Kashyap, Sonu

Renal artery stenosis (RAS) is a common disorder associated with both cardiovascular and renal morbidity and mortality. Recent studies have shown that stenting of the stenotic kidney fails to improve renal or cardiovascular outcomes, prompting a search for more effective therapies. Strain‐specific variability in renal outcomes is a well recognized problem associated with murine models of chronic injury. The objective of this study was to define strain specific differences in renal outcome and correlation with parameters of injury in a murine model of RAS. We used a 0.25 mm polytetrafluoroethylene cuff to establish RAS in mice (n=292 total). We examined the correlation between age, time following surgery, body weight, blood pressure, heart and kidney weights, ratio of STK/CLK weight, and plasma angiotensin level in 3 murine strains. Percent atrophy in the STK as a function of time following RAS surgery is summarized in the table below: Strain 1 Week 2 Week 4 Week 6 Week 129 21% 31% XX 45% C57BLKS/J 25% 51% 63% 72% C57BL/6J XX XX 39% 41% The right kidney weight and the STK/CLK weight ratio correlated with %atrophy in all strains, with the strongest correlation observed in C57BL/6J mice. In C57BL/6J mice, blood pressure correlated with %atrophy. CLK weight correlated with heart weight, with the strongest correlation observed in 129Sv mice. In BLKS mice, the time following surgery and age correlated with CLK weight and %atrophy in the STK; blood pressure correlated with plasma angiotensin level. We conclude that RAS produces severe chronic renal injury in the STK of all strains studied, with the most severe injury observed in C57BLKS/J mice.The STK/CLK weight ratio correlated most strongly with %atrophy and can be used to predict development of chronic renal damage in all strains. Hypertrophy of the CLK, as measured by CLK weight, strongly correlated with heart weight.

Aim The aim of this study was to define the functional correlates of reversibilitiy following restoration of blood flow in the murine 2K1C model of renovascular hypertension. Method : Renal artery stenosis surgery (RAS) was performed by placement of a polytetrafluoroethylene cuff on the right renal artery (N=18). Sham surgery was performed without cuff placement (N=6). In two groups the cuff was removed at 7 days (7D cuff‐off, N=6) and 14 days (14D cuff‐off, N=6) after surgery. In the RAS group, cuff was left in place for 28 days (28D RAS, N=6). Kidneys were harvested at 28 days for assessment of histology and microvessel density (MVD). Renal blood perfusion and oxygenation were measured using 16.4T MRI. Blood pressure measurements were done using tail cuff method. Results In 7D cuff‐off group, baseline intra‐renal perfusion (891±142.8 ml/100g/min) was significantly reduced (311±73.4 ml/100g/min, p<0.0001) following cuff placement. Perfusion was restored to normal level (810±81.2 ml/100g/min) at 28 days following cuff removal at 7 days after RAS. In 14D cuff‐off group, baseline intra‐renal perfusion (846±74.5 ml/100g/min) was also significantly reduced (304±45.9 ml/100g/min, p=0.0002) following cuff placement with non‐significant improvement (579±112.9 ml/100g/min) at 28 days after cuff removal at 14 days after RAS. Baseline intra‐renal perfusion (905±54.9 ml/100g/min) in the 28D RAS group was significantly reduced (455±56.4 ml/100g/min, p<0.0034) following cuff placement and the reduction was maintained throughout the study period (478±22.2 ml/100g/min). 7D cuff‐off group (142±8.2 mg) showed no significant difference in kidney weights measured at 28 days compared to the sham group (154±4.8 mg), but was significantly higher compared to the 28D RAS group (67±10.5 mg, p=0.0010). The kidney weights of the 14D cuff‐off group (86±18.3 mg) measured at 28 days was not significantly different from the 28D RAS group. The 7D cuff‐off group had a significantly lower % atrophy (7±2.2%, p=0.0042) and higher MVD count (152±6.5, p=0.0045) compared to 28D RAS group (% atrophy 66±13%, MVD count 78±15.2). The 14D cuff‐off group had severe atrophy (56±16.6%) and reduced MVD count (94±20.1) similar to that seen in 28D RAS group. 50% of mice that developed hypoxia at day 3 had an improvement in their oxygenation after cuff removal at day 7, whereas, mice undergoing cuff removal at day 14 and 28 day ras mice remained hypoxic. Conclusion Based on these results, we conclude that the renal architecture and microvessel density are preserved when the cuff is removed no longer than 7 days post surgery. We Identified that 2 weeks after cuff placement is the “point of no return” in the renal injury in murine RAS model due to their reduction in microvessel density. Studies to identify biomarkers associated with irreversible renal injury in this model are currently being performed. Support or Funding Information NIH AI 100911 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Recent studies have implicated CCR2 ‐ CCL2 signaling in macrophage influx and development of chronic renal disease in human and experimental renal artery stenosis (RAS). We sought to test the hypothesis that development of chronic renal disease would be abrogated in CCR2 knockout (KO) mice. We established RAS in CCR2 KO and WT mice (n=10) through placement of a cuff on the right renal artery We have shown that the cuff reproducibly reduces blood flow by 65% to the kidney. Sham mice underwent manipulation of the right renal artery without cuff placement. Both CCR2 KO and WT mice developed hypertension, which persisted throughout the 4 week study. There was no difference in renal atrophy between CCR2 KO (53+//‐14.6% cortical surface area) and WT (61.2%+/‐14%) mice. Furthermore, there were no significant differences in F4/80+ macrophage infiltration (12.78+/‐4.9% surface area for CCR2 KO mice and 13.13+/‐ 3.1% for WT mice) or CD3+ T cell infiltration. Infiltrating macrophages were predominantly iNOS+ M1 phenotype; few arginase+ cells were observed in any group. Urine protein excretion was similar in CCR2 KO and WT mice. CCL was induced to a similar extent in CCR2 KO and WT mice. Furthermore, no differences in CCL5, CCL7, CCR5, CCR1, or iNOS expression was observed between CCR2 KO and WT mice. We conclude that macrophage infiltration and chronic renal damage occurs in mice with deletion of CCR2. Future studies will identify signaling pathway(s) responsible for macrophage influx and chronic renal damage in this model. Grant Funding Source : AI‐100911

Background: Cardiac tissue possesses high energetic requirements. During development of heart failure there is a shift from fatty acid oxidation to the utilization of other substrates. This transition leads to the decline in NAD/NADH ratio, which increases susceptibility to stress. CD38 is the main NAD + -consuming enzyme responsible for NAD + levels regulation in tissue and therefore represents a possible therapeutic target in such conditions. Objective: To investigate the role of CD38 in cardiovascular performance and its potential use as a therapeutic target in obesity-induced heart failure. Results: When comparing adult Wild-Type (WT) and CD38 Knock-out (KO) mice we observed that the latter had higher exercise performance, increased heart rate variability (HRV) and decreased heart rate (HR). When comparing WT and CD38 catalytically inactive (CI) mice we observed the same results, showing its relation with CD38 catalytic activity. WT mice treated with an antibody which blocks CD38 activity showed improved exercise capacity and HRV. In both genetically modified or antibody treated groups, NAD+ levels were significantly increased in heart. In order to explore the NAD + role in these findings, we compared WT control and CD38KO treated with FK866 (Nampt inhibitor) and observed that NAD+ levels in heart decreased as well as exercise capacity. We then aimed to demonstrate CD38 role as a target for therapeutic intervention in a model of cardiac strain induced by obesity in aged mice. We compared WT and CD38 CI aged mice, placed in normal diet (ND) or high fat diet (HFD). WT animals fed HFD showed decreased exercise capacity and this effect is reversed in CD38 CI fed HFD. Similarly, WT placed in HFD developed diastolic dysfunction seen as a significant increase in E/e’ ratio, and CD38 CI placed in HFD were protect against the development of this dysfunction. Mechanistically, we observed that blockage of CD38 resulted in decreased protein acetylation and increased SERCA expression. Conclusion: CD38 plays a role in cardiovascular performance by regulation of NAD + homeostasis. In a model of obesity-induced diastolic dysfunction NAD+-boosting by blocking CD38 activity led to protection by up-regulating SERCA expression.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of Pkd1 RC/RC mice, a model of ADPKD. Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that cleaves inhibitory IGF binding proteins (IGFBPs), increasing the local bioactivity of IGF-1, was highly induced in the kidney of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed that PAPP-A transcription in ADPKD was mainly regulated through the cAMP/CREB/CBP/p300 pathway. Pappa deficiency effectively inhibited the development of cysts in the Pkd1 RC/RC mice. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with a monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1 RC/RC mice and ex vivo in embryonic kidneys. These data indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a therapeutic strategy for ADPKD that involves the inhibition of PAPP-A.

Purpose. Type 2 diabetes is the leading cause of end stage renal disease in the United States. Atherosclerotic renal artery stenosis is commonly observed in diabetic patients and impacts the rate of renal and cardiovascular disease progression. We hypothesized that renal artery stenosis contributes to bilateral renal disease in diabetics. In our original study, we found that leptin-deficient diabetic (db/db) mice subjected to RAS developed severe and bilateral renal disease, with the contralateral (uncuffed) kidney showing features reminiscent of progressive diabetic nephropathy. In non-diabetic mice (WT), the cuffed kidney developed progressive atrophy, but the contralateral kidney showed minimal histopathologic alterations. In doing these studies, we observed increased sudden death in db/db mice with RAS, but not in WT mice with RAS. The objective of this study was to characterize the aortic and cardiac phenotype of db/db mice subjected to RAS. Methods . We developed a murine model of renal artery stenosis by placement of a polytetrafluoroethylene cuff on the right renal artery in db/db mice. We studied 109 WT and 95 db/db mice subjected to Renal artery stenosis (RAS) or sham surgery. Results. The mortality rate of db/db RAS mice was about 23.5%, whereas only 1.5% deaths were observed in WT RAS mice. Interestingly, 60% of mortality in the db/db mice occurred in the first two weeks following RAS surgery. Necropsy showed massive intrathoracic hemorrhage associated with aortic dissection. Aortas from db/db RAS mice showed more smooth muscle dropout, medial disruption, and hemorrhage than aortas from WT mice with RAS. Cardiac tissue from db/db RAS mice had more fibrosis than did cardiac tissue from WT RAS mice. Conclusions . Db/db mice subjected to RAS are prone to develop fatal aortic dissection, which is not observed in WT mice with RAS. The db/db RAS model provides the basis for future studies directed towards defining basic mechanisms underlying the interaction of hypertension and diabetes on the development of aortic lesions.

Purpose. Type 2 diabetes is the leading cause of end stage renal disease in the United States. Atherosclerotic renal artery stenosis is commonly observed in diabetic patients and impacts the rate of renal and cardiovascular disease progression. We hypothesized that renal artery stenosis contributes to bilateral renal disease in diabetics. In our original study, we found that leptin-deficient diabetic (db/db) mice subjected to RAS developed severe and bilateral renal disease, with the contralateral (uncuffed) kidney showing features reminiscent of progressive diabetic nephropathy. In non-diabetic mice (WT), the cuffed kidney developed progressive atrophy, but the contralateral kidney showed minimal histopathologic alterations. In doing these studies, we observed increased sudden death in db/db mice with RAS, but not in WT mice with RAS. The objective of this study was to characterize the aortic and cardiac phenotype of db/db mice subjected to RAS. Methods . We developed a murine model of renal artery stenosis by placement of a polytetrafluoroethylene cuff on the right renal artery in db/db mice. We studied 109 WT and 95 db/db mice subjected to Renal artery stenosis (RAS) or sham surgery. Results. The mortality rate of db/db RAS mice was about 23.5%, whereas only 1.5% deaths were observed in WT RAS mice. Interestingly, 60% of mortality in the db/db mice occurred in the first two weeks following RAS surgery. Necropsy showed massive intrathoracic hemorrhage associated with aortic dissection. Aortas from db/db RAS mice showed more smooth muscle dropout, medial disruption, and hemorrhage than aortas from WT mice with RAS. Cardiac tissue from db/db RAS mice had more fibrosis than did cardiac tissue from WT RAS mice. Conclusions . Db/db mice subjected to RAS are prone to develop fatal aortic dissection, which is not observed in WT mice with RAS. The db/db RAS model provides the basis for future studies directed towards defining basic mechanisms underlying the interaction of hypertension and diabetes on the development of aortic lesions.

Purpose. Type 2 diabetes is the leading cause of end stage renal disease in the United States. Atherosclerotic renal artery stenosis is commonly observed in diabetic patients and impacts the rate of renal and cardiovascular disease progression. We sought to test the hypothesis that renovascular hypertension, induced by unilateral renal artery stenosis, exacerbates cardiac remodeling in leptin-deficient (db/db) mice, which serves as a model of human type II diabetes. Methods. We employed a murine model of renovascular hypertension through placement of a polytetrafluoroethylene cuff on the right renal artery in db/db mice. We studied 109 wild-type (non-diabetic, WT) and 95 db/db mice subjected to renal artery stenosis (RAS) or sham surgery studied at 1, 2, 4, and 6+ weeks following surgery. Cardiac remodeling was assessed by quantitative analysis of the percent of myocardial surface area occupied by interstitial fibrosis tissue, as delineated by trichrome stained slides. Aortic pathology was assessed by histologic sampling of grossly apparent structural abnormalities or by section of ascending aorta of vessels without apparent abnormalities. Results. We noted an increased mortality in db/db mice subjected to RAS. The mortality rate of db/db RAS mice was about 23.5%, whereas the mortality rate of WT RAS mice was only 1.5%. Over 60% of mortality in the db/db mice occurred in the first two weeks following RAS surgery. Necropsy showed massive intrathoracic hemorrhage associated with aortic dissection, predominantly in the ascending aorta and proximal descending aorta. Aortas from db/db RAS mice showed more smooth muscle dropout, loss of alpha smooth muscle actin expression, medial disruption, and hemorrhage than aortas from WT mice with RAS. Cardiac tissue from db/db RAS mice had more fibrosis than did cardiac tissue from WT RAS mice. Conclusions. db/db mice subjected to RAS are prone to develop fatal aortic dissection, which is not observed in WT mice with RAS. The db/db RAS model provides the basis for future studies directed towards defining basic mechanisms underlying the interaction of hypertension and diabetes on the development of aortic lesions.

Renovascular hypertension (RVH) is a common cause of both cardiovascular and renal morbidity and mortality. In renal artery stenosis (RAS), atrophy in the stenotic kidney is associated with an influx of macrophages and other mononuclear cells. We tested the hypothesis that chemokine receptor 2 (CCR2) inhibition would reduce chronic renal injury by reducing macrophage influx in the stenotic kidney of mice with RAS. We employed a well-established murine model of RVH to define the relationship between macrophage infiltration and development of renal atrophy in the stenotic kidney. To determine the role of chemokine ligand 2 (CCL2)/CCR2 signaling in the development of renal atrophy, mice were treated with the CCR2 inhibitor RS-102895 at the time of RAS surgery and followed for 4 wk. Renal tubular epithelial cells expressed CCL2 by 3 days following surgery, a time at which no significant light microscopic alterations, including interstitial inflammation, were identified. Macrophage influx increased with time following surgery. At 4 wk, the development of severe renal atrophy was accompanied by an influx of inducible nitric oxide synthase (iNOS)+ and CD206+ macrophages that coexpressed F4/80, with a modest increase in macrophages coexpressing arginase 1 and F4/80. The CCR2 inhibitor RS-102895 attenuated renal atrophy and significantly reduced the number of dual-stained F4/80+ iNOS+ and F4/80+ CD206+ but not F4/80+ arginase 1+ macrophages. CCR2 inhibition reduces iNOS+ and CD206+ macrophage accumulation that coexpress F4/80 and renal atrophy in experimental renal artery stenosis. CCR2 blockade may provide a novel therapeutic approach to humans with RVH.