bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP46: Knockout Of Add3 Impairs Autoregulation Of Renal Blood Flow And Promotes Hypertension-induced Renal Injury By Increasing Glomerular Capillary Pressure And Podocyte Loss
Authors: Fan Letao , Gao Wenjun , Liu Yedan , Nguyen Bond V , Jefferson Joshua R , Fan Fan , Roman Richard J
Published: September 2020

Recently, we reported that a mutation in γ-Adducin (ADD3) alters the actin cytoskeleton and is associated with an impaired myogenic response of the afferent arteriole and enhanced hypertension-induced renal disease. However, it remains to be determined whether the loss of ADD3 function promotes renal injury by increasing glomerular capillary pressure (Pgc) and podocyte loss or other mechanisms. The present study compared the time course of changes in renal hemodynamics and the progression of renal injury during the development of DOCA-salt hypertension in FHH 1
BN
rats (WT) with an intact myogenic response vs. FHH 1
BN
Add3KO rats (Add3KO) in which the myogenic response is impaired. When transmural pressure rose from 40 to 100 mmHg, the inner diameter of the preglomerular artery constricted by 19% (47.7 ± 4.3 to 38.4 ± 3.4 μm, n = 5) in WT, but it dilated by 28% (53.0 ± 2.2 to 67.9 ± 4.3 μm, n = 7) in Add3KO. Pgc was similar (50.1 ± 0.4 vs. 51.2 ± 0.8 mmHg, n = 6) at 100 mmHg, but rose by 6 and 14 mmHg in WT vs. Add3KO when perfusion pressure rose to 150 mmHg. Mean arterial pressure increased similarly and reached 177.7 ± 3.5 vs. 182.6 ± 2.3 mmHg (n = 9) after 3 weeks of DOCA-salt hypertension in WT vs. Add3KO. After 1 week of DOCA-salt hypertension, glomerular filtration rate (GFR) increased by 38% (1.2 ± 0.1 to 1.6 ± 0.1 ml/min/kidney, n = 6) and glomerular nephrin expression decreased by 20% (165.4 ± 4.5 to 131.614 ± 5.2 RFU, n = 7) in Add3KO. Both were unaltered in WT. Proteinuria increased 2 folds in WT (56.9 ± 4.7 to 168.6 ± 26.7 mg/day, n = 12) in the first week of hypertension vs. a 6-fold increase in Add3KO (64.6 ± 4.1 to 446.0 ± 41.9 mg/day, n = 9). After 3 weeks of hypertension, the degree of glomerulosclerosis (3.4 ± 0.1 vs. 2.4 ± 0.1 glomerular injury score, n = 9~12), protein cast formation (9.0% ± 0.8% vs. 4.8% ± 0.4% of area, n = 6), epithelial-mesenchymal transition, interstitial fibrosis (17.9% ± 0.8% vs. 9.5% ± 0.3% of area, n = 6), and inflammation was significantly greater in Add3KO vs. WT. GFR and Pgc were 28% and 19% lower in Add3KO than WT. These results indicate that the impaired myogenic response increases the transmission of pressure to the glomerulus to induce the loss of podocytes, which accelerates the progression of renal injury during the development of hypertension in Add3KO.
Abstract MP49: Central Angiotensin II Mediates Neurogenic Hypertension Through Hif1-Alpha/NMDAR Axis In The Paraventricular Nucleus Of The Hypothalamus (PVN)
Authors: Sharma Neeru , Haibara Andrea S , Katsurada Kenichi , Nandi Shyam S , Patel Kaushik P
Published: September 2020

Central activation of the renin-angiotensin system and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of hypertension. The specific neural substrates and the possible molecular mechanisms for the sympathoexcitation remain unclear. The glutamatergic activation via N-methyl-d-aspartate receptors (NMDAR) in the PVN enhances sympathetic outflow in hypertension. Previously, using Chromatin immunoprecipitation assay (ChiP) we have shown that Hypoxia-Inducible Factor-1 (heterodimer of HIF-1
α
and HIF-1
β subunits
) binds to HREs elements in the NMDA-NR1(structural subunit of NMDAR) promoter in nucleus and increases transcription. In this study, central infusion of Ang II (20 ng/min, 14days, 0.5μl/h, i.c.v) through osmotic mini-pumps in Sprague-Dawley rats increased renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max) and mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg). At the same time, there was an increased expression of HIF-1α mRNA (2.79 fold) and HIF-1α protein (1.54 fold), as well as the expression of NMDA-NR1 mRNA (3.17 fold) and NMDA-NR1 protein levels (1.52 fold) in the PVN of Ang II infused group. Direct application of Ang II (1μM) induced an increase in the expression of HIF-1α protein (0.74 ± 0.03* vs. 0.50 ± 0.04) as well as decreased expression of prolyl hydroxylase domain protein 2 (an enzyme hydroxylating the proline residues of HIF-α, inducing subsequent ubiquitination and proteasomal degradation) (1.47 ± 0.14* vs. 0.50 ± 0.03) in NG108-15 neuronal cells. Tandem Ubiquitin-Binding Entities assay showed decreased HIF-α -Ub conjugates in Ang II-treated cells (0.65 ± 0.09* vs. 1.31 ± 0.10) suggesting a role for Ang II in post-translational stabilization of HIF-α. Further, silencing of HIF-1α (~60%) with siRNA in NG108-15 cells leads to a decrease in the expression of NMDA-NR1 induced by Ang II, compared with scrambled siRNA (0.47 ± 0.05* vs 0.80 ± 0.04). Taken together, these studies suggest that elevated central Ang II upregulates the expression of HIF-1α at transcriptional as well as post-translational level, which potentiates glutamatergic tone by enhanced expression of NMDA-NR1 in the PVN leading to heightened glutamatergic tone with a consequent increase in sympathetic outflow resulting in hypertension.
Abstract P002: Soluble (pro)renin Receptor Contributes To Angiotensin II-Induced Hypertension In Mice
Authors: Feng Ye , Wang Fei , Peng Kexin , Chen Yanting , Luo Renfei , Zou Chang-jiang , Yang Tianxin
Published: September 2020

Activation of (pro)renin receptor (PRR) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during angiotensin II (AngII) infusion. Soluble PRR (sPRR), the extracellular domain of PRR, is generated by multiple proteases including furin or ADAM19, and recently site-1 protease (S1P). The goal of the present study was to test the role of S1P-derived sPRR mediated AngII-induced hypertension. F1 B6129SF1/J mice were infused for 6 days with control (CTR) or AngII at 300 ng/kg/day alone or in combination with S1P inhibitor PF-429242 (PF) and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated AngII-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histadine-tagged sPRR termed as sPRR-His. Mutagenesis of overlapping recognition site for S1P and furin in PRR for was generated in mice by CRISPR strategy (termed as mutant mice). Mutant mice were fertile and developed normally with a 50% reduction plasma sPRR. These mice exhibited blunted hypertensive response to AngII infusion accompanied with suppressed intrarenal renin levels. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mpkCCD cells exposed for 24 h to AngII, AngII + PF, or AngII + PF + sPRR-His. AngII-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that sPRR derived from S1P or in combination with furin mediates AngII-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.
Abstract P007: Overexpression Of Mitochondrial Transcription Factor A Attenuates Mitochondrial Damage And Hypertension-induced Aged Kidney Injury
Authors: Pushpakumar Sathnur , Juin Subir Kumar , Sen Utpal
Published: September 2020

Aging-induced changes in the kidney are associated with reduction of mitochondria and increased mitochondrial abnormalities. The presence of hypertension in the aging population has additional deleterious effects that alters redox balance and impairs mitochondrial function. Mitochondrial transcription factor A (TFAM) is an important regulator of mitochondrial genes that play crucial roles in maintaining mitochondrial stability and replication. In the kidney, the expression of TFAM decreases with aging to promote sclerosis; however, its role in combination with hypertension has not been studied. The purpose of the study was to investigate whether overexpression of TFAM in the aging mice mitigates hypertension-induced kidney damage. We treated 68-70 wk old, male C57BL/6J (WT) and transgenic mice overexpressing TFAM (Tg, based on C57 background) with Angiotensin-II (1000 ng/kg/min) for 4-week period. Tg mice had lower blood pressure, increased mitochondrial copy number, decreased mitochondrial lipid peroxidation, and better renal function compared to WT groups. The changes were associated with decreased inflammation, reduced wnt signaling and pro-fibrotic markers. Taken together, our results suggest that overexpression of TFAM and reduction of wnt activation reduces hypertension induced renal injury in aged mice by modulating mitochondrial bioenergetics and maintaining homeostasis between inflammation and fibrosis.
Abstract P013: Role Of Kir4.1 (
Kcnj10
) In The Regulation Of Salt-Induced Hypertension
Authors: Manis Anna D , Levchenko Vladislav , Hodges Matthew R , Palygin Oleg , Staruschenko Alexander
Published: September 2020

In the kidney, K
ir
4.1 (
Kcnj10
) and K
ir
5.1 (
Kcnj16
) are highly expressed in the aldosterone sensitive distal nephron, a major target for hormones controlling blood pressure. These basolateral inwardly rectifying potassium channel subunits assemble to form both heteromeric K
ir
4.1/K
ir
5.1 and homomeric K
ir
4.1 channels which control the transepithelial voltage, fine-tune renal electrolyte homeostasis, and contribute to long-term blood pressure control. To study the role of K
ir
4.1 in the renal control of blood pressure, renin-angiotensin-aldosterone system (RAAS) balance, and salt-sensitive hypertension, we created a
Kcnj10
knockout (SS
Kcnj10-/-
) on the Dahl SS rat background using CRISPR/Cas9 mutagenesis. Homozygous SS
Kcnj10-/-
rats were hypokalemic, had reduced heart, kidney, and body weights, and did not survive more than 3 weeks from birth. Mass spectrometry-based quantification of RAAS peptides revealed increased Ang I, Ang 1-5, and Ang 1-7 levels in SS
Kcnj10-/-
rats compared to age-matched SS
Kcnj10+/+
controls. There were no differences in Ang II, Ang III, Ang IV, or aldosterone levels. In addition, ACE activity was decreased in SS
Kcnj10-/-
rats. The observed increases in alternative RAS axis peptides and decrease in ACE activity in SS
Kcnj10-/-
rats are suggestive of a low blood pressure phenotype, however, limited survival precluded blood pressure measurement. Heterozygous (SS
Kcnj10+/-
) rats developed normally and showed no changes in serum electrolytes. Survival rate of SS
Kcnj10+/-
rats was lower than wild-type rats but improved with dietary K
+
supplementation (1.41% K
+
) allowing for telemetric blood pressure measurement. We found no differences in baseline blood pressure under control conditions, but salt-induced (4% NaCl) elevations in blood pressure were significantly attenuated in SS
Kcnj10+/-
rats. After 3 weeks on high salt, SS
Kcnj10+/-
rats showed decreased diuresis and improved albuminuria compared to wildtype. Our results demonstrate that renal K
ir
4.1 containing channels mediate salt-sensitive increases in blood pressure through regulation of potassium homeostasis, modulation of alternative RAS axis hormones, and control of renal salt handling in the ASDN.
Abstract P020: LOSS OF SOLUBLE (PRO)RENIN RECEPTOR ATTENUATES DOCA-SALT HYPERTENSION
Authors: Ramkumar Nirupama , Stuart Deborah , Peterson Caitlin S , Wheatley Will , Kohan Donald E
Published: September 2020

Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR), which maybe involved in mediating hypertension. We recently developed a novel mouse model with mutation in the cleavage site of the PRR using CRISPR/Cas9 such that sPRR is not generated and showed that absence of sPRR attenuated angiotensin-II induced hypertension and kidney damage. In this study, we examined if sPRR alters blood pressure (BP) in angiotensin-II independent hypertension using deoxycorticosterone acetate (DOCA)-salt treatment. Mutant sPRR mice and littermate controls were treated with DOCA (50 mg/kg) and high Na
+
diet for 3 weeks. BP was monitored by radio-telemetry and metabolic balance studies performed at the end of the study (Day 17-18). Compared to controls, male mutant sPRR mice had markedly lower plasma sPRR levels (control: 21.5 ± 2.5 vs mutant 0.2 ± 0.03 ng/ml) and baseline BP (systolic control: 122 ± 3 vs mutant 114 ± 3; diastolic control: 94 ±5 vs mutant 82 ± 3 mm Hg). BP remained low in mutant sPRR mice relative to controls following 12 days of DOCA-salt treatment (systolic control: 141 ± 2 vs mutant 132 ± 5; diastolic control: 110 ± 4 vs mutant 95 ± 5 mm Hg). Mutant sPRR mice had lower body weight but similar food intake and urinary albumin excretion compared to controls (Table 1). Mutant sPRR mice had lower urine volume, water intake and urinary K
+
but not Na
+
excretion. No differences in renal histology were noted between control and mutant sPRR mice.

Loss of sPRR attenuates DOCA-salt mediated hypertension. The mechanisms by which sPRR might regulate BP and water/Na
+
homeostasis in DOCA-salt hypertension are currently being investigated.
Abstract P026: The Accumulation Of Lysine In The Kidney Cortex Protects From Proximal Tubule Damage And Salt-Sensitive Hypertension.
Authors: Palygin Oleg , Rinschen Markus , Levchenko Vladislav , Spires Denisha , Siuzdak Gary , Staruschenko Alexander
Published: September 2020

The kidneys are highly sensitive to metabolic changes. Metabolic phenotyping uncovered a number of pathways associated with high blood pressure and kidney diseases, which sheds light on the complex pathophysiology of hypertension and associated kidney injury. However, the mechanisms underlying the effects of dietary interventions on kidney function and associated changes in cellular metabolic intermediates remain incompletely understood. L-lysine, one of the essential amino acids, is necessary for many physiological functions. Among all organs, the kidney is a key organ in lysine turnover and metabolism, in particular for modified lysines. Our recent metabolomics study revealed that intrarenal levels of L-Lysine are reduced in Dahl salt-sensitive (SS) rats fed an HS diet. This finding provided further evidence that L-Lysine is critical for metabolic states in the kidney. Using a well-established SS rat model of hypertension and kidney injury, we found that administration of lysine at a concentration of 17mg/ml in drinking water counteracted the lysine deficit in kidneys, restored albumin balance, triggered diuresis, and diminished the development of salt-sensitive hypertension (133±2 vs. 165±4 (136±4 vs. 166±7), mmHg in L-Lysine treated rats vs. control rats male (female) after 14 (21) days of an 8 (4) % NaCl diet). HPLC analysis revealed a significant elevation of lysine in the renal cortex of treated rats. In contrast, the concentration of other amino acids, such as glycine and arginine, did not change with increasing blood pressure. Overall, dietary lysine supplementation of SS rats on the HS diet leads to significant protection of proximal tubule damage, shown by Kim-1 immunostaining. Also, it restores kidney function by the rapid excretion of the pathologically accumulated renal albumin and rebalancing megalin abundance. Moreover, the analyses of glomerular damage showed improvement for the lysine treated group. In conclusion, dietary L-Lysine supplementation significantly enhanced cardiorenal protection via acute inhibition of metabolic load and oxidative stress triggered by hypertension.
Abstract P027: Hypertensive Cardiorenal Damage Is Aggravated In Axenic Mice
Authors: Bartolomaeus Hendrik , Avery Ellen G , Löber Ulrike , Bartolomaeus Theda U , Chen Chia-Yu , Tsvetkov Dmitriy , Kraeker Kristin , Geisberger Sabrina Yasmin , Markó Lajos , Balogh Andras , Forslund Sofia K , Mueller Dominik N , Wilck Nicola
Published: September 2020

Introduction:
The gut microbiota is suspected to play a role in hypertension and hypertensive end organ damage. In the present study, we used germ-free mice to demonstrate that microbial colonization modulates the response to a hypertensive stimulus.

Methods:
Four-week-old male germ-free C57BL6/J littermates were randomized to remain germ-free (GF) or to receive microbiota transfer from SPF donor mice to achieve full colonization status (COL). At 12 weeks, Angiotensin (Ang) II was infused s.c. for 14 days (1.44mg/kg/d, osmotic minipumps) and 1% NaCl added to the drinking water; sham-treated mice served as control. After 14 days of AngII we assessed inflammation and organ damage.

Results:
Fecal bacterial load in COL mice was similar to SPF donor mice (qPCR). Shotgun metagenomic sequencing of fecal samples revealed hypertension-induced alterations in microbiome composition confirming previous reports. Serum metabolome analysis (
Biocrates MxP Quant 500)
confirmed the absence of microbiota-dependent metabolites in GF. Interestingly, microbiota-dependent metabolites relevant for cardiovascular risk (TMAO, indoxyl sulfate) were elevated in hypertensive COL mice compared to sham-treated. Hypertensive kidney damage was aggravated in GF mice. However, marker genes for tubular damage (
Lcn2
), inflammation (
Ccl2
), and fibrosis (
Col1a3
) showed a stronger increase in GF mice (fold changes [fc] COL vs. GF: 7.5 vs 11.0, 1.2 vs 3.3, 1.3 vs 2.2, respectively). Albuminuria (fc 2 vs 25) and histology for kidney fibrosis (fc 1.1 vs 1.4) confirmed the aggravated kidney damage in GF mice. Similarly, we observed an aggravated cardiac damage in GF mice. Flow cytometry of splenic lymphocytes showed that the adaptive immune response to AngII + 1% NaCl, as evidenced by Th17 (fc 1.4 vs 2) and CD8+ central memory cells, was intensified in GF mice.
In vitro
, naïve T cells isolated from GF mice more readily polarized into Th17 (26 ± 5%) compared to T cells from SPF mice (19 ± 1%).

Conclusion:
The bacterial colonization status has potent effects on the phenotypic response to a hypertensive stimulus, evident to varying degrees in hearts and kidneys. The inflammatory response and the end organ damage in GF compared to COL mice demonstrates the importance of the gut microbiota in hypertension.
Abstract P036: Salt-inducible Kinase (SIK):
A Pharmacological Target Of Salt-sensitive Hypertension
Authors: Gomes Dayene S , Visniauskas Bruna , Prieto Minolfa C , Lara Lucienne d
Published: September 2020

Salt-inducible kinase (SIK) is a serine/threonine kinase involved in intracellular sensing network. However, its role in the development of salt-sensitive hypertension remains uncertain. We used a mouse model of salt-sensitive hypertension to test the hypothesis that SIK inhibition decreases systolic blood pressure (SBP) and renal injury. Male C57BL/6J mice (20-25g) were randomly fed either normal salt (NS; 0.5% NaCl) or high salt (HS; 4% NaCl) diet and daily treated or not with a SIK inhibitor (iSIK, YKL-05-099, 20 mg/Kg, via IP; n=6/group; CEUA: 013/19). In these mice: 1) SBP was measured by telemetry for 15 days; 2) Urine metabolic collection for 24 h was performed on day 14; and 3) Blood and kidney samples were collected after euthanasia on day 15. HS diet increased SBP during both day and night times (Day: from 114±1 to 136±1 mmHg; Night: from 127±3 to 141±4 mmHg; p<0.0001). Treatment with iSIK prevented increases in SBP (Day: 112±1 and Night: 123±2 mmHg). iSIK did not change SBP nor renal Na
+
handling in NS diet. In HS mice, iSIK prevented the augmentation of urine osmolality (HS: 1692±14
vs.
HS+iSIK: 1104±34 mOsm/Kg H
2
O; p=0.0019) but did not change increases in urine volume and Na
+
excretion neither decreases in urine creatinine. Moreover, HS mice displayed glomerular segmentation, tubular interstitial disruption, infiltration of inflammatory cells and collagen accumulation. These responses were attenuated by iSIK. Increased kidney SIK activity in HS mice were sensitive to iSIK (HS: 222.8±45
vs.
HS+iSIK: 19.10±15 pmol.mg
-1
.min
-1
; p=0.0026). The augmentation of (Na
+
+K
+
)ATPase activity in HS mice was exacerbated during iSIK (HS: 517±112 and HS+SIKi: 763±18 mmol.mg
-1
;.min
-1
p<0.0001). Equally important, SIK inhibition in HS mice: 1) Decreased plasma MCP-1 levels (HS: 79±7
vs.
HS+iSIK: 51±2 pg/dL; p=0.0019); 2) Prevented HS-dependent macrophages renal infiltration; 3) Decreased kidney ROS; and 4) Decreased kidney TGF-β protein expression (HS: 1.9±0.01
vs.
HS+iSIK: 1.4±0.3 au. p=0.0007). Thus, the data indicate that SIK inhibition attenuates salt-sensitive hypertension and prevents kidney injury, without altering Na
+
handling in response to HS. In perspective, SIK might be a pharmacological target for salt-sensitive hypertension treatment.
Abstract P041: Proximal Tubule-specific Deletion Of Angiotensin Ii Type 1a Receptors In The Kidney Lowers Basal Blood Pressure And Attenuates Angiotensin Ii-induced Hypertension By Increasing Glomerular Filtration And The Pressure-natriuresis Response
Authors: Li Xiao C , Leite Ana P , Zhang Liang , Zhuo Jia L
Published: September 2020

The present study tested the hypothesis that intratubular angiotensin II (Ang II) and AT
1a
receptors in the proximal tubules of the kidney plays an important role in basal blood pressure control and in the development of Ang II-induced hypertension. Mutant mice with proximal tubule-specific deletion of AT
1a
receptors in the kidney, PT-
Agtr1a
-/-
, were generated to test the hypothesis. Eight groups (n=7-12 per group) of adult male wild-type (WT) and PT-

Agtr1a
-/-

mice were infused with or without Ang II for 2 weeks (1.5 mg/kg, i.p.). Basal systolic, diastolic, and mean arterial pressures were ~13 ± 3 mmHg lower in PT-

Agtr1a
-/-

than WT mice (
P
<0.01). Basal glomerular filtration rate (GFR), as measured using transdermal FITC-sinistrin, was significantly higher in PT-

Agtr1a
-/-

mice (WT: 160.4 ± 7.0 μl/min vs. PT-

Agtr1a
-/-

: 186.0 ± 6.0 μl/min,
P
<0.05). Basal 24 h urinary Na
+
excretion (U
Na
V) was significantly higher in PT-

Agtr1a
-/-

than WT mice (
P
<0.01). In response to Ang II infusion, both WT and PT-

Agtr1a
-/-

mice developed hypertension, and the magnitude of the pressor response to Ang II was similar in WT (Δ43 ± 3 mmHg,
P
<0.01) and PT-

Agtr1a
-/-

mice (Δ39 ± 5 mmHg,
P
<0.01). However, the absolute blood pressure level was still 16 ± 3 mmHg lower in PT-

Agtr1a
-/-

mice (
P
<0.01). Ang II significantly decreased GFR to 132.2 ± 7.0 μl/min in WT mice (
P
<0.01), and to 129.4 ± 18.6 μl/min in PT-

Agtr1a
-/-

mice (
P
<0.01), respectively. In WT mice, U
Na
V increased from 139.3 ± 22.3 μmol/24 h in the control group to 196.4 ± 29.6 μmol/24 h in the Ang II-infused group (
P
<0.01). In PT-

Agtr1a
-/-

mice, U
Na
V increased from 172.0 ± 10.2 μmol/24 h in the control group to 264.7 ± 35.4 μmol/24 h in the Ang II-infused group (
P
<0.01). The pressor response to Ang II was attenuated, while the natriuretic response was augmented by losartan in WT and PT-

Agtr1a
-/-

mice (
P
<0.01). Finally, proximal tubule-specific deletion of AT
1a
receptors significantly augmented the pressure-natriuresis response and natriuretic responses to acute saline infusion (
P
<0.01) or a 2% high salt diet (
P
<0.01). We concluded that deletion of AT
1a
receptors selectively in the proximal tubules lowers basal blood pressure and attenuates Ang II-induced hypertension by increasing GFR and promoting the natriuretic response in PT-

Agtr1a
-/-

mice.
Abstract P042: Role Of Angiotensin In Determining Blood Flow Remaining Kidney Following Nephrectomy
Authors: Shimada Satoshi , Cowley Allen W
Published: September 2020

We have previously presented data obtained in anesthetized Sprague Dawley (SD) rats showing acute increases of the renal blood flow (RBF) in the remaining kidney after unilateral nephrectomy (UNx) which were prevented with the AT1 receptor blocker losartan (ARB). We now chronically monitored RBF for 7 days following UNx in unanesthetized SD rats (6 weeks of age) with daily ARB treatment (oral; 10 mg/Kg; n=6) or vehicle (Vhe; Nutella; n=7) administered every 12 hr, from 5 days before UNx and throughout the entire experimental period. Rats were first instrumented with a Transonic flow probe on the left renal artery and a femoral arterial catheter for 24 hr continuous monitoring of RBF and mean arterial pressure (MAP). A week later, rats were anesthetized with isoflurane, and the right kidney was removed (UNx). After rapid recovery (15 min), RBF and MAP were monitored continuously for the next 7 days. In ARB treated rats, MAP fell an average of 115 to 106 mmHg (p<0.05) and RBF increased from 6.2 to 7.4 ml/min (p=0.06). Following Unx, RBF of both the ARB and Vhe group gradually rose (P<0.05) to an average of 10.6 and 9.9 ml/min (p=0.48) in ARB and Vhe treated groups respectively at day 7 while MAP remained unchanged. A similar increase of kidney weight was observed in both groups. Other SD rats (n=7) were instrumented with a femoral arterial and venous catheter for measurements of GFR following UNx. Rats were trained to wear a jacket carrying a miniaturized light emitting diode for real-time transcutanous excitation and detection of the emitted light to determine disappearance curves following i.v. injection of FITC-Sinistrin. Total GFR decreased from 1.8 to 1.2 ml/min/100gbw 2 days after UNx (p<0.05) and remained at that level for 21 days following UNx. We conclude that following UNx the RBF of the remaining kidney increases independent of changes of the renin-angiotensin system and corresponds to kidney hypertrophy. GFR returned to nearly 70% of the 2 kidney filtration rate 2 days following UNx and remained unchanged for the next 21 days.
Abstract P047: Elastomer Grafted Induced Pluripotent Stem Cell Derived Micro Heart Muscles To Investigate Effects Of Afterload On Physiology
Authors: Guo Jingxuan , Simmons Daniel , Munsell Mary , Oguntuyo Kasoorelope , Rios Brandon , Pear Missy , Schuftan David , Huebsch Nathaniel
Published: September 2020

Mechanical loading (e.g. blood pressure) is a critical component of heart disease. Here, we developed a simple, scalable iPSC-derived micro heart muscle (μHM) model by grafting μHM on elastomeric substrates with different stiffnesses to simulate afterload. For tissue traction force microscopy (TFM), substrates were fabricated by spin coating a PDMS base layer followed by spin coating an ultra-thin PDMS/fluorescent bead (1-micron) layer. Substrates were then covalently grafted with fibronectin via glutaraldehyde. Sylgard 184 ‘dog-bone’ shaped stencils were added onto substrates to control tissue geometry. Both isogenic control (WTC) and PKP2
-/-
iPSC with GCaMP6f calcium indicator were differentiated using small molecule Wnt pathway modulators and were then purified (81±15% TNNT2 positive) for making tissue. Contractility was quantified using open source TFM software and calcium dynamics were measured using GCaMP6f intensity. At day 9, we observed a significant increase in contractility of isogenic control iPSC-μHM on stiffer substrates, the average traction from tissues formed on 5kPa, 15kPa and 30kPa substrates are 19.7μN, 28.7 μN and 36.7 μN. However, PKP2
-/-
iPSC-μHM showed a non-significant traction force increase from 5kPa (23.6 μN) to 15kPa (26.9 μN) although prolonged calcium transients were found on stiffer substrates for both WTC and PKP2
-/-
μHM, which may indicate calcium-contraction uncoupling of the PKP2
-/-
μHM. Immunostaining shows a slightly increase in cell size towards stiffer substrates. These data suggest that mechanical load (e.g. afterload) may be necessary to reveal genotype-phenotype relationships within
in vitro
iPSC-based models.
Abstract P049: Circadian Clock Dysfunction Impairs Mitochondrial Fitness And Contributes To Myocardial Ischemic Injury
Authors: Rabinovich-Nikitin Inna , Posen Illana , Margulets Victoria , Martino Tami A , Kirshenbaum Lorrie A
Published: September 2020

Cardiac function is highly reliant on mitochondrial oxidative metabolism and fitness. The circadian clock is critically linked to vital physiological process including mitochondrial fission, fusion and quality control mechanisms. However, little is known of how the circadian clock regulates these vital processes in the heart. Herein, we identified a putative circadian Clock - mitochondrial interactome that gates an adaptive stress response for cell viability during myocardial ischemia reperfusion (I-R) injury. We show that Clock transcriptionally coordinates expression of mitochondrial dynamic fusion and fission, bioenergetic and quality control proteins in cardiac myocytes. Transcriptome and gene ontology mapping revealed Clock defective hearts subjected to I-R exhibited major transcriptional deficits in several key survival processes including mitochondrial dynamics, bioenergetics and autophagy that were reduced further following I-R. Gain of function of Clock activity re-established gene transcription of mitochondrial respiratory complex activity, quality control mechanisms and cell viability. Collectively, our data show that mitochondrial fitness and cell survival is mutually dependent upon and obligatorily linked to transcription of the circadian Clock gene in cardiac myocytes. Our data suggest the functional loss of Clock activity predisposes cardiac myocytes to metabolic catastrophe. Hence, therapeutic strategies designed to preserve circadian clock activity in the hearts may prove beneficial in reducing morbidity and mortality following ischemia -related pathologies such as myocardial infarction.
Abstract P055: Smooth Muscle Cell-specific Conditional Knockout Of
Lysine (K)-specific Demethylase 6a
Causes Endothelial Dysfunction, Increased Blood Pressure And Arterial Stiffness
Authors: Myers Rebecca L , Chen Kai , Sun Zhongjie
Published: September 2020

Arterial stiffening is the progressive loss of elasticity of the large vessels resulting from age. Increased arterial stiffening has been correlated with hypertension and other cardiovascular co-morbidities. KDM6A is a histone 3 lysine demethylase whose activity increases expression of target genes via removal of silencing methylation marks on lysine 27 of histone 3 (H3K27). The precise role of H3K27me3, or its absence in aortic vascular smooth muscle cells (VSMCs), remains to be investigated. The objectives of this study were to determine the effects of loss of KDM6A on arterial stiffness, blood pressure, and vascular cell function. We generated VSMC-specific
Kdm6a
knockout mice of C57BL/6 background and confirmed knockout by western blot. Knockout of
Kdm6a
resulted in increased resting systolic blood pressure and pulse wave velocity in male mice. Pulse wave velocity (PWV) is the gold-standard method for determination of arterial stiffness. PWVs of the knockout mice were significantly increased compared to controls four weeks after
Kdm6a
knockout (Controls 2.62
+
0.69 mm/ms; KO 3.13
+
0.30 mm/ms; P=0.0228). Systolic blood pressure was directly obtained for each mouse via carotid cannulation. The systolic blood pressures of knockout mice were significantly increased. Mesenteric arteries were isolated from three control and three
Kdm6a
knockout mice and mounted onto a wire myograph. Vessels were pre-constricted with norepinephrine and vasodilation was measured by treatment with increasing doses of acetylcholine and sodium nitroprusside to assess endothelial and VSMC function. Interestingly, despite
Kdm6a
knockout in VSMCs, vasodilation of VSMCs was not different between control and knockout arteries, but relaxation in response to acetylcholine, which stimulates the release of nitric oxide from endothelial cells, was decreased. This suggests endothelial dysfunction. These results indicate that loss of KDM6A in VSMCs leads to elevated systolic blood pressure and arterial stiffness.
Abstract P059: Association Of Uric Acid With Change In Arterial Stiffness And Blood Pressure Over Time In Type 1 Diabetes Mellitus: The SEARCH For Diabetes In Youth Study
Authors: South Andrew M , Rigdon Joseph , Stafford Jeanette M , Dabelea Dana , Lawrence Jean , Marcovina Santica M , Mottl Amy , Pihoker Catherine , Voruganti Saroja , Urbina Elaine M , Jensen Elizabeth T
Published: September 2020

Background:
Type 1 diabetes (T1D) increases CVD risk, and early detection of CV changes could mitigate CVD risk. Increased arterial stiffness is an early marker of adverse CV health and precedes hypertension. Uric acid has been associated with arterial stiffness, hypertension, and CVD in select conditions such as obesity, but its relationship to T1D is undefined. We hypothesized that uric acid predicts increased arterial stiffness and BP over time in youth with T1D, especially those with overweight/obesity.

Methods:
Prospective cohort of 451 participants with T1D from the SEARCH for Diabetes in Youth Study. We measured uric acid, BP, and arterial stiffness assessed by pulse wave velocity (PWV) in the arm (PWV-R), trunk (PWV-T), and lower extremity (PWV-LE). BP and PWV were repeated a mean of 4.5 years later (±1.1). We defined high BP as ≥90
th
%ile for age/sex/height (<13 years) and ≥120/80 mmHg (≥13 years). We applied multivariable regression models to estimate the association of uric acid with annualized changes in each outcome. Directed acyclic graph-informed models were adjusted for age, sex, race/ethnicity, health insurance, family history of CVD, A1c, antihypertensive use, T1D duration, dyslipidemia, and study site. We assessed for effect modification by overweight/obesity.

Results:
Mean age was 18.1 years (±4.5), 50.8% (229/451) were female, 66.1% (298/451) were White, and 42.4% (191/451) had overweight/obesity. A 1 mg/dl increase in uric acid was associated with increased PWV-R (adjusted
β
: 0.02 m/s/year, 95% CI -0.01 to 0.06), decreased SBP (-0.45 mmHg/year, -0.73 to -0.18), and sustained high BP (referenced to normal BP, adjusted OR 1.28, 0.91 to 1.79), but not PWV-T or PWV-LE. Compared to normal BMI, those with overweight/obesity demonstrated somewhat stronger associations between uric acid and PWV-R (0.05 m/s/year, 0.0 to 0.1 vs. 0.0 m/s/year, -0.05 to 0.05, interaction
p
=0.17) and sustained high BP (1.58, 0.99 to 2.51 vs. 0.79, 0.44 to 1.4, interaction
p
=0.06).

Discussion:
Among participants with T1D, uric acid was modestly associated with changes in PWV-R and BP over time, particularly in those with overweight/obesity. Further investigations are warranted to determine uric acid’s role in hypertension and arterial stiffness in T1D.
Abstract P062: Trimethylamine N-oxide Impairs Endothelium-dependent Relaxation In Aorta Of Non-diabetic And Diabetic Mice
Authors: Priviero Fernanda , Dela Justina Vanessa , Arishe Olufunke O , Webb R Clinton
Published: September 2020

Trimethylamine N-oxide (TMAO) is a gut microbiota metabolite related with cardiovascular diseases. Circulating levels of TMAO are increased in diabetes and TMAO is associated with the development of insulin resistance. We aimed to investigate if TMAO impairs vascular reactivity in diabetes. The aorta of 16-week old male diabetic (db/db) and db/+ mice were mounted in a myograph and concentration-response curves to acetylcholine (ACh; 1 nM - 10 μM), sodium nitroprusside (SNP; 0.1 nM - 1 μM) and phenylephrine (PE; 1 nM - 10 μM) were obtained in the presence of TMAO (100 mM, 30 min), repeated in the presence of the antioxidant tempol (10 μM), the NLRP3 inhibitor (MCC950, 1 μM) or IL-1β receptor antagonist (IL1RA, 20 ng/mL). TMAO did not affect the contraction of PE or relaxation to SNP in aorta of db/db or db/+ mice, but decreased the potency of ACh in the aorta of db/+ (pEC
50
: 7.96
+
0.22 vs 7.25
+
0.16, lean or TMAO, respectively) and db/db (pEC
50
: 7.91
+
0.10 vs 7.17
+
0.09, db/db vs TMAO, respectively). In db/+ mice, tempol improved the potency of ACh (pEC
50
- TMAO: 7.05
+
0.21 vs TMAO + tempol 7.42
+
0.15) while further impairment in the relaxation to ACh was seen in the presence of MCC950 or IL1RA. In conclusion, TMAO impairs endothelium-dependent relaxation of aorta through the production of reactive oxygen species.
Abstract P063: Pressor Response To Acute Stress Is Regulated By Histamine In Humanized Sickle Cell Mice
Authors: Molina Patrick A , Fox Brandon M , Sedaka Randee , Botta Davide , Becker Bryan , Allan John , Kasztan Malgorzata , Pollock David M , Pollock Jennifer
Published: September 2020

Acute psychosocial stress has been linked to the onset of vaso-occlusive pain crises in patients with sickle cell disease (SCD), however mechanistic insight is lacking. Studies suggest that histamine is a stress-responsive factor and promotes inflammation. In SCD, histamine levels are elevated in association with vaso-occulsive crisis, however, the impact of acute stress is unknown. We hypothesized that acute stress in a humanized SCD mouse model stimulates the histamine pathway, inflammatory mediator release, and a pressor response. Acute stress was induced using cage switch stress (CSS) in male humanized SCD (HbSS) or control (HbAA) mice (n=6-8/group) with blood pressure (BP) monitored by radiotelemetry and mice terminated at baseline (BL, no CSS) and 30 min post-CSS for plasma measurements. Plasma histamine was unchanged in HbAA mice but was elevated in HbSS in response to CSS (nM; HbAA: 90.2±21.3; 104.3±10.2; HbSS: 91.1±13.6; 174.3±19.2*, *p<0.01 vs genotype BL). HbSS mice have significantly higher IL-6 levels at BL and post-CSS (ug/mL; HbAA: 20±4, 23±7; HbSS: 37±8, 65±5*, p
genotype
=0.0001, *p<0.05 vs genotype BL). Significant stress-by-genotype effects were found for E-selectin (ug/mL; BL vs CSS: HbAA: 7.9±0.9, 6.4±0.8; HbSS: 10±0.7, 13±1.4*, *p
genotype
<0.001) and P-selectin (ug/mL; BL vs CSS: HbAA: 29±2.7, 25±2.1; HbSS: 33±3.0, 42±4.1*, *p
genotype
<0.01). The peak mean arterial pressure response to CSS was blunted in HbSS versus HbAA mice and was driven by lower diastolic BP (DBP), not systolic BP. Mice were treated with H1 and H2 receptor antagonists, diphenhydramine and cimetidine, to examine the histamine pathway in the BP response to CSS. Following H1/H2 receptor antagonism, the CSS-induced peak DBP response was similar in HbAA mice, but was elevated in HbSS mice (change in BP, mmHg; vehicle vs H1/H2 blockade: HbAA: 32±2, 30±1; HbSS: 23±2, 31±2*, *p<0.05 vs genotype vehicle). These data reveal that acute stress stimulates histamine release and inflammatory mediators in HbSS mice but not HbAA. Further, the stress-induced DBP response is dependent on the histamine H1/H2 pathway in HbSS mice. These data identify that in SCD the histamine pathway may be stress-responsive and represents a link between acute stress and vaso-occlusive crises.
Abstract P068: Endothelial Mineralocorticoid Receptor Activation Promotes Endothelial And Vascular Stiffening In Western Diet Fed Female Mice Through Sgk1 Mediated Activation Of The Endothelial Sodium Channel
Authors: Yang Yan , Sun Zhe , Aroor Annayya , Zhang Liping , Jia Guanghong , Bonnard Benjamin , Jaisser Frederic , Sowers James R , Hill Michael A
Published: September 2020

Over-nutrition/obesity predisposes persons, particularly women, to endothelial dysfunction and vascular stiffening. We have employed a clinically relevant model using female mice fed a high fat and high fructose diet (western diet, WD). These mice display high plasma aldosterone levels, endothelial stiffness and dysfunction and increased mineralocorticoid receptor (MR) expression in the vasculature. One potential mechanism by which MR activation may promote endothelial stiffness is through increased expression and activation of epithelial sodium channel (EnNaC) in endothelial cells (ECs) through mTOR2 mediated activation of serum and glucocorticoid regulated kinase 1(SGK1). In this investigation we observed that WD feeding in female mice for 16 wks caused endothelial (atomic force microscopy (AFM)), and aortic stiffening (PW analysis) in concert with increased expression of EnNaC and SGK1 in the endothelium and EnNaC activation in ECs. Further, amelioration of WD induced EC and vascular stiffness was accomplished by EnNaC inhibition with low dose amiloride (1mg/kg/day in drinking water) over the 16 wks of WD. We then explored the idea that inhibition of SGK1 as well as specific deletion of ECMR and EnNaC decreases vascular EC stiffness accompanied by decreased sodium current in isolated lung ECs. Accordingly, female wild type and ECMR and EnNaC KO mice were fed a WD or control diet (CD) for 16 wks. Aortic and coronary artery EC stiffness, measured ex vivo by AFM, was increased in WD fed mice and this was prevented in ECMR and EnNaC KO models. Both ECMR and EnNaC KO mice fed a WD showed decreased amiloride sensitive sodium current in isolated ECs. Further, in cultured ECs , inhibition of SGK1 by a chemical inhibitor attenuated aldosterone mediated sodium currents. Collectively, these findings support the notion that a WD promotes ECMR mediated increases in SGK1 and associated EnNaC activity in ECs together with increased endothelial and vascular stiffness in females.
Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction
Authors: Garcia Vinicius P , Hijmans Jamie G , Stockelman Kelly A , Brewster Madden , Fandl Hannah , Rahaman Clay , Maly Lukas , Greiner Jared , Desouza Christopher
Published: September 2020

Introduction:
Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production.

Methods:
Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N
G
-Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144
+
) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated.

Results:
EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs.

Conclusion:
eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.
Abstract P071: Transglutaminases Are Active In Perivascular Adipose Tissue
Authors: Orr Alexis , Thompson Janice , Lyttle Janae , Watts Stephanie W
Published: September 2020

Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model in which to test interactions of amines and TGs. We hypothesized that TG2 and FXIII are active in PVAT. Sprague-Dawley rat aortic, superior mesenteric (SMA), and mesenteric resistance artery (MR) PVAT express TG2 and blood coagulation factor XIII (FXIII) mRNA (Figure 1A). Consistent with this, immunohistochemical analyses support that PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs and a catalyzing calcium solution, visualized with TRITC fluorescence (Figure 1B,C). Both TG2 and FXIII are active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT by 6.14% of total normalized signal (p<0.0001, N=7). Further Western blot analysis proved that experimentally added 5-HT competitively inhibits incorporation of experimentally added BAP into MRPVAT adipocytes, reducing total normalized signal by 10.75% (p=0.001, N=4). Further studies to determine what proteins TGs are amidating will give insight into how these enzymes contribute to the development of hypertension.
Abstract P074: Mitochondrial Isolevuglandins Contribute To Vascular Oxidative Stress And Mitochondria-targeted Scavenger Of Isolevuglandins Reduces Mitochondrial Dysfunction And Hypertension
Authors: Dikalova Anna , Mayorov Vladimir , Xiao Liang , Panov Alexander , Amarnath Venkataraman , Zagol-Ikapitte Irene , Vergeade Aurelia , Ao Mingfang , Yermalitsky Valery , Nazarewicz Rafal , Boutaud Olivier , Lopez Marcos G , Billings Frederic T , Davies Sean S , Roberts L. Jackson , Harrison David G , Dikalov Sergey I
Published: September 2020

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products isolevuglandins (isoLGs) and that scavenging of mitochondrial isoLG improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isoLG-protein adducts in human patients with essential hypertension and angiotensin II mouse model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isoLG was tested by the novel mitochondria-targeted isoLG scavenger, mito2HOBA. Mitochondrial isoLG in arterioles isolated from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects improved deacetylation of a key mitochondrial antioxidant, superoxide dismutase 2 (SOD2). In human aortic endothelial cells, mito2HOBA diminished mitochondrial superoxide and inhibited cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In angiotensin II-infused mice, mito2HOBA prevented accumulation of mitochondrial isoLG-protein adducts, improved Sirt3 mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in angiotensin II-infused mice. These data support the role of mitochondrial isoLGs in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isoLGs may have therapeutic potential in treatment of vascular dysfunction and hypertension.