Fawn-hooded Hypertensive Rat Research Articles

Empagliflozin Is Not Renoprotective in Non-Diabetic Rat Models of Chronic Kidney Disease.

Gliflozins (sodium-glucose transporter-2 inhibitors) exhibited renoprotective effects not only in diabetic but also in non-diabetic patients with chronic kidney disease (CKD). Controversial results were reported in experimental non-diabetic models of CKD. Therefore, we examined empagliflozin effects in three CKD models, namely, in fawn-hooded hypertensive (FHH) rats, uninephrectomized salt-loaded (UNX + HS) rats, and in rats with Goldblatt hypertension (two-kidney, one-clip 2K1C) that were either untreated or treated with empagliflozin (10 mg/kg/day) for eight weeks. Plethysmography blood pressure (BP) was recorded weekly, and renal parameters (proteinuria, plasma urea, creatinine clearance, and sodium excretion) were analyzed three times during the experiment. At the end of the study, blood pressure was also measured directly. Markers of oxidative stress (TBARS) and inflammation (MCP-1) were analyzed in kidney and plasma, respectively. Body weight and visceral adiposity were reduced by empagliflozin in FHH rats, without a significant effect on BP. Experimentally induced CKD (UNX + HS and 2K1C) was associated with a substantial increase in BP and relative heart and kidney weights. Empagliflozin influenced neither visceral adiposity nor BP in these two models. Although empagliflozin increased sodium excretion, suggesting effective SGLT-2 inhibition, it did not affect diuresis in any experimental model. Unexpectedly, empagliflozin did not provide renoprotection because proteinuria, plasma urea, and plasma creatinine were not lowered by empagliflozin treatment in all three CKD models. In line with these results, empagliflozin treatment did not decrease TBARS or MCP-1 levels in either model. In conclusion, empagliflozin did not provide the expected beneficial effects on kidney function in experimental models of CKD.

The adducin saga: pleiotropic genomic targets for precision medicine in human hypertension-vascular, renal, and cognitive diseases.

Hypertension is a leading risk factor for stroke, heart disease, chronic kidney disease, vascular cognitive impairment, and Alzheimer's disease. Previous genetic studies have nominated hundreds of genes linked to hypertension, and renal and cognitive diseases. Some have been advanced as candidate genes by showing that they can alter blood pressure or renal and cerebral vascular function in knockout animals; however, final validation of the causal variants and underlying mechanisms has remained elusive. This review chronicles 40 years of work, from the initial identification of adducin (ADD) as an ACTIN-binding protein suggested to increase blood pressure in Milan hypertensive rats, to the discovery of a mutation in ADD1 as a candidate gene for hypertension in rats that were subsequently linked to hypertension in man. More recently, a recessive K572Q mutation in ADD3 was identified in Fawn-Hooded Hypertensive (FHH) and Milan Normotensive (MNS) rats that develop renal disease, which is absent in resistant strains. ADD3 dimerizes with ADD1 to form functional ADD protein. The mutation in ADD3 disrupts a critical ACTIN-binding site necessary for its interactions with actin and spectrin to regulate the cytoskeleton. Studies using Add3 KO and transgenic strains, as well as a genetic complementation study in FHH and MNS rats, confirmed that the K572Q mutation in ADD3 plays a causal role in altering the myogenic response and autoregulation of renal and cerebral blood flow, resulting in increased susceptibility to hypertension-induced renal disease and cerebral vascular and cognitive dysfunction.

Effects of renal sympathetic denervation on the course of congestive heart failure combined with chronic kidney disease: Insight from studies with fawn-hooded hypertensive rats with volume overload induced using aorto-caval fistula

ABSTRACT Background: The coincidence of congestive heart failure (CHF) and chronic kidney disease (CKD) results in poor survival rate. The aim of the study was to examine if renal denervation (RDN) would improve the survival rate in CHF induced by creation of aorto-caval fistula (ACF). Methods: Fawn-hooded hypertensive rats (FHH), a genetic model of spontaneous hypertension associated with CKD development, were used. Fawn-hooded low-pressure rats (FHL), without CKD, served as controls. RDN was performed 4 weeks after creation of ACF and the follow-up period was 10 weeks. Results: We found that intact (non-denervated) ACF FHH exhibited survival rate of 58.8% (20 out of 34 rats), significantly lower than in intact ACF FHL (81.3%, 26/32 rats). In intact ACF FHL albuminuria remained stable throughout the study, whereas in ACF FHH it increased significantly, up to a level 40-fold higher than the basal values. ACF FHL did not show increases in renal glomerular and tubulointerstitial injury as compared with FHL, while ACF FHH exhibited marked increases in kidney injury as compared with FHH. RDN did not improve the survival rate in either ACF FHL or ACF FHH and did not alter the course of albuminuria in ACF FHL. RDN attenuated the albuminuria, but did not reduce the kidney injury in ACF FHH. Conclusions: Our present results support the notion that even modest CKD increases CHF-related mortality. RDN did not attenuate CHF-dependent mortality in ACF FHH, it delayed the progressive rise in albuminuria, but it did not reduce the degree of kidney injury.

Knockout of γ-Adducin Promotes NG-Nitro-L-Arginine-Methyl-Ester-Induced Hypertensive Renal Injury.

Previous studies identified a region on chromosome 1 associated with NG-nitro-L-arginine methyl ester (L-NAME) hypertension-induced renal disease in fawn-hooded hypertensive (FHH) rats. This region contains a mutant γ-adducin (Add3) gene that impairs renal blood flow (RBF) autoregulation, but its contribution to renal injury is unknown. The present study evaluated the hypothesis that knockout (KO) of Add3 impairs the renal vasoconstrictor response to the blockade of nitric oxide synthase and enhances hypertension-induced renal injury after chronic administration of L-NAME plus a high-salt diet. The acute hemodynamic effect of L-NAME and its chronic effects on hypertension and renal injury were compared in FHH 1Brown Norway (FHH 1BN) congenic rats (WT) expressing wild-type Add3 gene versus FHH 1BN Add3 KO rats. RBF was well autoregulated in WT rats but impaired in Add3 KO rats. Acute administration of L-NAME (10 mg/kg) raised mean arterial pressure (MAP) similarly in both strains, but RBF and glomerular filtration rate (GFR) fell by 38% in WT versus 15% in Add3 KO rats. MAP increased similarly in both strains after chronic administration of L-NAME and a high-salt diet; however, proteinuria and renal injury were greater in Add3 KO rats than in WT rats. Surprisingly, RBF, GFR, and glomerular capillary pressure were 41%, 82%, and 13% higher in L-NAME-treated Add3 KO rats than in WT rats. Hypertensive Add3 KO rats exhibited greater loss of podocytes and glomerular nephrin expression and increased interstitial fibrosis than in WT rats. These findings indicate that loss of ADD3 promotes L-NAME-induced renal injury by altering renal hemodynamics and enhancing the transmission of pressure to glomeruli. SIGNIFICANCE STATEMENT: A mutation in the γ-adducin (Add3) gene in fawn-hooded hypertensive rats that impairs autoregulation of renal blood flow is in a region of rat chromosome 1 homologous to a locus on human chromosome 10 associated with diabetic nephropathy. The present results indicate that loss of ADD3 enhanced NG-nitro-L-arginine methyl ester-induced hypertensive renal injury by altering the transmission of pressure to the glomerulus.

Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

We recently reported that the enhanced susceptibility to chronic kidney disease (CKD) in the fawn-hooded hypertensive (FHH) rat is caused, at least in part, by a mutation in γ-adducin (ADD3) that attenuates renal vascular function. The present study explored whether Add3 contributes to the modulation of podocyte structure and function using FHH and FHH.Add3 transgenic rats. The expression of ADD3 on the membrane of primary podocytes isolated from FHH was reduced compared with FHH.Add3 transgenic rats. We found that F-actin nets, which are typically localized in the lamellipodia, replaced unbranched stress fibers in conditionally immortalized mouse podocytes transfected with Add3 Dicer-substrate short interfering RNA (DsiRNA) and primary podocytes isolated from FHH rats. There were increased F/G-actin ratios and expression of the Arp2/3 complexes throughout FHH podocytes in association with reduced synaptopodin and RhoA but enhanced Rac1 and CDC42 expression in the renal cortex, glomeruli, and podocytes of FHH rats. The expression of nephrin at the slit diaphragm and the levels of focal adhesion proteins integrin-α3 and integrin-β1 were decreased in the glomeruli of FHH rats. Cell migration was enhanced and adhesion was reduced in podocytes of FHH rats as well as in immortalized mouse podocytes transfected with Add3 DsiRNA. Mean arterial pressures were similar in FHH and FHH.Add3 transgenic rats at 16 wk of age; however, FHH rats exhibited enhanced proteinuria associated with podocyte foot process effacement. These results demonstrate that reduced ADD3 function in FHH rats alters baseline podocyte pathophysiology by rearrangement of the actin cytoskeleton at the onset of proteinuria in young animals.

Impaired renal hemodynamics and glomerular hyperfiltration contribute to hypertension-induced renal injury

Recently, we reported a mutation in γ-adducin (ADD3) was associated with an impaired myogenic response of the afferent arteriole and hypertension-induced chronic kidney disease (CKD) in fawn hooded hypertensive (FHH) rats. However, the mechanisms by which altered renal blood flow (RBF) autoregulation promotes hypertension-induced renal injury remain to be determined. The present study compared the time course of changes in renal hemodynamics and the progression of CKD during the development of DOCA-salt hypertension in FHH 1BN congenic rats [wild-type (WT)] with an intact myogenic response versus FHH 1BN Add3KO (Add3KO) rats, which have impaired myogenic response. RBF was well autoregulated in WT rats but not in Add3KO rats. Glomerular capillary pressure rose by 6 versus 14 mmHg in WT versus Add3KO rats when blood pressure increased from 100 to 150 mmHg. After 1 wk of hypertension, glomerular filtration rate increased by 38% and glomerular nephrin expression decreased by 20% in Add3KO rats. Neither were altered in WT rats. Proteinuria doubled in WT rats versus a sixfold increase in Add3KO rats. The degree of renal injury was greater in Add3KO than WT rats after 3 wk of hypertension. RBF, glomerular filtration rate, and glomerular capillary pressure were lower by 20%, 28%, and 19% in Add3KO rats than in WT rats, which was associated with glomerular matrix expansion and loss of capillary filtration area. The results indicated that impaired RBF autoregulation and eutrophic remodeling of preglomerular arterioles increase the transmission of pressure to glomeruli, which induces podocyte loss and accelerates the progression of CKD in hypertensive Add3KO rats.

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

Pulling the Hood off Genetic Susceptibility to Hypertensive Renal Disease

A Mutation in γ-Adducin Impairs Autoregulation of Renal Blood Flow and Promotes the Development of Kidney Disease.

The genes and mechanisms involved in the association between diabetes or hypertension and CKD risk are unclear. Previous studies have implicated a role for γ-adducin (ADD3), a cytoskeletal protein encoded by Add3. We investigated renal vascular function in vitro and in vivo and the susceptibility to CKD in rats with wild-type or mutated Add3 and in genetically modified rats with overexpression or knockout of ADD3. We also studied glomeruli and primary renal vascular smooth muscle cells isolated from these rats. This study identified a K572Q mutation in ADD3 in fawn-hooded hypertensive (FHH) rats-a mutation previously reported in Milan normotensive (MNS) rats that also develop kidney disease. Using molecular dynamic simulations, we found that this mutation destabilizes a critical ADD3-ACTIN binding site. A reduction of ADD3 expression in membrane fractions prepared from the kidney and renal vascular smooth muscle cells of FHH rats was associated with the disruption of the F-actin cytoskeleton. Compared with renal vascular smooth muscle cells from Add3 transgenic rats, those from FHH rats had elevated membrane expression of BKα and BK channel current. FHH and Add3 knockout rats exhibited impairments in the myogenic response of afferent arterioles and in renal blood flow autoregulation, which were rescued in Add3 transgenic rats. We confirmed these findings in a genetic complementation study that involved crossing FHH and MNS rats that share the ADD3 mutation. Add3 transgenic rats showed attenuation of proteinuria, glomerular injury, and kidney fibrosis with aging and mineralocorticoid-induced hypertension. This is the first report that a mutation in ADD3 that alters ACTIN binding causes renal vascular dysfunction and promotes the susceptibility to kidney disease.

Abstract 130: Down Regulation of Gamma-Adducin Diminishes Glomerular Function and Promotes Hypertension Related Chronic Kidney Disease

Fawn Hooded Hypertensive (FHH) rats are a genetic model of hypertension-induced chronic kidney disease (CKD) in association with podocyte dysfunction. We previously identified an inactivating mutation of γ-Adducin (ADD3) is associated with the development of proteinuria in FHH rats and that knockdown of ADD3 disrupts actin cytoskeleton structure in the vascular smooth muscle cells. However, whether loss of ADD3 function also alters podocyte function in FHH rats has not been studied. The present study examined whether loss of ADD3 function in FHH also alters the actin cytoskeleton and podocyte function and contribute to the development of CKD. Knockdown of Add3 in the conditionally immortalized mouse podocytes using Add3 DsiRNA disrupted the actin cytoskeleton and increased F/G actin ratio using immunostaining and Western blots. Mean arterial pressure was similar, but proteinuria increased to a lesser extent in the DOCA/salt hypertensive FHH.Add3 transgenic (259 ± 5 mg/day, n=12) than FHH (484 ± 78 mg/day, n=21) rats. The convective permeability of isolated glomeruli to albumin (Palb) was lower in FHH.Add3 (0.13 ± 0.01, n=6, 157) than FHH (0.24 ± 0.02, n=4, 173) rats as assessed by a fluorescence dilution technique. Moreover, Palb increased to greater extent in FHH (0.36 ± 0.03, n=5, 140) than in FHH.Add3 (0.21 ± 0.03, n=3, 75) rats after DOCA/salt hypertension. Hypertensive FHH.Add3 rats exhibited less podocyte foot process effacement than FHH rats. The glomerular injury score averaged 2.50 ± 0.03 (n=9, 270) in DOCA/salt hypertensive FHH.Add3 versus 3.31 ± 0.01 (n=9, 270) in FHH rats. The percentage of fibrosis in the renal cortex was significantly lower in hypertensive FHH.Add3 than age-matched FHH rats. These results indicate that loss of ADD3 function contributes to the development of proteinuria and CKD in FHH rats by altering the actin cytoskeleton in podocytes, leading to podocyte effacement and glomerular barrier dysfunction.

Sex Differences in the Progression of Type 2 Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of chronic renal disease. To explore the age‐related progression and sexual dimorphisms in the development of DN, we used a rat model of type 2 diabetic nephropathy (T2DN). The T2DN rat was previously created by introgression of the mitochondria and some passenger loci from the Fawn Hooded Hypertensive rat into the background of the Goto‐Kakizaki (GK; type 2 diabetic) rat. Type 2 diabetes in T2DN rats is accompanied by renal histologic abnormalities that are characteristic of DN, similar to clinical observations in human patients. The experimental groups were Wistar (non‐diabetic controls), GK, or T2DN rats at the early stages of DN (12 weeks old) or the late stages of the disease (>40 weeks of age). At the early stage of DN, male T2DN and GK rats showed significant increases in blood glucose (374 ± 96 and 329 ± 45 vs. 280 ± 24 mg/dL in Wistar, respectively) and kidneys to body weight ratio (10.05 ± 1.19 and 9.39 ± 0.78 vs. 7.31 ± 1.13 mg/g, respectively). In the late stage of the disease progression, T2DN rats had a significant increase in renal damage compared with GK and Wistar rats, indicated by progressive albuminuria, diuresis, a high prevalence of medullary protein cats, and glomerular lesions. Besides a striking DN phenotype in the T2DN model, we found significant sex differences in the development and progression of the disease. The male weight was double that of females (414 ± 27 vs. 264 ± 19 g, respectively), which correlates with a significant difference in postprandial blood glucose levels (435 ± 50 vs. 353 ± 46 mg/dL, respectively). To further evaluate changes in type 2 diabetes between the sexes, we performed the glucose tolerance test (GTT) in fasting animals. The male GTT indicates significant impairment in the body's response to sugar load compared to the female (155 ± 18 vs. 118 ± 5 at control and 378 ± 19 vs. 183 ± 48 mg/dL after oral glucose gavage; male vs female respectively). Blood tests also show elevated levels of serum alkaline phosphatase (158 ± 32 U/L) and cholesterol in males, similar to the clinical observations in patients with diabetes mellitus. Analysis of the renal damage between the sexes reveals significant increases in protein cast formation and glomerular abnormalities indicated by a high percentage of damaged glomeruli in males (28% vs. 4% in females). Aged male T2DN rats also had increases in nephrin shedding in their urine, which correlates with the dramatic differences in albuminuria. In T2DN males, there was an inverse correlation in serum levels (2.5 ± 0.2 vs. 3.3 ± 0.1 g/dL) and urinary excretion of albumin (11.8 ± 5.7 vs. 0.98 ± 0.64 Alb/Cre ratio) in comparison to females of similar age. Here we report that T2DN rat develops renal and physiological lesions to those seen in patients with type 2 diabetes, and there is a dramatic sex difference in the progression of renal damage.Support or Funding InformationNHLBI R35 HL135749 and P01 HL116264AHA 17SDG33660149 and 18PRE34030127ADA 1‐15‐BS‐172This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacological Blockade of Soluble Epoxide Hydrolase Attenuates the Progression of Congestive Heart Failure Combined With Chronic Kidney Disease: Insights From Studies With Fawn-Hooded Hypertensive Rats.

An association between congestive heart failure (CHF) and chronic kidney disease (CKD) results in extremely poor patient survival rates. Previous studies have shown that increasing kidney epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, improves the survival rate in CHF induced by aorto-caval fistula (ACF) and attenuates CKD progression. This prompted us to examine if sEH inhibitor treatment would improve the outcome if both experimental conditions are combined. Fawn-hooded hypertensive (FHH) rats, a genetic model showing early CKD development was employed, and CHF was induced by ACF. Treatment with an sEH inhibitor was initiated 4 weeks after ACF creation, in FHH and in fawn-hooded low-pressure (FHL) rats, a control strain without renal damage. The follow-up period was 20 weeks. We found that ACF FHH rats exhibited substantially lower survival rates (all the animals died by week 14) as compared with the 64% survival rate observed in ACF FHL rats. The former group showed pronounced albuminuria (almost 30-fold higher than in FHL) and reduced intrarenal EET concentrations. The sEH inhibitor treatment improved survival rate and distinctly reduced increases in albuminuria in ACF FHH and in ACF FHL rats, however, all the beneficial actions were more pronounced in the hypertensive strain. These data indicate that pharmacological blockade of sEH could be a novel therapeutic approach for the treatment of CHF, particularly under conditions when it is associated with CKD.

Down‐Regulation of Gamma‐Adducin Disrupts the Actin Cytoskeleton in FHH rats and May Contribute to the Development of Hypertension‐induced Renal Injury

Fawn Hooded Hypertensive (FHH) rat develops chronic kidney disease after onset of hypertension. We previously reported that the expression of Add3 is decreased in various tissues, including kidney and renal vessels in FHH rats. More recently, we demonstrated that Add3 dysfunction contributes to renal vascular dysfunction in FHH rats, and they exhibit enhanced glomerular injury, interstitial fibrosis and proteinuria after the development of hypertension. Add3 is an actin‐binding protein that regulates actin capping and polymerization. The tail of the dimerized adducin protein interacts with actin and spectrin. However, it remains to be elucidated whether Add3 dysfunction alters actin cytoskeleton and contributes to renal microvascular dysfunction in FHH rats. The present study examined the role of Add3 on the regulation of the actin cytoskeleton in a vascular smooth muscle (VSMC) cell line treated with an Add3 DsiRNA, and in primary VSMCs isolated from FHH and FHH. Add3 transgenic rats. Downregulation of Add3 disrupted the actin cytoskeletal structural integrity in VSMC cells transfected with an Add3 DsiRNA (20 nM) in comparison to untreated or scramble siRNA treated cells. F‐actin distribution was significantly reduced in VSMCs isolated from the renal vasculature of FHH (2.4 ± 0.3%/cell, n = 6) vs. FHH. Add3 transgenic rats (11.4 ± 1.7%/cell, n = 6) consistent with disruption of the actin cytoskeleton. In addition, we observed the formation of an F‐actin net in primary renal VSMCs isolated from FHH rats, and reduced expression of the myosin regulatory light chains (MYL12A/B) that regulate actin polymerization and vascular contraction upon phosphorylation. These results suggest that down‐regulation of Add3 disrupts the actin cytoskeleton and the expression of MYL12A/B protein in renal VSMCs. These effects combine to impair the myogenic response of the Af‐art and autoregulation of renal blood flow. This increases transmission of pressure to the glomerulus, which contributes to the development of chronic kidney disease induced by hypertension in the FHH rat.Support or Funding InformationThis study was supported by grants HL36279 (RJR) and DK104184 (RJR), AG050049 (FF), P20GM104357 (RJR, FF ) from the National Institutes of Health; 16GRNT31200036 (FF) from the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 067: Genetic Complementation Establishes That a K572Q Mutation in Gamma-adducin Plays a Causal Role in Renal Microvascular Dysfunction in FHH and MNS Rats

The Fawn-Hooded hypertensive (FHH) rat is a genetic model of hypertension-induced renal disease. However, the causal genes and pathways involved are unclear. We previously reported that the transfer of a small region in Chr. 1 of Brown-Norway (BN) rats which contains 15 genes, including gamma-Adducin (Add3), into the FHH background restores renal microvascular function and attenuates the development of proteinuria in FHH rats. Our further work identified a K572Q mutation in Add3 in FHH rats as a potential candidate variant in the pathogenesis of renal disease. The present study examined the role of Add3 in the impaired myogenic response of the afferent artery (Af-art) and autoregulation of renal blood flow (RBF) using transgenic and KO rats. RBF increased by 21.5 ± 3.0% in SD. Add3 KO rats (n=7) when mean arterial pressure (MAP) was increased from 100 to 150 mmHg. In contrast, RBF only increased by 3.5 ± 0.9% in wildtype SD rats (n=13). The diameters of the renal Af-art decreased by 12.9 ± 0.8% in SD rats when perfusion pressure was increased from 60 to 120 mmHg, but it increased in SD.Add3 KO rats. The myogenic response of the Af-art in FHH rats was markedly impaired and increased by 8 ± 1.2% when the pressure was increased by from 60 to120 mmHg. The myogenic response was restored, and the diameters of the Af-art decreased by 12 ± 0.7% and 7 ± 1.0% in FHH. 1 BN congenic rats (n=27) and FHH.Add3 transgenic rats that express wt-Add3. RBF increased by 35.1 ± 3.0% when MAP was increased from 100 to 150 mmHg in FHH rats (n=15) versus 7.5 ± 1.7% and 6.0+ 1.3% in FHH.1 BN or a F1 cross of FHH and FHH.1 BN rats (n=8) and FHH.Add3 transgenic rats. The myogenic response of Af-art and autoregulation of RBF were also impaired in MNS rats (n=6) that carry the same K572Q mutation in Add3 as FHH rats. These phenotypes were complemented in a F1 cross of FHH and MNS rats (n=7), but the myogenic response and autoregulation of RBF were restored in an F1 cross of FHH and FHH.1 BN rats with one copy of wt-Add3. These results indicate that the recessive K572Q mutation of Add3 in FHH and MNS rats plays a causal role in renal microvascular dysfunction, which may contribute to the development of chronic kidney disease induced by hypertension in these models.

Abstract P124: Role of Add3 Dysfunction in Renal Epithelial Cells in the Development of Renal Injury in Fhh Rats

The Fawn Hooded Hypertensive Rat (FHH) is a genetic model of hypertension, in which Add3 has been identified as a candidate gene for renal injury but the mechanism is unknown. The present study examined the effects of knockdown of Add3 on rat renal epithelial (NRK) cells which are the model system for podocytes and proximal tubular cells. Knockdown of the expression of Add3 using a 27-mer Dicer-substrate RNAi (DsiRNA) decreased cell viability using an MTS assay by 50% in comparison to cells treated with vehicle or scrambled DSiRNA (Add3DsiRNA, OD 0.5±0.1; untreated cells 0.9±0.1; NC-1 0.9±0.1; n=8, P<0.001). F-actin immunostaining revealed that Add3 DSiRNA markedly disrupted the cytoskeleton, and the F-actin staining intensity/cell ratio was significantly reduced (untreated 543±47, n=552 cells; NC-1 592±39, n=566 cells; Add3DsiRNA 430±58, n=385 cells, P<0.05). The maximum mitochondrial respiratory rate was reduced in Add3 DsiRNA treated cells (untreated 444±42; NC-1 414±32; Add3 DsiRNA 275±16 pmole/min/μg, P<0.01). Proton leak was higher (untreated 42±3; NC-1 40±4; Add3 DsiRNA 63±5 pmole/min/μg, P<0.002) and oxygen consumption coupled to ATP production was decreased in Add3 DsiRNA treated cells (untreated 229±12, n=9; NC-1 200±12, n=8; Add3 DsiRNA 146±11 pmole/min/μg, n=9, P<0.001). Spare respiratory capacity, an index of cell fitness, was reduced by knockdown of Add3 (untreated 154±42, n=9: NC-1 143±32, n=8; Add3DsiRNA 33±16 pmole/min/μg, n=9, P<0.001). Non-mitochondrial oxygen consumption rate (OCR/μg protein) increased in cells treated with Add3 DsiRNA (untreated 57±7; NC-1 80±11; Add3DsiRNA 116±15 pmole/min/μg, P<0.01) suggesting a possible metabolic switch to glycolytic oxygen consumption. In conclusion, knockdown of Add3 in NRK cells disrupts the actin cytoskeleton, promotes mitochondrial dysfunction and decreases cell viability. Loss of Add3 function may contribute to proteinuria in FHH rats by altering the cytoskeleton and promoting effacement and loss of podocytes and the reuptake of filtered protein in the proximal tubule.

Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells.

The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium–dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes. Patch clamp recording of single-channel current activity identified the presence of a single-channel cationic current with unitary conductance of ~85 pS and ~96 pS at hyperpolarizing and depolarizing potentials, respectively, that was inhibited by the TRPV4 channel antagonist RN 1734 or HC 067074 and activated by the potent TRPV4 channel agonist GSK1016790A. Application of negative pressure via the interior of the patch pipette increased the NPo of the TRPV4-like single-channel cationic current recorded in cell-attached patches at a patch potential of 60 mV that was inhibited by prior application of the TRPV4 channel antagonist RN 1734 or HC 067047. Treatment with the TRPV4 channel agonist GSK1016790A caused concentration-dependent increase in the NPo of KCa single-channel current recorded in cell-attached patches of cerebral arterial myocytes at a patch potential of 40 mV, which was not influenced by pretreatment with the voltage-gated L-type Ca2+ channel blocker nifedipine or the T-type Ca2+ channel blocker Ni2+. These findings demonstrate that FHH rat cerebral arterial myocytes express mRNA transcript and translated protein for TRPV4 channel and display TRPV4-like single-channel cationic current activity that was stretch-sensitive and activation of which increased the open state probability of KCa single-channel current in these arterial myocytes.

Dynamic Bayesian Graphical Modeling to Predict Regulatory Networks in Hypertensive Rats

We have previously used Bayesian graphical methods to detect networks of interacting genes relevant to the development of salt sensitive hypertension. However, these previous approaches are “static” or time‐insensitive methods that cannot infer causality to gene interactions. Here, we present a dynamic Bayesian graphical modeling approach which is used to connect potential target genes to other genes and networks.Dynamic Bayesian graphical networking approaches require large amounts of time‐series data, in addition to being computationally complex. Data was generated by isolating medullary thick ascending limb (mTAL) cells from 3 rat strains: the Dahl Salt‐sensitive rat (SS), the Fawn Hooded Hypertensive rat (FHH) and the Spontaneously Hypertensive Rat (SHR). After isolation, the freshly isolated mTAL cells were exposed to one of two stimuli: H2O2 (500 nM) or TNF‐α (50 ng/mL). RNA was then collected at 30 minute intervals for 8 hours, resulting in a 16‐point time‐series for each strain and stimulus. Each time‐series was duplicated, and duplicates were pooled to reduce overall measurement noise. As there are 6 combinations of strains (3) and stimuli (2) this procedure resulted in 96 individual time‐points. RNA was then isolated and sequenced on an Illumina Hi‐Seq 2500.The Dynamic Bayesian graphical model attempts to find connections between genes by looking at the sequence of activation/inactivation of genes across the entire 8 hour time‐series. For example, in a time‐series generated by treatment of SS mTAL cells with 500 nM H2O2, we find that the gene Dusp5 undergoes a 4‐fold increase in RNA expression one hour before a similar 4‐fold increase in the gene Rasgrp3, a GWAS gene for hypertension that has no known function in the mTAL. The Bayesian graphical modeling algorithm looks at such changes across each time‐series for the different rat strains and stimuli, and detects the most probable connections between the analyzed genes.This analysis represents the first attempt to create a dynamic Bayesian graph in non‐cancer mammalian cells. The use of such a graphical modeling approach in mTAL, a cell type of particular relevance to the pathophysiology of hypertension, will give us insight into the regulation of critical genes in hypertension and allow us to better understand, and potentially treat, this important disease.Support or Funding InformationA. Dayton supported by NIH Fellowship: 1F30HL127979‐01A1.Work funded by NIH grant: 4P01HL116264.

Unintended Consequences: Renal Denervation Attenuates Hypertension Development but Impairs Glucose Metabolism in Female Fawn‐Hooded Hypertensive Rats

For both men and women, hypertension (HTN) is the highest risk factor for cardiovascular disease and stroke – the two leading causes of morbidity and mortality throughout the world. Though HTN etiology remains debated, altered sympathetic nervous activity, particularly to the kidney (RSNA), is regarded as a primary contributor to the development and maintenance of HTN. Ablation of renal sympathetic nerves (RDNx) reportedly prevents and/or reverses several forms of experimental hypertension. Several off‐target improvements are also reported, such as glucose metabolism. However, an overwhelming majority of these experimental models and mechanisms focus only on responses in male subjects. Moreover, the sex differences in sympathetic control of blood pressure in hypertension remain poorly defined. In this study, we assessed the role of RDNx to prevent hypertension development and metabolic dysfunction in the Fawn‐Hooded Hypertensive (FHH) rat model. We hypothesized that RDNx would attenuate the development of HTN, and improve glucose handling in female and male rats. FHH rats (10 weeks old) underwent sham or bilateral RDNx surgeries (n=5/group). For the following six weeks, 24‐hour mean arterial pressure (MAP) was monitored by radiotelemetry. Glucose handling was assessed biweekly by glucose tolerance test (GTT). Cardiovascular data was averaged weekly, and analyzed by two‐way ANOVA with a Bonferroni post‐hoc test (α=.05). Data presented as mean ± SEM. Six weeks post‐treatment, RDNx attenuated the HTN in the females (p<.05) vs. sham controls (107±5 vs. 118±3). Interestingly, no difference in MAP between RDNx and sham males was observed (129±2 vs. 125±1mmHg). Regarding glucose metabolism, baseline fasting blood glucose (FBG) and glucose tolerance (GT) were similar between males and females prior to RDNx. After six weeks, FBG in female sham FHH (100.9±2.3mg/dL) was higher (p<.05) than male shams (89.2±2.7mg/dL). Likewise, GT was impaired in female rats compared to male shams. In contrast to our hypothesis, RDNx worsened the FBG (110±2.6mg/dL) and GT in female vs. female shams. No effect of time or RDNx on glucose handling was observed males. In summary, RDNx attenuated HTN development in female FHH rats; however, glucose metabolism was impaired by RDNx in females but not in males. These results suggest that sex may need to be considered in the use of RDNx to treat HTN. These findings are clinically important in that RDNx responses may be partially dependent on sex, and may result in negative outcomes such as impaired glucose metabolism in females. Further examination of sex differences in the responses to RDNx is underway.Support or Funding InformationNIH 5R01HL116476; NIH 2T32HL7741

Deficiency of the Angiotensinase Aminopeptidase A Increases Susceptibility to Glomerular Injury.

Aminopeptidase A (APA) is expressed in glomerular podocytes and tubular epithelia and metabolizes angiotensin II (AngII), a peptide known to promote glomerulosclerosis. In this study, we tested whether APA expression changes in response to progressive nephron loss or whether APA exerts a protective role against glomerular damage and during AngII-mediated hypertensive kidney injury. At advanced stages of FSGS, fawn-hooded hypertensive rat kidneys exhibited distinctly increased APA staining in areas of intact glomerular capillary loops. Moreover, BALB/c APA-knockout (KO) mice injected with a nephrotoxic serum showed persistent glomerular hyalinosis and albuminuria 96 hours after injection, whereas wild-type controls achieved virtually full recovery. We then tested the effect of 4-week infusion of AngII (400 ng/kg per minute) in APA-KO and wild-type mice. Although we observed no significant difference in achieved systolic BP, AngII-treated APA-KO mice developed a significant rise in albuminuria not observed in AngII-treated wild-type mice along with increased segmental and global sclerosis and/or collapse of juxtamedullary glomeruli, microcystic tubular dilation, and tubulointerstitial fibrosis. In parallel, AngII treatment significantly increased the kidney AngII content and attenuated the expression of podocyte nephrin in APA-KO mice but not in wild-type controls. These data show that deficiency of APA increases susceptibility to glomerular injury in BALB/c mice. The augmented AngII-mediated kidney injury observed in association with increased intrarenal AngII accumulation in the absence of APA suggests a protective metabolizing role of APA in AngII-mediated glomerular diseases.

Impaired myogenic response of the afferent arteriole contributes to the increased susceptibility to renal disease in Milan normotensive rats.

Milan normotensive (MNS) rats are more susceptible to the development of renal disease than Milan hypertensive (MHS) rats, but the genes and pathways involved are unknown. This study compared the myogenic response of isolated perfused afferent arterioles (Af‐Art) and autoregulation of renal blood flow (RBF) and glomerular capillary pressure (Pgc) in 6–9‐week‐old MNS and MHS rats. The diameter of the Af‐Art of MHS rats decreased significantly from 14.3 ± 0.5 to 11.5 ± 0.6 μm when perfusion pressure was elevated from 60 to 120 mmHg. In contrast, the diameter of Af‐Art of MNS rats did not decrease. RBF was well autoregulated in MHS rats, but it increased by 26% in MNS rats. Pgc rose by 11 mmHg when renal perfusion pressure (RPP) was increased from 100 to 140 mmHg in MNS but not in MHS rats. Protein excretion increased from 10 ± 1 to 245 ± 36 mg/day in MNS rats as they aged from 3 to 11 months but it did not increase in MHS rats. We also compared the development of proteinuria in MNS and MHS rats following the induction of diabetes with streptozotocin. Protein excretion rose from 16 ± 3 to 234 ± 43 mg/day in MNS rats, but it remained unaltered in MHS rats. These data indicate that the myogenic response of the Af‐art is impaired in MNS rats and increased transmission of pressure to the glomerulus may contribute to renal injury in MNS rats similar to what is seen in fawn‐hooded hypertensive and Dahl salt‐sensitive rats.

Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries.

We have reported that the myogenic response of the renal afferent arteriole (Af-art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow are impaired in Fawn-Hooded Hypertensive (FHH) rats. Transfer of a region of chromosome 1 containing γ-adducin (Add3) from the Brown Norway rat rescued the vascular dysfunction and the development of renal disease. To examine whether Add3 is a viable candidate gene altering renal and cerebral hemodynamics in FHH rats, we knocked down the expression of Add3 in rat Af-arts and MCAs cultured for 36-h using a 27-mer Dicer-substrate short interfering RNA (DsiRNA). Control Af-arts constricted by 10 ± 1% in response to an elevation in pressure from 60 to 120 mmHg but dilated by 4 ± 3% when treated with Add3 DsiRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to norepinephrine (10-7 M). Add3 DsiRNA had a similar effect on the attenuation of the myogenic response in the MCA. Peak potassium currents were threefold higher in smooth muscle cells isolated from Af-arts or MCAs transfected with Add3 DsiRNA than in nontransfected cells isolated from the same vessels. This is the first study demonstrating that Add3 plays a role in the regulation of potassium channel function and vascular reactivity. It supports the hypothesis that sequence variants in Add3, which we previously identified in FHH rats, may play a causal role in the impaired myogenic response and autoregulation in the renal and cerebral circulation.