2K1C Mice Research Articles

Mechanism Actions of Coniferyl Alcohol in Improving Cardiac Dysfunction in Renovascular Hypertension Studied by Experimental Verification and Network Pharmacology.

Renovascular hypertension (RH), a secondary hypertension, can significantly impact heart health, resulting in heart damage and dysfunction, thereby elevating the risk of cardiovascular diseases. Coniferol (CA), which has vascular relaxation properties, is expected to be able to treat hypertension-related diseases. However, its potential effects on cardiac function after RH remain unclear. In this study, in combination with network pharmacology, the antihypertensive and cardioprotective effects of CA in a two-kidney, one-clip (2K1C) mice model and its ability to mitigate angiotensin II (Ang II)-induced hypertrophy in H9C2 cells were investigated. The findings revealed that CA effectively reduced blood pressure, myocardial tissue damage, and inflammation after RH. The possible targets of CA for RH treatment were screened by network pharmacology. The interleukin-17 (IL-17) and tumor necrosis factor (TNF) signaling pathways were identified using a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The inflammatory response was identified using a Gene Ontology (GO) enrichment analysis. Western blot analysis confirmed that CA reduced the expression of IL-17, matrix metallopeptidase 9 (MMP9), cyclooxygenase 2 (COX2), and TNF α in heart tissues and the H9C2 cells. In summary, CA inhibited cardiac inflammation and fibrohypertrophy following RH. This effect was closely linked to the expression of MMP9/COX2/TNF α/IL-17. This study sheds light on the therapeutic potential of CA for treating RH-induced myocardial hypertrophy and provides insights into its underlying mechanisms, positioning CA as a promising candidate for future drug development.

OXGR1-Dependent (Pro)Renin Receptor Upregulation in Collecting Ducts of the Clipped Kidney Contributes to Na+ Balance in Goldblatt Hypertensive Mice.

The two-kidney, one-clip (2K1C) Goldblatt rodent model elicits a reduction in renal blood flow (RBF) in the clipped kidney (CK). The reduced RBF and oxygen bio-ability causes the accumulation of the tricarboxylic cycle intermediary, α-ketoglutarate, which activates the oxoglutarate receptor-1 (OXGR1). In the kidney, OXGR1 is abundantly expressed in intercalated cells (ICs) of the collecting duct (CD), thus contributing to sodium transport and electrolyte balance. The (pro)renin receptor (PRR), a member of the renin-angiotensin system (RAS), is a key regulator of sodium reabsorption and blood pressure (BP) that is expressed in ICs. The PRR is upregulated in 2K1C rats. Here, we tested the hypothesis that chronic reduction in RBF in the CK leads to OXGR1-dependent PRR upregulation in the CD and alters sodium balance and BP in 2K1C mice. To determine the role of OXGR1 in regulating the PRR in the CDs during renovascular hypertension, we performed 2K1C Goldblatt surgery (clip = 0.13 mm internal gap, 14 days) in two groups of male mice: (1) mice treated with Montelukast (OXGR1 antagonist; 5 mg/Kg/day); (2) OXGR1-/- knockout mice. Wild-type and sham-operated mice were used as controls. After 14 days, 2K1C mice showed increased systolic BP (SBP) (108 ± 11 vs. control 82 ± 5 mmHg, p < 0.01) and a lower natriuretic response after the saline challenge test. The CK group showed upregulation of erythropoietin, augmented α-ketoglutarate, and increased PRR expression in the renal medulla. The CK of OXGR1 knockout mice and mice subjected to the OXGR1 antagonist elicited impaired PRR upregulation, attenuated SBP, and better natriuretic responses. In 2K1C mice, the effect of reduced RBF on the OXGR1-dependent PRR upregulation in the CK may contribute to the anti-natriuretic and increased SBP responses.

P100 ALPHA KETOGLUTARATE RECEPTOR 1 REGULATES THE EXPRESSION OF THE (PRO)RENIN RECEPTOR IN MEDULLARY RENAL TISSUES, MODULATES SODIUM EXCRETION AND ATTENUATES TWO-KIDNEY, ONE-CLIP GOLDBLATT HYPERTENSION IN MICE

Background: The two-kidney, one-clip (2K1C) Goldblatt model elicits a reduction of renal blood flow (RBF) in the clipped kidney (CK). The reduced RBF and oxygen bio ability causes the accumulation of the intermediary of the tricarboxylic cycle, α-ketoglutarate, which activates the oxoglutarate receptor-1 (OXGR1). In the kidney, the OXGR1 is abundantly expressed in intercalated cells (IC) of the collecting duct (CD), which may contribute to Na+ transport and electrolyte balance. The (pro)renin receptor (PRR), a member of the renin-angiotensin-aldosterone system is a key regulator of Na+ reabsorption, and blood pressure (BP) and is also expressed in IC. PRR is upregulated in 2K1C. Here, we tested the hypothesis that reduction of RBF in the CK leads to OXGR1-dependent PRR upregulation in the CD and alterations of Na+ balance and BP in 2K1C mice. Methods: To determine the role of OXGR1 in regulating PRR in the CDs during renovascular hypertension, we used 2K1C Goldblatt surgery (clip= 0.13 mm internal gap) in two groups of male mice: 1) Oxgr1-/- knockout (n=6) mice; and 2) mice treated with Montelukast (OXGR1 antagonist; 5 mg/Kg/day, n=6). WT and sham-operated mice were used as controls. Results: 2K1C mice showed increased systolic BP (SBP) (108 ± 11 vs. control 82 ± 5 mmHg, p&lt;0.01) and a lower natriuretic response after the saline challenge test. The CK showed augmented levels of α-ketoglutarate, erythropoietin mRNA levels, PRR (mRNA and protein abundances) in the renal medulla. Non-CK showed PRR upregulation in the CDs. However, the CK of Oxgr1-/- knockout mice, and from those subjected to OXGR1 antagonism, elicited impaired PRR upregulation, attenuated SBP (Oxgr1-/- 2K1C: 90 ± 8 mmHg, p=non-significant vs. control; Montelukast 2K1C: 95 ± 3 mmHg, p&lt;0.05 vs. control) and better natriuretic responses. Conclusion: In 2K1C mice, the effect of reduced RBF on the OXGR1-dependent PRR upregulation in the CK may contribute to the anti-natriuretic and increased SBP responses.

B cell-derived anti-beta 2 glycoprotein I antibody mediates hyperhomocysteinemia-aggravated hypertensive glomerular lesions by triggering ferroptosis

Hyperhomocysteinemia (HHcy) is a risk factor for chronic kidney diseases (CKDs) that affects about 85% CKD patients. HHcy stimulates B cells to secrete pathological antibodies, although it is unknown whether this pathway mediates kidney injury. In HHcy-treated 2-kidney, 1-clip (2K1C) hypertensive murine model, HHcy-activated B cells secreted anti-beta 2 glycoprotein I (β2GPI) antibodies that deposited in glomerular endothelial cells (GECs), exacerbating glomerulosclerosis and reducing renal function. Mechanistically, HHcy 2K1C mice increased phosphatidylethanolamine (PE) (18:0/20:4, 18:0/22:6, 16:0/20:4) in kidney tissue, as determined by lipidomics. GECs oxidative lipidomics validated the increase of oxidized phospholipids upon Hcy-activated B cells culture medium (Hcy-B CM) treatment, including PE (18:0/20:4 + 3[O], PE (18:0a/22:4 + 1[O], PE (18:0/22:4 + 2[O] and PE (18:0/22:4 + 3[O]). PE synthases ethanolamine kinase 2 (etnk2) and ethanolamine-phosphate cytidylyltransferase 2 (pcyt2) were increased in the kidney GECs of HHcy 2K1C mice and facilitated polyunsaturated PE synthesis to act as lipid peroxidation substrates. In HHcy 2K1C mice and Hcy-B CM-treated GECs, the oxidative environment induced by iron accumulation and the insufficient clearance of lipid peroxides caused by transferrin receptor (TFR) elevation and down-regulation of SLC7A11/glutathione peroxidase 4 (GPX4) contributed to GECs ferroptosis of the kidneys. In vivo, pharmacological depletion of B cells or inhibition of ferroptosis mitigated the HHcy-aggravated hypertensive renal injury. Consequently, our findings uncovered a novel mechanism by which B cell-derived pathogenic anti-β2GPI IgG generated by HHcy exacerbated hypertensive kidney damage by inducing GECs ferroptosis. Targeting B cells or ferroptosis may be viable therapeutic strategies for ameliorating lipid peroxidative renal injury in HHcy patients with hypertensive nephropathy.

Renomedullary exosomes produce antihypertensive effects in reversible two-kidney one-clip renovascular hypertensive mice

The rapid fall in blood pressure following unclipping of the stenotic renal artery in the Goldblatt two-kidney one-clip (2K1C) model of renovascular hypertension is proposed to be due to release of renomedullary vasodepressor lipids, but the mechanism has remained unclear. In this study, we hypothesized that the hypotensive response to unclipping is mediated by exosomes released from the renal medulla. In male C57BL6/J mice made hypertensive by the 2K1C surgery, unclipping of the renal artery after 10 days decreased mean arterial pressure (MAP) by 23 mmHg one hr after unclipping. This effect was accompanied by a 556% increase in the concentration of exosomes in plasma as observed by nanoparticle tracking analysis. Immunohistochemical analysis of exosome markers, CD63 and AnnexinII, showed increased staining in interstitial cells of the inner medulla of stenotic but not contralateral control kidney of clipped 2K1C mice. Treatment with rapamycin, an inducer of exosome release, blunted the hypertensive response to clipping, whereas GW-4869, an exosome biosynthesis inhibitor, prevented both the clipping-induced increase in inner medullary exosome marker staining and the unclipping-induced fall in MAP. Plasma exosomes isolated from unclipped 2K1C mice showed elevated neutral lipid content compared to sham mouse exosomes by flow cytometric analysis after Nile red staining. Exosomes from 2K1C but not sham control mice exerted potent MAP-lowering and diuretic-natriuretic effects in both 2K1C and angiotensin II-infused hypertensive mice. These results are consistent with increased renomedullary synthesis and release of exosomes with elevated antihypertensive neutral lipids in response to increased renal perfusion pressure.

Antihypertensive Action of Exosomes Released from the Renal Medulla after Unclipping of the Renal Artery of 2 Kidney‐1 Clip Hypertension Mice

The renal medulla has been long known to exert antihypertensive action by releasing different bioactive substances. However, so far it remains unknown what is the identity of such renal medullary antihypertensive substances. In the present study, we hypothesized that exosomes, but not a single molecule released from the renal medulla are importantly implicated in the renal medullary antihypertensive action. To test this hypothesis, we first produced the 2 kidney‐one clip (2K1C) hypertension mouse model by clipping the right renal artery (127.9 ± 1.40 mmHg vs. 94.8 ± 1.75 mmHg of sham mice). After 1 week, we unclipped the right renal artery and monitored the mean arterial blood pressure (MAP) for 1 hour and at the same time collected blood samples for analysis of exosomes. It was found that unclipping the right renal artery of 2K1C mice significanty reduced MAP by 21.2 ± 2.65 mmHg but increased the plasma exosome concentrations by more than 5 folds (before unclipping 2.9 ± 0.7 vs. 3.3±0.5, N.S., after unclipping 4±0.5 vs. 17±5× 108particles/mL, p&lt;0.05). Immunohistochemical (IHC) assessments showed that the clipped kidneys had dramatic increase in exosome markers (CD63 and Annexin II) in the renal medulla of 2K1C mice compared to contralateral kidneys or sham mouse kidneys. These functional and IHC changes in 2K1C mice were almost completely blocked by pretreatment of mice with an exosome biogenesis inhibitor, GW‐4869 (1 mg/kg/day, i.p., n=8). We also found that rapamycin, a compound that increases autophagy and lysosome trafficking (6 mg/kg/day, i.p., n=8) substantially attenuated exosome release from the renal medulla and associated antihypertensive action in 2K1C with unclipping the right renal artery, even though it had some effects to reduce MAP and increase exosome release before the unclipping. In another series of studies using 2K1C hypertensive mice, we extracted and purified exosomes from the blood of mice with unclipping of the right renal artery and then infused these exosomes intravenously to another group of 2K1C mice. It was found that plasma exosomes from 2K1C mice with unlipping of the right renal artery signicantly reduced MAP when they are infused into recipient 2K1C mice (at a concentration of 3 x 108 exosomes) compard to exosomes from sham mice. Urinary flow and sodium excretion of recipient mice also increased slightly by infusion of exosomes from 2K1C mice. These results suggest that exosomes are enriched in the renal medulla of the clipped kidney in 2K1C mice and they can be released into the blood if the renal artery clip is released. These exosomes have strong effects to lower arterial blood pressure.

Characterization and Ischemic Response of Telecytes in the Mouse Renal Medulla

Telocytes are currently defined as a novel type of interstitial (stromal) cells with very long and thin prolongations, and these cells can produce exosomes to transmit signals to different cells such as vascular smooth muscle cells, tubular cells and telocytes themselves. So far it remains unclear whether telocytes are present in the renal medulla and whether they are able to regulate renal medullary function. In the present study, we first characterized telocytes in the kidney, in particular, in the renal medulla, given that the renal medullary interstial cells (RMICs) are able to sense pressure and release bioactive lipids lowering arterial blood pressure. Using two selective markers of telocytes, CD34 and c‐kit for immunohistochemical analysis, we found that telocytes are present in the renal medullary interstitium between tubules, vessels or tubular‐blood vessels. Compared to the renal cortex, telocytes are more enriched in the renal medulla. Their long prolongations could be seen in the interstitium along the tubules or blood vessels. Using confocal microscopy, colocalization of CD34 and c‐kit are mainly detected around different tubules or vessels, but not in tubular or vascular cells. Electron microscopy found that many renal medullary telocytes had at least 3 prolongations, where a number of telopods were shown. In some interstitial space with enriched telopods, moreover, a large number of exosomes could be found. We also isolated and cultured telocytes, which were purified from renal medullary interstitial cells used in our laboratories by MiniMACS Cell Separator with anti‐c‐kit antibody beads‐loaded column. In culture, these cells showed typical telocyte features such as more than 3 prolongations, telopods and project connections between cells. Confocal microscopy showed that most cells through MiniMacs column could be double stained by antibodies against c‐kit and CD34, but the original renal medullary institial cells had much fewer cells stained, which was also confirmed by flow cytometry. In a group of mice with 2 kidney‐1 clip (2K1C) hypertensive model, we found that compared to the unclipped kidney, the clipped kidney had largely increased CD34 and c‐kit staining in the renal medullary interstitium, but not much in the renal cortex. These results suggest that telocytes are present in the renal medullary interstitium and in response to chronic ischemia produced by partially clipping renal artery, telocytes are increased. Given that exosomes are substantially enriched in the renal medulla of 2K1C mice, telocytes may be one of the important source for these exosomes that have antihypertensive action (Supported by NIH grants DK054927 and DK120491).

Use of kefir peptide (Kef-1) as an emerging approach for the treatment of oxidative stress and inflammation in 2K1C mice.

The benefits of kefir consumption are partially due to the rich composition of bioactive molecules released from its fermentation. Angiotensin-converting enzyme (ACE) inhibitors are bioactive molecules with potential use in the treatment or prevention of hypertension, heart failure, and myocardial infarction. Here, the in vivo actions of the Kef-1 peptide, an ACE inhibitor derived from kefir, were evaluated in an angiotensin II-dependent hypertension model. The Kef-1 peptide showed a potential anti-hypertensive effect. Additionally, Kef-1 exhibited systemic antioxidant and anti-inflammatory activities. In smooth muscle cells (SMCs), the Kef-1 peptide decreased ROS production through the reduced participation of NADPH oxidase and mitochondria. The aorta of 2K1C mice treated with Kef-1 showed lesser wall-thickening and partial restoration of the endothelial structure. In conclusion, these novel findings highlight the in vivo biological potential of this peptide demonstrating that Kef-1 may be a relevant nutraceutical treatment for cardiovascular diseases.

Angiotensin II augments renal vascular smooth muscle soluble GC expression via an AT1 receptor-forkhead box subclass O transcription factor signalling axis.

Reduced renal blood flow triggers activation of the renin-angiotensin-aldosterone system (RAAS) leading to renovascular hypertension. Renal vascular smooth muscle expression of the NO receptor, soluble GC (sGC), modulates the vasodilator response needed to control renal vascular tone and blood flow. Here, we tested if angiotensin II (Ang II) affects sGC expression via an AT1 receptor-forkhead box subclass O (FoxO) transcription factor dependent mechanism. Using a murine two-kidney-one-clip (2K1C) renovascular hypertension model, we measured renal artery vasodilatory function and sGC expression. Additionally, we conducted cell culture studies using rat renal pre-glomerular smooth muscle cells (RPGSMCs) to test the in vitro mechanistic effects of Ang II treatment on sGC expression and downstream function. Contralateral, unclipped renal arteries in 2K1C mice showed increased NO-dependent vasorelaxation compared to sham control mice. Immunofluorescence studies revealed increased sGC protein expression in 2K1C contralateral renal arteries over sham controls. RPGSMCs treated with Ang II caused a significant up-regulation of sGC mRNA and protein expression as well as downstream sGC-dependent signalling. Ang II signalling effects on sGC expression occurred through an AT1 receptor and FoxO transcription factor-dependent mechanism at both the mRNA and protein expression levels. Renal artery smooth muscle, in vivo and in vitro, up-regulates expression of sGC following RAAS activity. In both cases, up-regulation of sGC leads to increased downstream cGMP signalling, suggesting a previously unrecognized protective mechanism to improve renal blood flow in the uninjured contralateral renal artery. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Effects of Angiotensin II Type 1A Receptor on ACE2, Neprilysin and KIM-1 in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension.

Activation of the renin angiotensin system plays a pivotal role in the regulation of blood pressure, which is mainly attributed to the formation of angiotensin-II (Ang II). The actions of Ang II are mediated through binding to the Ang-II type 1 receptor (AT1R) which leads to increased blood pressure, fluid retention, and aldosterone secretion. In addition, Ang II is also involved in cell injury, vascular remodeling, and inflammation. The actions of Ang II could be antagonized by its conversion to the vasodilator peptide Ang (1–7), partly generated by the action of angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Previous studies demonstrated increased urinary ACE2 shedding in the db/db mouse model of diabetic kidney disease. The aim of the study was to investigate whether renal and urinary ACE2 and NEP are altered in the 2K1C Goldblatt hypertensive mice. Since AT1R is highly expressed in the kidney, we also researched the effect of global deletion of AT1R on renal and urinary ACE2, NEP, and kidney injury marker (KIM-1). Hypertension and albuminuria were induced in AT1R knock out (AT1RKO) and WT mice by unilateral constriction of the renal artery of one kidney. The 24 h mean arterial blood pressure (MAP) was measured using radio-telemetry. Two weeks after 2K1C surgery, MAP and albuminuria were significantly increased in WT mice compared to AT1RKO mice. Results demonstrated a correlation between MAP and albuminuria. Unlike db/db diabetic mice, ACE2 and NEP expression and activities were significantly decreased in the clipped kidney of WT and AT1RKO compared with the contralateral kidney and sham control (p < 0.05). There was no detectable urinary ACE2 and NEP expression and activity in 2K1C mice. KIM-1 was significantly increased in the clipped kidney of WT and AT1KO (p < 0.05). Deletion of AT1R has no effect on the increased urinary KIM-1 excretion detected in 2K1C mice. In conclusion, renal injury in 2K1C Goldblatt mouse model is associated with loss of renal ACE2 and NEP expression and activity. Urinary KIM-1 could serve as an early indicator of acute kidney injury. Deletion of AT1R attenuates albuminuria and hypertension without affecting renal ACE2, NEP, and KIM-1 expression.

Excessive dietary salt promotes aortic stiffness in murine renovascular hypertension.

Renovascular hypertension is characterized by activation of the renin-angiotensin-aldosterone system, blunted natriuretic responses, and elevated sympathetic nerve activity. Excess dietary salt intake exaggerates arterial blood pressure (ABP) in multiple models of experimental hypertension. The present study tested whether a high-salt diet exaggerated ABP and vascular dysfunction in a 2-kidney, 1-clip (2K1C) murine model. Male C57BL/6J mice (8–12 wk) were randomly assigned, and fed a 0.1% or 4.0% NaCl diet, and instrumented with telemetry units to measure ABP. Then, the 2K1C model was produced by placing a cuff around the right renal artery. Systolic, diastolic, and mean ABP were significantly higher in mice fed 4.0% vs. 0.1% NaCl at 1 wk but not after 3 wk. Interestingly, 2K1C hypertension progressively increased arterial pulse pressure in both groups; however, the magnitude was significantly greater in mice fed 4.0% vs. 0.1% NaCl at 3 wk. Moreover, pulse wave velocity was significantly greater in 2K1C mice fed 4.0% vs. 0.1% NaCl diet or sham-operated mice fed either diet. Histological assessment of aortas indicated no structural differences among groups. Finally, endothelium-dependent vasodilation was significantly and selectively attenuated in the aorta but not mesenteric arteries of 2K1C mice fed 4.0% NaCl vs. 0.1% NaCl or sham-operated control mice. The findings suggest that dietary salt loading transiently exaggerates 2K1C renovascular hypertension but promotes chronic aortic stiffness and selective aortic vascular dysfunction.NEW & NOTEWORTHY High dietary salt exaggerates hypertension in multiple experimental models. Here we demonstrate that a high-salt diet produces a greater increase in arterial blood pressure at 1 wk after induction of 2-kidney, 1-clip (2K1C) hypertension but not at 3 wk. Interestingly, 2K1C mice fed a high-salt diet displayed an exaggerated pulse pressure, elevated pulse wave velocity, and reduced endothelium-dependent vasodilation of the aorta but not mesenteric arteries. These findings suggest that dietary salt may interact with underlying cardiovascular disease to promote selective vascular dysfunction and aortic stiffness.

Abstract P2036: Effects of Angiotensin II Type 1 A Receptor (AT1aR) on Renal and Urinary Biomarkers of Acute Kidney Injury in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension

Renin angiotensin system (RAS) plays a key role in the pathogenesis and progression of hypertension and renal injury. Angiotensin-II (Ang-II) type 1 receptor (AT1R) mediates the actions of Ang II to increase blood pressure, fluid retention, and aldosterone secretion. Ang II is involved in cell injury, vascular remodeling, and inflammation by releasing inflammatory and pro-inflammatory mediators. The effects of Ang II could be counteracted through its catabolism into the vasodilator peptide Ang (1-7) by angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Diabetes and high blood pressure are the major causes of chronic kidney disease which could lead to end stage renal disease. Our previous studies demonstrated that shedding of renal ACE2 and NEP in the urine of db / db diabetic mice could act as biomarkers for diabetic kidney disease. The aim of the study is to test the hypothesis that there is increased renal ACE2 and NEP shedding in the urine of two-kidney one clip (2K1C) model of renovascular hypertension. In addition, we investigated the effects of deleting AT1aR on these RAS components and on kidney injury molecule-1 (KIM-1). Renovascular hypertension was induced in AT1aR knock out (KO) and WT mice by 2K1C surgery. Blood pressure (BP) measurement by radio-telemetry revealed significant increase of 46.1± 3.6 mmHg in 2K1C mice compared to control mice (p&lt;0.001). Deleting AT1aR significantly decreased the BP in 2K1C mice compared to 2K1C WT mice (69.86 ± 0.19 vs. 151.69 ± 12.22 mmHg, p&lt;0.001). Renal expression and activity of ACE2 and NEP were significantly decreased in the clipped kidney of WT and AT1KO group (p&lt;0.05). In contrast to diabetes, there was no detectable urinary ACE2 and NEP expression and activity in 2KIC mice. In addition, urinary expression of KIM-1 was increased in the clipped kidney. Although deleting AT1R attenuated albuminuria and hypertension, it did not altered urinary expression of KIM-1. These results suggest that the decreased renal NEP and ACE2 in the clipped kidney of 2K1C model may thus worsen kidney injury via impairment of Ang (1-7) formation. In conclusion, urinary KIM-1 could serve as an early indicator of non-diabetic acute kidney injury. Urinary ACE2 and NEP could be used as biomarker of diabetic kidney disease.

Renal sensory nerves increase sympathetic nerve activity and blood pressure in 2-kidney 1-clip hypertensive mice.

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to define the function, anatomy, and contribution of mouse renal sensory neurons to a renal nerve-dependent model of hypertension. First, electrical stimulation of mouse renal afferent nerves produced frequency-dependent increases in ABP that were eliminated by ganglionic blockade. Stimulus-triggered averaging revealed renal afferent stimulation significantly increased splanchnic, renal, and lumbar sympathetic nerve activity (SNA). Second, kidney injection of wheat germ agglutinin into male C57Bl6 mice (12-14 wk; Jackson Laboratories) produced ipsilateral labeling in the T11-L2 dorsal root ganglia. Next, 2-kidney 1-clip (2K1C) hypertension was produced in male C57Bl6 mice (12-14 wk; Jackson Laboratories) by placement of a 0.5-mm length of polytetrafluoroethylene tubing around the left renal artery. 2K1C mice displayed an elevated ABP measured via telemetry and a greater fall in mean ABP to ganglionic blockade at day 14 or 21 vs. day 0. Renal afferent discharge was significantly higher in 2K1C-clipped vs. 2K1C-unclipped or sham kidneys. In addition, 2K1C-clipped vs. 2K1C-unclipped or sham kidneys had lower renal mass and higher mRNA levels of several proinflammatory cytokines. Finally, both ipsilateral renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33 mM capsaicin) at time of clipping resulted in lower ABP of 2K1C mice at day 14 or 21. These findings suggest mouse renal sensory neurons are activated to increase SNA and ABP in 2K1C hypertension. NEW & NOTEWORTHY This study documents the function, anatomy, and contribution of mouse renal sensory nerves to neurogenic hypertension produced by renal stenosis. Activation of renal afferents increased sympathetic nerve activity and blood pressure. Renal afferent activity was elevated in hypertensive mice, and renal afferent denervation lowered blood pressure. Clinically, patients with renal stenosis have been excluded from clinical trials for renal denervation, but this study highlights the potential therapeutic efficacy to target renal nerves in these patients.

EXCESSIVE DIETARY SALT PROMOTES AORTIC STIFFNESS IN 2K1C HYPERTENSION

Renal artery stenosis is the most common cause of secondary hypertension. The 2‐kidney‐1‐clip (2K1C) model of renal stenosis is associated with elevated arterial blood pressure (ABP), activation of the sympathetic nervous system, and blunted reno‐renal reflexes and natriuretic responses. Excess dietary salt intake has multiple actions on the cardiovascular system and exacerbates ABP in some models of hypertension. The present study tested the hypothesis that 2K1C mice exhibit salt sensitive hypertension and worsening cardiovascular responses to high salt feeding. Male C57Bl6 mice (8–12 weeks) were fed 0.1% NaCl or 4% NaCl diets and ABP was continuously monitored using telemetry. 2K1C hypertension was produced by placing a 0.5mm length of PTFE tubing around the right renal artery. Baseline 24‐h mean ABP was not different between 0.1% versus 4.0% NaCl fed mice (101±1 vs 99±1 mmHg, respectively; n=8/group). 2K1C significantly increased ABP in both groups; however, 24‐h mean ABP at 3 weeks post‐renal clipping was not different between mice fed 0.1% vs 4.0% NaCl (122±3 vs. 124±6 mmHg, respectively; n=8/group). In addition, renal artery stenosis significantly increased arterial pulse pressure (systolic‐diastolic) in 0.1% NaCl (Day 0: 35±1 vs Day 21: 42±1 mmHg, P&lt;0.05) and 4.0% NaCl (Day 0: 36±1 vs Day 21: 49±3 mmHg, P&lt;0.05). Interestingly, the magnitude of the increase in arterial pulse pressure was significantly greater in mice fed 4.0% vs 0.1% NaCl (Δ14±3 vs Δ7±2 mmHg, P&lt;0.05). This suggests dietary salt intake promotes aortic stiffness in the 2K1C model. To test this, endothelial‐dependent vasodilatory responses were measured using myography in sham and 2K1C mice fed 0.1% and 4.0% NaCl diets for 3 weeks. 4.0% NaCl 2K1C mice exhibited significantly blunted aortic vasodilation responses versus 2K1C mice fed 0.1% NaCl or sham mice fed 0.1% or 4.0% NaCl. These effects were not observed in the mesenteric arteries of the same animal. In vivo, pulse wave velocity (PWV) measurements revealed a significantly higher PWV value in 2K1C mice fed 4.0% NaCl versus 2K1C fed 0.1% NaCl (3.97±0.19; n=7 vs. 3.18±0.19; n=8, P&lt;0.01) or sham fed 0.1% or 4.0% NaCl (3.21±0.19; n=7 vs. 3.01±0.19; n=7). We conclude that high dietary salt does not exaggerate the chronic hypertension in 2K1C mice but produces a specific endothelial‐dependent aortic stiffness.Support or Funding InformationRO1 HL128388This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Sensory Nerves Increase Blood Pressure and Sympathetic Nerve Activity in 2‐Kidney 1‐Clip Hypertensive Mice

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to determine whether renal sensory nerves contribute to the 2‐Kidney‐1‐Clip (2K1C) model of hypertension. 2K1C hypertension was produced in male C57B16 mice (12–14 weeks, Jackson Laboratories) by placement of a 0.5mm length of PTFE tubing (ID: 0.008” x OD: 0.014”) around the left renal artery. 2K1C mice (n=10) displayed an elevated ABP measured via telemetry (Day 0: 96 ± 1mmHg vs Day 14: 120 ± 3mmHg; P&lt;0.01). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 14 vs Day 0 (Day 0: −35 ± 2mmHg vs Day 14: −57 ± 5mmHg; P&lt;0.01). 2K1C‐clipped kidneys vs 2K1C‐unclipped kidneys had lower mass (105 ± 14mg vs 173 ± 8mg, respectively; P&lt;0.05) and higher mRNA levels of several pro‐inflammatory cytokines (IL1B, IL2, IL10, TNFa; P&lt;0.05). Renal afferent discharge was significantly higher in 2K1C‐clipped kidneys vs 2K1C‐unclipped vs sham mice (2K1C‐clipped: 3.4 ± 0.9 Hz vs 2K1C‐unclipped: 0.7 ± 0.3Hz vs control: 0.3 ± 0.1 Hz, n=6/group; P&lt;0.05). Furthermore, electrical stimulation of renal afferent nerves in control mice (n=6) produced a frequency‐dependent increase in ABP (5Hz: 4 ± 1mmHg, 10Hz: 6 ± 1mmHg, 20Hz: 11 ± 2mmHg; P&lt;0.05). These responses were eliminated after ganglionic blockade with 5mg/kg chlorisondamine (5Hz: 1 ± 1mmHg, 10Hz: 1 ± 1mmHg, 20Hz: 1 ± 1mmHg, n=4/group; P&lt;0.05). Stimulus‐triggered averaging of SNA during stimulation of renal afferent nerves (1 Hz, 200uA) revealed significant (P&lt;0.05, n=4/group) increases in splanchnic (179 ± 11%), renal (195 ± 15%), and lumbar (167 ± 9%) SNA. Interestingly, the latency to the peak SNA (142 ± 10ms) suggests supraspinal pathways mediate the sympathoexcitatory response. Finally, both total renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33mM capsaicin) at time of clipping resulted in lower ABP than 2K1C mice at Day 14 (2K1C: 120 ± 3mmHg, phenol: 106 ± 3mmHg, capsaicin: 108 ± 3mmHg, n=6–10/group; P&lt;0.05). These findings suggest 2K1C hypertension depends on renal sensory nerves and elevated SNA via supraspinal pathways.Support or Funding InformationNIH grant HL113270, AHA Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of Bone Marrow-Derived Cells Angiotensin (Ang) II Type 1 Receptor by Ang II Promotes Atherosclerotic Plaque Vulnerability.

Angiotensin (Ang) II triggers vulnerable atherosclerotic plaque development. Bone marrow (BM)-derived cells are key players in atherogenesis but whether Ang II induces plaque vulnerability directly through Ang II type 1 receptor (AT1R) activation on these cells remains to be clarified. In the present study, we investigated whether a lack of AT1R on BM-derived cells might affect Ang II-mediated vulnerable plaque development. The 2-kidney, 1-clip (2K1C) model (Ang II-dependent mouse model of advanced atherosclerosis and vulnerable plaques) was generated in ApoE−/− mice transplanted with AT1aR−/− or AT1aR+/+ BM. Plasma cholesterol as well as hepatic mRNA expression levels of genes involved in cholesterol metabolism were significantly lower in 2K1C mice transplanted with AT1aR−/− BM than in controls. Atherosclerotic lesions were significantly smaller in AT1aR−/− BM 2K1C mice (−79% in the aortic sinus and −71% in whole aorta compared to controls). Plaques from AT1aR−/− BM 2K1C mice exhibited reduced lipid core/fibrous cap and macrophage/smooth muscle cells ratios (−82% and −88%, respectively), and increased collagen content (+70%), indicating a more stable phenotype. Moreover, aortic mRNA levels of pro-inflammatory cytokines IL-12p35, IL-1β, and TNF-α were significantly reduced in AT1aR−/− BM 2K1C mice. No significant differences in either the number of circulating Ly6Chigh inflammatory monocytes and Ly6Clow resident anti-inflammatory monocyte subsets, or in mRNA levels of aortic M1 or M2 macrophage markers were observed between the two groups. No significant differences were observed in splenic mRNA levels of T cell subsets (Th1, Th2, Th17 and Treg) markers between the two groups. In conclusion, direct AT1R activation by Ang II on BM-derived cells promotes hepatic mRNA expression of cholesterol-metabolism-related genes and vascular mRNA expression of pro-inflammatory cytokines that may lead to plaque instability.

Abstract 081: Renal Sensory Nerves Increase Blood Pressure and Sympathetic Nerve Activity in 2-Kidney 1-Clip Hypertensive Mice

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to determine whether renal sensory nerves contributed to the 2-Kidney-1-Clip (2K1C) model of hypertension. 2K1C hypertension was produced in male C57Bl6 mice (12-14 weeks, Jackson Laboratories) by placement of a 0.5mm length of PTFE tubing (ID: 0.008” x OD: 0.014”) around the left renal artery. 2K1C mice (n=6) displayed an elevated ABP measured via telemetry (Day 0: 96±4mmHg vs Day 14: 115±3mmHg, P&lt;0.05). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 14 vs Day 0 (Day 0: -38±4mmHg vs Day 14: -51±4mmHg, P&lt;0.05). Ipsilateral vs contralateral kidneys of 2K1C mice had lower mass (0.072±0.01g vs 0.163±0.02g, respectively; P&lt;0.05) and higher mRNA levels of several pro-inflammatory cytokines (IL1B, IL2, IL10, TNFa; P&lt;0.05). Both total renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33mM capsaicin) at time of clipping resulted in a lower ABP than 2K1C mice at Day 14 (2K1C: 115±3mmHg, phenol: 104±2mmHg, capsaicin: 105±3mmHg; P&lt;0.05). Direct recording of renal afferent nerve activity showed significantly greater discharge in 2K1C versus control mice (control: 2.2±1.3Hz vs 2K1C: 61±12Hz, n=3/group; P&lt;0.05). Furthermore, electrical stimulation of renal afferent nerves in control mice produced a frequency-dependent increase in ABP (5Hz: 3±1mmHg, 10Hz: 7±1mmHg, 20Hz: 12±2mmHg, n=4/group; P&lt;0.05). These responses were eliminated after ganglionic blockade with 5mg/kg chlorisondamine (5Hz: 1±1mmHg, 10Hz: 1±1mmHg, 20Hz: 1±1mmHg; P&lt;0.05). Stimulus-triggered averaging of SNA during stimulation of renal afferent nerves (1 Hz, 200uA) revealed significant (P&lt;0.05, n=4/group) increases in splanchnic (221±28%), renal (195+15%), and lumbar (234±32%) SNA. Interestingly, the latency to the peak SNA (142±10ms) suggests supraspinal pathways mediate the sympathoexcitatory response. These findings suggest 2K1C hypertension depends on renal sensory nerves and elevated SNA via supraspinal pathways.

Abstract P239: Toll-Like Receptors 9 (TLR9) Play a Key Role in Cardiac and Vascular Dysfunction Associated With 2-Kydney 1-Clip (2K1C) Hypertension in Mice

Introduction: Renovascular hypertension is the leading cause of secondary hypertension and is also involved in the physiopathogenesis of resistant hypertension. The activation of toll-like receptors 9 (TLR9) was demonstrated to contribute to the increase in arterial pressure (AP) in spontaneously hypertensive rats. We hypothesize that the TLR9 are also activated in two-kidney one- clip (2K1C) hypertensive mice, eliciting cardiac and vascular dysfunction; therefore, contributing to the increase in AP. Methods and Results: C57BL (WT) and TLR9 knockout (TLR9_KO) mice were anesthetized with isoflurane and submitted to 2K1C hypertension by placing a silver clip (0.12 mm) around the left renal artery. After 4 weeks, the cardiac function of mice was evaluated by echocardiography in anesthetized subjects. Following, the direct AP measurement was carried out and the third-order mesenteric resistance arteries (MRA) were removed for vascular function evaluation. All data were compared to Sham-operated WT mice. The AP was remarkably elevated in 2K1C WT mice (133±2 vs 93 ± 4 mmHg), whereas this increase was partially prevented by the absence of the TLR9 (114±5 mmHg). 2K1C hypertension caused cardiac dysfunction in WT mice, displaying decreased ejection fraction (42.6±3.1 vs 52.7±1.5%), fractional shortening (10.1±0.7 vs 13.7±0.9%), stroke volume (25.9±2.2 vs 37.9±1.9 μL) and cardiac output (9.2±0.8 vs 14.6±0.8 mL/min); however, the 2K1K hypertension did not affect the cardiac function in TLR9_KO mice: ejection fraction (54.5±1.6%), fractional shortening (14.5±1.1%), stroke volume (43.4±4.6 μL) and cardiac output (15.2±1.6 mL/min). Vascular dysfunction, characterized by increased contractile response to phenylephrine (Emax: 167.2±2.2 vs 125.9±3.5%) and reduced relaxation to acetylcholine (Emax: 69.3±1.6 vs 87.6±1.4%) and sodium nitroprusside (pD2: 7.3±0.07 vs 7.9±0.05), was observed in 2K1C WT mice, but not in 2K1C TLR9_KO mice (Emax: 127.7±2.9%, 88.5±2.0% and pD2: 7.78±0.06, to phenylephrine, acetylcholine and sodium nitroprusside). Conclusions: TLR9 is involved in both cardiac and vascular dysfunctions of 2K1C mice and also in the increase of AP of this experimental model.

Abstract P422: Two-kidney One Clip (2k1c) Hypertension Reduces Complexity of Heart Rate Variability in Mice

Introduction: Hypertension is the most common chronic cardiovascular disease, being multifactorial in origin and an important cause of morbidity and mortality worldwide. Complex behaviors of heart rate series have been widely recognized and the loss of complexity in heart rate variability (HRV) has been shown to predict adverse cardiovascular outcomes. We hypothesized that two-kidney one clip (2K1C) hypertension reduces the HRV complexity in mice. Methods and Results: C57BL/6 mice were anesthetized with isoflurane and submitted to 2K1C hypertension by placing a silver clip (0.12 mm) around left renal artery. After 4 weeks, mice were implanted with subcutaneous electrocardiogram (ECG) electrodes and allowed to recover for 48 h. On the day of the experiment, the ECG was recorded for 30 minutes in conscious, unrestrained mice. At the end of the recording, arterial pressure (AP) was directly measured in each mouse under isoflurane anesthesia. RR interval time series were generated and the complexity of HRV was determined using detrending fluctuation analysis (DFA) and multiscale entropy (MSE). Mean AP was higher in 2K1C mice (133±2 vs 93±4 mmHg) while the HR was similar between groups. DFA scaling exponents were calculated in short (5 to 15), mid (30 to 200) and long (200 to 1500) window sizes, but only the long-term exponent was different between groups (1.27±0.09 vs 0.89±0.08 in 2K1C and sham mice, respectively). MSE was calculated up to scale 20 and averaged in short (1 to 5) and long (6 to 20) time scales. In both short (0.75±0.16 vs 1.25±0.11) and long (0.76±0.17 vs 1.22±0.09) ranges, entropy is lower in hypertensive mice. Conclusions: The complexity of HRV dynamics was found lower in renovascular hypertensive mice. Both sympathetic and vagal control of the heart seems to be involved in this process, as predictability (MSE) and fractality (DFA) is affected in various temporal scales. Nevertheless, the greatest entropy difference between groups is found at scale 6, which is closely related to respiration.

Running Exercise and Angiotensin II Type I Receptor Blocker Telmisartan Are Equally Effective in Preventing Angiotensin II-Mediated Vulnerable Atherosclerotic Lesions.

The present study was conducted to directly compare the efficacy of running exercise and telmisartan treatment on angiotensin (Ang) II-mediated atherosclerosis and plaque vulnerability. Apolipoprotein E-deficient (ApoE-/-) mice with Ang II-mediated atherosclerosis (2-kidney, 1-clip [2K1C] renovascular hypertension model) were randomized into 3 groups: treadmill running exercise (RUN), telmisartan treatment (TEL), and sedentary untreated controls (SED) for 5 weeks. Atherosclerosis was assessed using histological and immunohistochemical analyses. Gene expression was determined by real-time reverse transcription polymerase chain reaction. TEL but not RUN mice significantly decreased (50%) atherosclerotic lesion size compared to SED. RUN and TEL promoted plaque stabilization to a similar degree in ApoE-/- 2K1C mice. However, plaque composition and vascular inflammatory markers were differently affected: RUN decreased plaque macrophage infiltration (35%), whereas TEL reduced lipid core size (88%); RUN significantly increased aortic peroxisome proliferator-activated receptor (PPAR)-α, -δ, and -γ expression, whereas TEL significantly modulated T-helper 1/T-helper 2 (Th1/Th2) aortic response toward an anti-inflammatory state (decreased aortic interleukin [IL] 2 to IL-10 and IL-2 to IL-13 expression ratios). Plaque smooth muscle cell content was similarly increased (128% and 141%, respectively). Aortic AT1 and AT2 receptor expression as well as aortic CD11c/CD206 and IL-1β/IL-1ra expression ratios were not significantly modulated by either RUN or TEL. Running exercise and telmisartan treatment are equally effective in preventing Ang II-mediated plaque vulnerability but through distinct cellular and molecular mechanisms. Our findings further support the use of exercise training and selective AT1 receptor blocker therapies for atherosclerotic cardiovascular disease prevention.