreated controls. The anti-inflammatory effect of AT2R activation was abolished in the presence of neutralizing IL-10 antibody (1 μg/ml), indicating a central role for IL-10 in mediating the beneficial response to C21 in LPS activated macrophages. Further, inhibition of nitric oxide (NO) by L-NAME prior to C21 pre-treatment also prevented the decrease in TNF-α and increase in IL-10 in response to AT2R agonist, which suggests that the anti-inflammatory response to C21 may be mediated via increase in NO production prior to LPS activation of macrophages. In conclusion, AT2R stimulation may potentially suppress the inflammatory response of macrophages to LPS by shifting the balance from pro- to anti-inflammatory cytokine production and may prove to be beneficial in the control of the inflammatory component of stroke and hypertension.
Abstract 622: Role of Endothelial Nitric Oxide Synthase in a Capsaicin Diet-Induced Alleviation of Blood Pressure-Elevation in 2-Kidney, 1-Clip Hypertensive Rats
Authors: Segawa Yukiko , Hashimoto Hiroko , Osera Tomoko , Kurihara Nobutaka
Published: July 2021

Objective:
Capsaicin directly acts on the sensory nerve ending and releases neuropeptides, which activate a vasodilator, nitric oxide (NO) and decrease blood pressure (BP). We demonstrated that BP elevation was alleviated by consuming a low concentration of capsaicin diet in 2-kidney, 1-clip (2K1C) hypertensive rats. We also observed that when 2K1C rats took the NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester, during the protocol, the effect of alleviating hypertension by dietary capsaicin was diminished. Thus, we hypothesized that NO is involved in the effect of capsaicin in 2K1C rats. In this study, to test the hypothesis, we observed endothelial NOS (eNOS) expression in 2K1C rats fed a diet containing capsaicin.

Methods:
Six-week old male Sprague-Dawley rats were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). One week after the surgery, each group of rats were further divided into 2 groups randomly, which received either a control diet (CTL) or a diet containing 0.006% capsaicin (CAP) for 6 weeks. The systolic BP (SBP) was measured by a tail-cuff method once per week throughout the protocol. At the end of the protocol, MAP was measured in each rat under anesthesia. After euthanasia, the thoracic aortas were collected for extracting mRNA. Then, eNOS mRNA expression in aorta was evaluated in each group of rats by reverse transcriptase PCR.

Results and Discussion:
The present data of BP confirmed that capsaicin diet alleviates BP elevation in 2K1C rats. At six weeks after the surgery, SBP in 2K1C-CTL rats (183 ± 6 mmHg) was significantly higher than SHAM-CTL (124 ± 6 mmHg, p < 0.05), but SBP in 2K1C-CAP (140 ± 4 mmHg) was significantly lower than that in 2K1C-CTL. SHAM-CAP (129 ± 8 mmHg) did not show a significant difference in SBP with SHAM-CTL. MAP at the end of the protocol showed the same tendency as SBP. Moreover, eNOS mRNA expression was enhanced in 2K1C-CTL (0.39 ± 0.06) compared to SHAM-CTL (0.31 ± 0.04, p < 0.05). Although it was not enhanced in SHAM-CAP (0.30 ± 0.01) compared to SHAM-CTL, it was tended to be enhanced in 2K1C-CAP (0.43 ± 0.02) compared to 2K1C-CTL. These data suggested that dietary capsaicin decreased BP with elevation of eNOS mRNA expression in 2K1C rats, but did not change eNOS mRNA nor BP in SHAM normotensive rats.
Abstract 63: ACE2 Deficiency Reduces Insulin Content of Isolated Pancreatic Islets and Plasma Insulin Concentrations Post-Glucose Challenge in Obese C57BL/6 Mice
Authors: Shoemaker Robin C , Cassis Lisa A
Published: July 2021

Objective:
Diet-induced obesity promotes type 2 diabetes (T2D). Drugs that inhibit the renin-angiotensin system (RAS) have been demonstrated in clinical trials to decrease the onset of T2D. Angiotensin converting enzyme 2 (ACE2) negatively regulates the RAS by catabolizing angiotensin II (AngII). Preliminary data indicate that ACE2 deficient mice display impairments in glucose homeostasis at 8 weeks of age. We tested the hypothesis that ACE2 deficiency promotes the development of glucose intolerance and β-cell dysfunction in mice with diet-induced obesity.

Methods and Results:
Male

Ace2
+/y

or

-/y

mice were fed a low fat (LF, 10% kcal as fat) or high fat (HF, 60% kcal as fat) diet for 5 or 17 weeks. After 5 weeks, plasma insulin concentrations (0, 30 min) following a glucose challenge were significantly greater in HF
versus
(
vs)
LF-fed mice. However, glucose-stimulated increases in plasma insulin concentrations were decreased in HF-fed ACE2 deficient mice compared to controls (2.96 ± 0.18
vs
4.44 ± 0.40 ng/ul, respectively; P<0.01). Surprisingly, isolated pancreatic islets from HF-fed mice of either genotype released similar concentrations of insulin in response to glucose. However, mRNA abundance of insulin was significantly reduced in islets from HF-fed

Ace2
-/y

compared to

+/y

mice (1.76 ± 0.17
vs
2.54 ± 0.18 insulin/18S ratio; P<0.05). After 17 weeks, the plasma insulin response to glucose was further reduced in the HF-fed ACE2 deficient mice compared to controls (8.07 ± 0.98
vs
13.90 ± 1.10 ng/ul; P<0.01). Further, LF-fed ACE2 deficient mice also displayed reductions in plasma glucose-stimulated insulin concentrations (1.92 ± 0.98
vs
3.09 ± 0.98 ng/ul; P<0.01). Islets from HF-fed wild type mice displayed reduced ACE2 gene expression compared to LF (0.069 ± 0.009
vs
0.169 ± 0.01, ACE2/18S ratio; P<0.001) and AngII totally suppressed islet glucose-stimulated insulin secretion compared to vehicle (-0.16 ± 0.18
vs
0.9 ± 0.26, fold change over basal; P<0.05).

Conclusions:
These results demonstrate that ACE2 deficiency promotes the development of T2D by regulating islet insulin content. Moreover, diet-induced obesity reduces islet ACE2 gene expression with augmented AngII-induced impairment of insulin secretion.
Abstract 79: Caveolin-1 Knockout Mice Have Salt-Sensitive Hypertension and Dopamine-1 Receptor Defects in Renal Cortex and Isolated Renal Proximal Tubule Cells
Authors: Gildea John J , Howell Nancy L , Van Sciver Robert E , Kemp Brandon A , Carey Robert M , Felder Robin A
Published: July 2021

Dopamine-1 receptors (D1R) are necessary for kidney proximal tubule-dependent natriuresis and maintenance of normal blood pressure, especially under high salt conditions. G-protein coupled receptor kinase 4 (GRK4) is a negative regulator of D1R function and single nucleotide polymorphisms in GRK4 have been associated with both hypertension and salt sensitivity in humans. Caveolin-1 (CAV1) directly binds to GRK4 and decreases kinase activity. We hypothesized that CAV1 knockout mice (CAV1KO) would have increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the D1R. Mean arterial blood pressure (MAP, in mmHg ±SEM) measured over 5 days was not significantly different for Wild-type mice (WT, 128.9±4.2 mmHg, n=4) vs CAV1KO (129.5±3.5 mmHg, n=4) on normal chow (0.3% sodium). However, on a 4% high sodium diet, the MAP of CAV1KO mice increased in just 2 days by 20.1±4.2 mmHg (p<0.05 vs either Day 0 CAV1KO or Day 2 WT, n=4). The CAV1KO MAP increased by 25.9±6.6 mmHg by day 7 (p<0.05 vs either Day 0 CAV1KO or Day 7 WT, n=4). Hyperphosphorylation and inactivation of the D1R in renal cortex was examined by looking at phospho-serine D1R by immuno-precipitation and Western dot blotting. A 92.5% ± 18.8 SEM increase in phospho-D1R was found in the CAV1KO renal cortex (n=4, p<0.01 vs WT; 14,574/7570 RFU). Cortical slices were made and incubated for 30 minutes with fenoldopam (FEN, 10 μM) with or without LE300 (D1R-like antagonist, 10 μM) or vehicle (VEH). Cyclic AMP was measured by TR-FRET (Lance, Perkin Elmer). FEN significantly increased cAMP 5.6 fold ± 1.2 SEM (n=4, p<0.01 vs VEH; 7.84/1.4 pmole/mg protein) in WT but not in CAV1KO slices, and this effect was completely blocked by LE300. Primary mouse CAV1KO and WT renal proximal tubule cell lines were established and monensin (sodium ionophore, 5 μM, 30 minutes)-induced plasma membrane D1R recruitment increased as measured by confocal microscopy in WT (30.4% ± 7.4 SEM, n=11, p<0.01 vs VEH; 8990/6894 RFU) but not in CAV1KO proximal tubule cells. In summary, CAV1 is necessary in high salt conditions for maintaining normal blood pressure in mice and for preserving normal D1R function in kidney cortex and in mouse renal proximal tubule cells.
Abstract 80: Abnormal Water Balance in Salt-Sensitivity of Blood Pressure
Authors: Laffer Cheryl L , Scott Robert C , Titze Jens M , Elijovich Fernando
Published: July 2021

We studied body weight (BW), serum Na, osmolality (Osm), urine sodium excretion (UNaV) and sodium balance (NaBal) responses to a 24-hr 460 mEq sodium load in 53 subjects (32 hypertensive HTN, 21 normotensive NTN) classified as salt-resistant (SR) or salt-sensitive (SS) with an in-patient protocol. Cutoff for SS was a fall in mean daytime systolic BP (Spacelabs 90207) of ≥10 mmHg from high- to low-salt (10 mEq Na diet plus furosemide 40 mg x 3). UNaV (SR 362±22 vs SS 353±21 mEq/day) and NaBal (SR 98±22 vs SS 107±21 mEq/day) did not differ between groups. In contrast, SR had non-significant BW loss (-0.2±0.2 kg) whereas SS gained BW (0.8±0.2, p<0.001), suggesting different water balances; supported by a differential trend of changes in Na between SR (0.6±0.4 mEq) and SS (-0.3±0.5, p~0.07) and lack of change of Osm in SR (0.04±0.86 mOsm) versus a significant reduction in SS (-1.70±0.87, p<0.03). These observations were similar in HTN and NTN. We also measured hemodynamics (echocardiogram) in NTN during the high- and low-salt days. On low-salt, cardiac output (CO, 3.7±0.2 vs 4.0±0.5 L/min), mean arterial pressure (MAP, 85±2 vs 83±3 mmHg), and total peripheral resistance (TPR, 1896±92 vs 1767±163 dyn*cm
-5
*sec
-1
), were not different between SR and SS. On high-salt, SR significantly reduced TPR (-347±84 dyn*cm
-5
*sec
-1
; p<0.001) and maintained MAP (Δ -1±3 mmHg), whereas SS failed to vasodilate (ΔTPR +24.3±97.4, ns) and increased MAP (Δ 14±3; p<0.001). Atrial filling pressures increased equally with high-salt in both groups. In contrast, left ventricular (LV) filling pressures (E/e’ ratios) were higher in SS than in SR on both days (low-salt 9.0±0.9 vs 5.4±0.4, p<0.005, and high-salt 9.8±0.7 vs 7.7±0.3, p<0.03). Our data suggest that: 1. SR are able to store Na without concomitant water in an undefined compartment whereas this storage is impaired in SS; 2. water retention by SS is not intravascular because despite small effects on Na and Osm, their cardiac output response to salt was no greater than that in SR; and 3. inability of SS to vasodilate in response to salt and increased LV filling pressures regardless of salt-balance suggest a role for abnormal water metabolism in vascular smooth muscle and cardiomyocyte dysfunction, the former probably mediating pressure natriuresis.
Abstract 88: Single Nucleotide Polymorphisms of the Dopamine D2 Receptor Increase Inflammation and Fibrosis in Human Renal Proximal Tubule Cells
Authors: Jiang Xiaoliang , Konkalmatt Prasad , Gildea John , Jones John , Felder Robin A , Jose Pedro A , Armando Ines
Published: July 2021

We have reported that lack or downregulation of dopamine D(2) receptor (D
2
R) function in mice increases the vulnerability to renal inflammation independent of blood pressure and that the D
2
R negatively regulates inflammation and prevents injury in mouse renal proximal tubule cells (RPTCs). Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277 and 1800497) in the human
DRD2
gene are associated with decreased D
2
R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs expressing these SNPs have increased expression of inflammatory markers and injury. We studied immortalized human RPTCs isolated from normal tissue of nephrectomies which were genotyped for the D
2
R SNPs; 10 cell lines were selected: wild type (WT) n=5; heterozygous for both rs6276 and 6277 n=3; heterozygous for both rs6276 and 1800497 n=2. All 5 cell lines expressing SNPs (hRPTC-SNP) had decreased D
2
R mRNA (0.52±0.03 vs. 1±0.02 (WT) fold; P<0.02 ) and protein (62±4 vs. 100±6%; P<0.04) and were pooled together for analysis. D
2
R function was also decreased as determined by the decreased ability of a D
2
R agonist to increase PMA-induced cAMP accumulation (hRPTC-SNP: 201±16; hRPTC-WT 270 ±5 %; n=4; P<0.05). The expression of the pro-inflammatory cytokines TNFα (2.5 ± 0.3 fold; P<0.04) and CD40L (2.3 ± 0.5 fold; P<0.05), as well as the inflammation marker CRP (1.5 ± 0.2 fold; P<0.04) was increased in hRPTC-SNP. The expression of the pro-fibrotic TGFβ1 (1.8 ± 0.4 fold; P<0.04) and its signaling targets PAI-1 (6.3± 0.5 fold; P<0.01), SMAD3 (2.8± 0.4 fold; P<0.02) and VEGFa (2.3± 0.6 fold; P<0.04) were also increased. Furthermore hRPTC-SNP showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins as shown by increased fibronectin -1 (3.1± 0.7 fold; P<0.03), col 1a (1.6± 0.2 fold; P<0.05), col 3a (2.2± 0.5 fold; P<0.05), MMP3 (5.6± 0.8 fold; P<0.04), and MMP9 (3.5± 0.5 fold; P<0.04). These data support the hypothesis that D
2
R function has protective effects in human RPTCs and suggest that carriers of these SNPs may be prone to chronic renal disease and high blood pressure.