Kidneys Of Spontaneously Hypertensive Rats Research Articles

Differential gene expression in the kidneys of SHR and WKY rats after intravenous administration of Akkermansia muciniphila-derived extracellular vesicles

Although Akkermansia muciniphila (Am) plays a beneficial role as a probiotic in the treatment of metabolic syndrome, the mechanisms remain elusive. We tested the hypothesis that Am extracellular vesicles (AmEVs) protect against hypertension through modulation of gene expression in the kidneys of spontaneously hypertensive rats (SHRs). Extracellular vesicles purified from anaerobically cultured Am (1.0 × 108 or 1.0 × 109 particles/kg) or vehicles were injected into the tail veins of Wistar-Kyoto rats (WKYs) and SHRs weekly for 4 weeks. Renal cortical tissues isolated from both rat strains were analyzed by trichrome stain and RT-qPCR. AmEVs protect against the development of hypertension in SHRs without a serious adverse reaction. AmEVs increased the expression of vasocontracting Agt and At1ar as well as vasodilating At2r, Mas1 and Nos2 in the kidneys of both strains. These results indicate that AmEVs have a protective effect against hypertension without a serious adverse reaction. Therefore, it is foreseen that AmEVs may be utilized as a novel therapeutic for the treatment of hypertension.

3-Demethyl-2-geranyl-4-prenylbellidifoline, a natural xanthone with diuretic and kidney protective properties.

The diuretic and kidney protective effect of the 3-demethyl-2-geranyl-4-prenylbellidifoline (DGP) were evaluated in rats. The normotensive (NTR) and spontaneously hypertensive rats (SHR) received, once a day for 7 days, oral treatment with DGP (0.1 mg/kg), hydrochlorothiazide (10 mg/kg), or vehicle (10 ml/kg). Urine, blood, and kidney samples were collected for further analysis. The urine and Na+ elimination content were significantly higher in the groups that received DGP. Furthermore, a Ca2+-sparing action was detected in the urine of DGP-treated groups, which was consistent with the reduction in calcium oxalate crystal formation. Relevantly, the treatment did not change the parameters examined in the blood. Concerning the renal analyses, DGP treatment recovered the morphological damages of the kidney corpuscle area of SHR. In addition to the differences observed between the NTR and SHR vehicle groups, DGP augmented the amount of reduced glutathione and the activity of glutathione S-transferase GST while reducing the catalase and N-acetyl-β-D-glucosaminidase activity and nitrite levels. Together, this study displayed the prolonged diuretic action of DGP and its natriuretic, Ca2+-sparing, and antiurolytic effects. The antioxidative and anti-inflammatory effects of DGP were evidenced in SHR kidneys, opening perspectives for further studies regarding the benefits of this xanthone.

Tissue-specific expression of senescence biomarkers in spontaneously hypertensive rats: evidence of premature aging in hypertension.

Cellular senescence is an age-related physiological process that contributes to tissue dysfunction and accelerated onset of chronic metabolic diseases including hypertension. Indeed, elevation of blood pressure in hypertension coincides with premature vascular aging and dysfunction. In addition, onsets of metabolic disturbance and osteopenia in patients with hypertension have also been reported. It is possible that hypertension enhances premature aging and causes progressive loss of function in multiple organs. However, the landscape of cellular senescence in critical tissues affected by hypertension remains elusive. Heart, liver, bone, hypothalamus, and kidney were collected from spontaneously hypertensive rats (SHR) and age- and sex-matched normotensive Wistar rats (WT) at 6, 12, 24 and 36 weeks of age (n = 10 animals/group). Changes in mRNA levels of senescence biomarkers namely cyclin-dependent kinase (CDK) inhibitors (CDKIs), i.e., Cdkn2a (encoding p16Ink4a) and Cdkn1a (encoding p21cip1) as well as senescence-associated secretory phenotypes (SASPs), i.e., Timp1, Mmp12, Il6 and Cxcl1, were determined. Additionally, bone collagen alignment and hydroxy apatite crystal dimensions were determined by synchrotron radiation small- and wide-angle X-ray scattering (SAXS/WAXS) techniques. Real-time PCR revealed that transcript levels of genes encoding CDKIs and SASPs in the heart and liver were upregulated in SHR from 6 to 36 weeks of age. Expression of Timp1 and Cxcl1 was increased in bone tissues isolated from 36-week-old SHR. In contrast, we found that expression levels of Timp1 and Il6 mRNA were decreased in hypothalamus and kidney of SHR in all age groups. Simultaneous SAXS/WAXS analysis also revealed misalignment of bone collagen fibers in SHR as compared to WT. Premature aging was identified in an organ directly affected by high blood pressure (i.e., heart) and those with known functional defects in SHR (i.e., liver and bone). Cellular senescence was not evident in organs with autoregulation of blood pressure (i.e., brain and kidney). Our study suggested that cellular senescence is induced by persistently elevated blood pressure and in part, leading to organ dysfunction. Therefore, interventions that can both lower blood pressure and prevent cellular senescence should provide therapeutic benefits for treatment of cardiovascular and metabolic consequences.

Abstract P436: Increased West Nile Virus Infection And Pathology In The Spontaneously Hypertensive Rat

Introduction: West Nile virus (WNV), a mosquito-borne flavivirus, causes grave neuroinvasive disease and death, and has been reported in over 55,000 people in the US since 1999. Patients >55 years and those with hypertension are most at risk for WNV-associated mortality. Understanding how hypertension correlates with WNV morbidity and mortality may reduce the burden of this disease. Investigation of WNV in hypertensive people is limited to basic epidemiology. Without an animal model, understanding the causal relationship between hypertension and WNV-associated pathology remains elusive. Here, we tested a novel hypothesis that hypertension will promote WNV infection and pathology in a rodent model of age-dependent chronic hypertension, the spontaneously hypertensive rat (SHR). Methods: Male adult SHR and normotensive WKY rats were implanted with radiotelemetry transmitters for measurements of blood pressure and heart rate. Following baseline measurements, SHR and WKY rats (N=9/gp) were injected with WNV (1 X 10 4 PFU/ml, i.p.). Vehicle injection (PBS, i.p.) was performed in additional WKY and SHR (N=2-3/gp). Each rat was observed for clinical abnormalities, and body weight, blood pressure, and heart rate were measured daily. Rats were sacrificed on days 9 and 10 post-infection (PI). At endpoint, several tissues were processed for measurement of viral load by RT-qPCR and histopathology. Bone marrow and circulating immune cells (CD4 + , CD8 + , CD68 + ) were quantified at endpoint by flow cytometry. Results: One SHR rat developed neurological symptoms and was euthanized on day 4 PI. Viral load was significantly increased in the SHR kidney and spleen (P<0.001) in SHR compared to WKY rats. In addition, WNV-infection elevated heart rate in the SHR but not the WKY (P<0.0001). SHR also showed elevated circulating CD68 + monocytes (P<0.05), and a significant increase (P<0.05) in renal pathology was observed in the SHR following WNV infection. Conclusions: We demonstrate, for the first time, a positive interaction between high blood pressure and severity of WNV infection in a rodent model of chronic hypertension. Further studies are needed to elucidate the mechanisms of this interaction for new therapeutic targets of WNV disease.

Inhibition of endoplasmic reticulum stress restores the balance of renal RAS components and lowers blood pressure in the spontaneously hypertensive rats

ABSTRACT Background Endoplasmic reticulum (ER) stress has been shown to play a critical role in the pathogenesis of hypertension. However, the underlying mechanisms for lowering blood pressure (BP) by suppressing ER stress remain unclear. Here, we hypothesized that inhibition of ER stress could restore the balance between RAS components and lower BP in spontaneously hypertensive rats (SHRs). Methods Wistar-Kyoto (WKY) rats and SHRs received vehicle or 4-PBA, an ER stress inhibitor, in the drinking water for 4 weeks. BP was measured by tail-cuff plethysmography, and the expression of RAS components was examined by Western blot. Results Compared with vehicle-treated WKY rats, vehicle-treated SHRs exhibited higher blood pressure and increased renal ER stress and oxidative stress, accompanied by impaired diuresis and natriuresis. Moreover, SHRs had higher ACE and AT1R and lower AT2R, ACE2, and MasR expressions in the kidney. Interestingly, 4-PBA treatment improved impaired diuresis and natriuresis and lowered blood pressure in SHRs, accompanied by reducing ACE and AT1R protein expression and increasing AT2R, ACE2, and MasR expression in the kidneys of SHRs. In addition, these changes were associated with the reduction of ER stress and oxidative stress. Conclusions These results suggest that the imbalance of renal RAS components was associated with increased ER stress in SHRs. Inhibition of ER stress with 4-PBA reversed the imbalance of renal RAS components and restored the impaired diuresis and natriuresis, which, at least in part, explains the blood pressure-lowering effects of 4-PBA in hypertension.

Mangiferin alleviates renal inflammatory injury in spontaneously hypertensive rats by inhibiting MCP-1/CCR2 signaling pathway

ObjectiveHypertension is a low-grade inflammation state of the disease and was easily complicated by kidneys’ inflammatory response. Mangiferin (MGF), a pharmacologically active compound in various plants including Mangifera indica, has a strong anti-inflammatory activity. However, the effects of MGF on renal inflammatory injury in spontaneously hypertensive rats (SHRs) remain unclear. The purpose of this study was to investigate the protective effects and mechanisms of MGF on renal inflammatory injury in SHRs. MethodsMGF was used in SHRs at the doses of 10, 20, 40 mg/kg/d for 8 weeks consecutively. The blood and urine were collected for assessment of renal function. Renal tissues were collected for histological, immunohistochemistry, ELISA, Western blot and real time reverse transcription PCR (RT-PCR) analysis. ResultsThe results showed that the levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and recombinant chemokine C-C-Motif receptor 2 (CCR2) were increased in SHRs, meanwhile, the level of IL-10 was decreased in SHR. Treatment of MGF inhibited the expression of IL-6, TNF-α, MCP-1 and CCR2, and promoted the expression of IL-10. Furthermore, the content of blood urea nitrogen (BUN) and serum uric acid (SUA) was significantly increased in the model group, and treatment of MGF had no obvious effects on these parameters at all dose levels. ConclusionOur study proved that the kidneys of SHRs had significant inflammatory injury, and MGF had the protective effects on renal inflammatory injury in SHRs; The protective mechanism may be mediated partly by the MCP-1/CCR2 signaling pathway. Thus, it is a potential new drug for the treatment of hypertension.

S-38-2: PREVENTION OF EXPERIMENTAL GENETIC HYPERTENSION - GENOMIC CLUES FROM THE KIDNEY

Brief treatment with renin-angiotensin system (RAS) inhibitors permanently reduces the genetic hypertension of the spontaneously hypertensive rat (SHR). The kidney is involved in this reprogramming, but relevant molecular genetic changes are unknown. We studied SHR kidney RNA differential expression (controlling for non-specific effects of lower BP) and DNA methylation 6 weeks after treatment with the angiotensin receptor blocker losartan from 10 to 14 weeks of age. RNA sequencing revealed a significant 6-fold (P < 0.0001) increase in renin gene (Ren) expression during treatment. Six weeks later direct mean arterial pressure remained lower than untreated SHR (123 vs 140 mmHg, P = 0.001) and kidney Ren expression was reduced by 23% (P = 0.03) and DNA methylation within the Ren promoter region was significantly increased (P = 0.04). Experiments with the ACE inhibitor perindopril and renin immunohistochemical analyses confirmed significant long-term reduction in kidney Ren expression following RAS inhibition. Genomic RNA sequencing analysis identified 13 candidate genes (Grhl1, Ammecr1l, Hs6st1, Nfil3, Fam221a, Lmo4, Adamts1, Cish, Hif3a, Bcl6, Rad54l2, Adap1, Dok4) and the miRNA miR-145-3p that were significantly differentially expressed and correlated closely with decreased Ren expression. Ten of the candidates were central to gene networks that exhibited significant enrichment for genes relevant to BP and the RAS. We propose that reduced renin gene expression is a legacy of early RAS inhibition and responsible for the persistent reduction in BP in SHR.

Persistent vascular congestion in male spontaneously hypertensive rats contributes to delayed recovery of renal function following renal ischemia perfusion compared with females.

Acute kidney injury (AKI) due to ischemia is a serious and frequent clinical complication with mortality rates as high as 80%. Vascular congestion in the renal outer medulla occurs early after ischemia reperfusion (IR) injury, and congestion has been linked to worsened outcomes following IR. There is evidence implicating both male sex and preexisting hypertension as risk factors for poor outcomes following IR. The present study tested the hypothesis that male spontaneously hypertensive rats (SHR) have greater vascular congestion and impaired renal recovery following renal IR vs. female SHR and normotensive male Sprague-Dawley rats (SD). Thirteen-week-old male and female SHR and SD were subjected to sham surgery or 30 min of warm bilateral ischemia followed by reperfusion. Rats were euthanized 24 h or 7 days post-IR. IR increased renal injury in all groups vs. sham controls at 24 h. At 7 days post-IR, injury remained elevated only in male SHR. Histological examination of SD and SHR kidneys 24 h post-IR showed vascular congestion in males and females. Vascular congestion was sustained only in male SHR 7 days post-IR. To assess the role of vascular congestion on impaired recovery following IR, additional male and female SHR were pretreated with heparin (200 U/kg) prior to IR. Heparin pretreatment reduced IR-induced vascular congestion and improved renal function in male SHR 7 days post-IR. Interestingly, preventing increases in blood pressure (BP) in male SHR did not alter sustained vascular congestion. Our data demonstrate that IR-induced vascular congestion is a major driving factor for impaired renal recovery in male SHR.

Effects of renal denervation on endogenous ouabain in spontaneously hypertensive rats.

To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR). Sixteen 12-week-old male SHR were randomly separated into the renal denervation group (RDNX group) and sham operation group (sham group), and eight age-matched Wistar Kyoto rats (WKY) were served as control group. EO concentrations, the Na+- K+-ATPaseactivity, and the expression of Na+-K+-ATPase were assessed. EO levels in serum, kidneys and hypothalamus of sham group were higher than in RDNX group (p < 0.05). Renal Na+-K+-ATPase activity subjected to denervation surgery showed significantly reduction when compared with the sham groups (p < 0.05). A positive correlation existed between norepinephrine (NE) content and Na+-K+-ATPase activity in the kidney (r2 = 0.579). Renal Na+-K+-ATPase α1 subunit mRNA expression was down-regulated in the RDNX group compared with the sham group (P < 0.05), while renal Na+-K+-ATPase α1 subunit mRNA expression was no statistical significance between the groups (P = 0.63). Immunohistochemical analysis showed that there were significant differences in the renal expression of Na+-K+-ATPasebetween the three groups (P < 0.05). These experiments demonstrate that RDN exerted an anti-hypertensive effect with reduction of EO levels and Na+-K+-ATPase activity and Na+-K+-ATPase α1 subunit expression of kidney in SHR.

Morphology and cyclooxygenase-2 and renin expression in the kidney of young spontaneously hypertensive rats.

The kidneys play an important role in blood pressure regulation under normal and pathological conditions. We examined the histological changes and expression patterns of cyclooxygenase-2, renin, and (pro)renin receptor (PRR) in the renal cortex of prehypertensive spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs). Moreover, blood pressure and plasma urea, creatinine, angiotensin II, and angiotensin (1-7) levels were measured. The results showed that both strains had similar blood pressure and plasma urea and creatinine levels. The glomerular area, basement membrane thickness, collagen fiber content, and arterial wall thickness were greater in SHRs than in WKYs. By immunohistochemistry, cyclooxygenase-2 was localized in the macula densa and renal tubules of both strains. In SHRs, cyclooxygenase-2 was detected in a larger number of tubules, and the cortical expression of cyclooxygenase-2 was also increased. In both strains, PRR and renin were localized in the tubular epithelium and juxtaglomerular cells, respectively. In SHRs, PRR immunolocalization was increased in the glomerulus. The cortical expression of immature renin was markedly increased in SHRs compared to that in WKYs, while renin was significantly decreased. These changes were associated with higher plasma angiotensin II levels and lower plasma angiotensin (1-7) levels in SHRs. The results indicate that the kidneys of SHRs showed morphological changes and variations in cortical expression patterns of PRR, cyclooxygenase-2, and renin before the development of hypertension.

Tengdan Capsule Prevents Hypertensive Kidney Damage in SHR by Inhibiting Periostin-Mediated Renal Fibrosis.

BACKGROUND: Hypertension-induced renal damage is a serious and complex condition that has not been effectively treated by conventional blood pressure-lowering drugs. Tengdan capsule (TDC) is a China FDA-approved compound herbal medicine for treating hypertension; however, its chemical basis and pharmacological efficacy have not been fully investigated in a preclinical setting. METHODS: High-performance liquid chromatography (HPLC) was used to identify and quantify the major chemical components of TDC extracted from ultrapure water. Adult spontaneously hypertensive rats (SHR) and age/sex-matched Wistar Kyoto normotensive rats (WKY) were both treated with TDC, losartan, or saline for one month, and their blood pressure (BP) was monitored at the same time by tail-cuff BP system. Biochemical indexes such as urine creatinine (CRE) and blood urea nitrogen (BUN) were determined. Kidney tissue sections were examined with (H&E), and Masson staining to evaluate the pathological effect of TDC on SHR’s kidneys. After TDC treatment, the differentially expressed proteins in the kidneys of SHR were identified by the TMT-based quantitative proteomics analysis, which may provide the targets and possible mechanisms of TDC action. In addition, Western blot analysis, RT-qPCR, and ELISA assays were carried out to further verify the proteomics findings. Finally, two different models involving in vitro renal injuries were established using human kidney HEK293 cells; and the molecular mechanism of TDC kidney protection was demonstrated. RESULTS: Seven chemical compounds, namely Notoginsenoside R1, Ginsenoside RG1, Ginsenoside Re, Ginsenoside Rb1, Sodium Danshensu, Protocatechualdehyde, and Salvianolic acid B, were identified and quantified from the water-soluble extracts of TDC by HPLC. In vivo study using rats showed that TDC effectively reduced BP, BUN, and CRE levels and attenuated renal fibrosis in SHR, and ameliorated damage to the kidneys. Proteomics and subsequent bioinformatics analyses indicated that periostin-mediated inflammatory response and TGFβ/Smad signaling pathway proteins were closely related to the therapeutic effect of TDC in rat kidneys. Western blot analysis and RT-qPCR showed that TDC markedly downregulated the mRNA and protein expression of periostin in renal tissues compared to the untreated SHR. In addition, TGF-β and COL1A1 mRNA levels also decreased in SHR renal tissues following TDC treatment. In vitro studies showed that low to medium doses of TDC down-regulated the expression of periostin in the injury model of HEK293 cell. In addition, medium to high doses of TDC significantly inhibited collagen deposition in TGFβ1-induced HEK293 cell fibrosis. CONCLUSIONS: Major components from the compound herbal medicine Tengdan Capsule are identified and quantified. TDC effectively lowers blood pressure and protects against renal damage caused by hypertension in SHR. Mechanistically, TDC blocks periostin by regulating the TGF-β/Smad signaling pathway in the kidney, both in vivo and in vitro. Preventing periostin-mediated renal fibrosis and inflammation might be a promising strategy for treating a hypertensive renal injury.

Attenuating Effects of Dieckol on Hypertensive Nephropathy in Spontaneously Hypertensive Rats.

Hypertension induces renal fibrosis or tubular interstitial fibrosis, which eventually results in end-stage renal disease. Epithelial-to-mesenchymal transition (EMT) is one of the underlying mechanisms of renal fibrosis. Though previous studies showed that Ecklonia cava extracts (ECE) and dieckol (DK) had inhibitory action on angiotensin (Ang) I-converting enzyme, which converts Ang I to Ang II. It is known that Ang II is involved in renal fibrosis; however, it was not evaluated whether ECE or DK attenuated hypertensive nephropathy by decreasing EMT. In this study, the effect of ECE and DK on decreasing Ang II and its down signal pathway of angiotensin type 1 receptor (AT1R)/TGFβ/SMAD, which is related with the EMT and restoring renal function in spontaneously hypertensive rats (SHRs), was investigated. Either ECE or DK significantly decreased the serum level of Ang II in the SHRs. Moreover, the renal expression of AT1R/TGFβ/SMAD was decreased by the administration of either ECE or DK. The mesenchymal cell markers in the kidney of SHRs was significantly decreased by ECE or DK. The fibrotic tissue of the kidney of SHRs was also significantly decreased by ECE or DK. The ratio of urine albumin/creatinine of SHRs was significantly decreased by ECE or DK. Overall, the results of this study indicate that ECE and DK decreased the serum levels of Ang II and expression of AT1R/TGFβ/SMAD, and then decreased the EMT and renal fibrosis in SHRs. Furthermore, the decrease in EMT and renal fibrosis could lead to the restoration of renal function. It seems that ECE or DK could be beneficial for decreasing hypertensive nephropathy by decreasing EMT and renal fibrosis.

The Long-Term Study of Urinary Biomarkers of Renal Injury in Spontaneously Hypertensive Rats

Background: The age-related increase in blood pressure in spontaneously hypertensive rats (SHRs) is associated to cardiac hypertrophy, heart failure, and renal injury. Here, we investigated for the first time the urinary enzymatic activities of glutamil aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), and Klotho urinary levels, proteins that are strongly expressed in the kidney, as early biomarkers of renal injury in SHRs. Methods: Male SHR and Wistar Kyoto (WKY) rats were studied from 2 to 8 months old. Systolic blood pressure (SBP), the heart rate (HR), metabolic variables, and urinary markers were measured monthly. At the end of the study, a histopathological evaluation of the kidney was performed. Results: Kidneys of SHR did not develop signs of relevant histopathological changes, but showed increased glomerular area and cellularity. Plasma creatinine was decreased, and creatinine clearance was augmented in SHR at the end of the study. Urinary excretion of Klotho was higher in SHR at 5 and 8 months old, whereas plasma Klotho levels were similar to WKY. GluAp, AlaAp, and DPP4 urinary activities were increased in SHR throughout the time-course study. A positive correlation between glomerular area and cellularity with creatinine clearance was observed. Urinary GluAp, AlaAp, DPP4, and Klotho showed positive correlations with SBP. Conclusions: GluAp, AlaAp, DPP4, and Klotho in the urine are useful tools for the evaluation of renal damage at early stages, before the whole histopathological and biochemical manifestations of renal disease are established. Moreover, these observations may represent a novel and noninvasive diagnostic approach to assess the evolution of kidney function in hypertension and other chronic diseases.

Identification of Antihypertensive Mechanisms ofFicus deltoidea kunstleri; in Spontaneously Hypertensive Rats Using Metabolomics

Blood pressure lowering effect of Ficus deltoidea kunstleri (FDK) has been associated with changes in the Renin-Angiotensin-Aldosterone System (RAAS) and endothelial function in Spontaneously Hypertensive Rats (SHR). Whether it also involves other systems is unclear. This study therefore screened for other potential BP lowering mechanisms in FDK-treated SHR based on urine metabolite profile obtained using 1H-NMR spectrometry and multivariate data analysis. Male SHR were administered orally, either 0.5 mL of distilled water (control), or 1000 mg.kg-1 body weight of FDK or 10 mg.kg-1 body weight of losartan once daily for 4 weeks. BP was measured weekly and 24-hr urine was collected using metabolic chambers at the end of the 4th week. Animals were then euthanised and blood was collected for estimation of total anti-oxidant capacity TAC. Kidneys were harvested for antioxidant enzyme gene expression studies. BP in FDK- and losartan-treated SHR was significantly lower, whereas serum TAC was significantly higher than those in the controls. Orthogonal partial least square analysis indicated that FDK-treated SHR were separated from controls by the presence of a larger number of ketone body and amino acid metabolism intermediates. Superoxide dismutase-2, catalase and fork head box gene expressions were higher in kidneys of FDK-treated SHR than those in the controls. It appears that apart from RAAS, the BP lowering effect of standardized aqueous-ethanolic extract of leaves of FDK in SHR is also associated with increased TAC, antioxidant enzyme expressions and probably also involves multiple metabolic pathways, including those involving ketone bodies, amino acids and energy metabolism

Identification of Clock Genes Related to Hypertension in Kidney From Spontaneously Hypertensive Rats.

BackgroundThere is a diurnal variation in the blood pressure fluctuation of hypertension, and blood pressure fluctuation abnormality is considered to be an independent risk factor for organ damage including cardiovascular complications. In the current study, we tried to identify molecules responsible for blood pressure circadian rhythm formation under the control of the kidney biological clock in hypertension.MethodsDNA microarray analysis was performed in kidneys from 5-week-old spontaneously hypertensive rats (SHRs)/Izm, stroke-prone SHR rats (SHRSP)/Izm, and Wistar Kyoto (WKY)/Izm rats. To detect variation, mouse tubular epithelial cells (TCMK-1) were stimulated with dexamethasone. We performed immunostaining and western blot analysis in the renal medulla of kidney from 5-week-old WKY rats and SHRs.ResultsWe extracted 1,032 genes with E-box, a binding sequence for BMAL1 and CLOCK using a Gene Set Enrichment Analysis. In a microarray analysis, we identified 12 genes increased as more than 2-fold in the kidneys of SHRs and SHRSP in comparison to WKY rats. In a periodic regression analysis, phosphoribosyl pyrophosphate amidotransferase (Ppat) and fragile X mental retardation, autosomal homolog 1 (Fxr1) showed circadian rhythm. Immunocytochemistry revealed PPAT-positivity in nuclei and cytoplasm in the tubules, and FXR1-positivity in the cytoplasm of TCMK-1. In 5-week-old WKY rat and SHR kidneys, PPAT was localized in the nucleus and cytoplasm of the proximal and distal tubules, and FXR1 was localized to the cytoplasm of the proximal and distal tubules.ConclusionsPPAT and FXR1 are pivotal molecules in the control of blood pressure circadian rhythm by the kidney in hypertension.

Hypoxia-mediated regulation of mitochondrial transcription factors in renal epithelial cells: implications for hypertensive renal physiology.

Kidneys have a high resting metabolic rate and low partial pressure of oxygen due to enhanced mitochondrial oxygen consumption and ATP production needed for active solute transport. Heightened mitochondrial activity leads to progressively increasing hypoxia from the renal cortex to the renal medulla. Renal hypoxia is prominent in hypertensive rats due to increased sodium reabsorption within the nephrons, which demands higher energy production by oxidative phosphorylation (OXPHOS). Consequently, spontaneously hypertensive rats (SHR) display greater oxygen deficiency (hypoxia) than normotensive Wistar Kyoto rats (WKY). Here, we sought to investigate the expression of key proteins for mitochondrial biogenesis in SHR and WKY, and study the regulation of mitochondrial transcription factors (mtTFs) under in vitro hypoxic conditions in renal epithelial cells. We report that renal expressions of hypoxia-inducible factor-1-alpha (HIF-1α), peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1α), mtTFs, and OXPHOS proteins are elevated in SHR compared to WKY. In addition, our experiments in cultured kidney cells demonstrate that acute hypoxia augments the expression of these genes. Furthermore, we show that the transcripts of HIF-1α and mtTFs are positively correlated in various human tissues. We reveal, for the first time to our knowledge, that HIF-1α transactivates mtTF genes by direct interaction with their promoters in rat kidney epithelial cells (NRK-52E) under acute hypoxia. Concomitant increases in the mitochondrial DNA and RNA, and OXPHOS proteins are observed. Taken together, this study suggests that hypoxia within the renal epithelial cells may enhance mitochondrial function to meet the energy demand in proximal tubular cells during prehypertensive stages in kidneys of young SHR.

Brain and kidney GHS-R1a underexpression is associated with changes in renal function and hemodynamics during neurogenic hypertension

Ghrelin is a peptide hormone whose effects are mediated by the growth hormone secretagogue receptor subtype 1a (GHS-R1a), mainly expressed in the brain but also in kidneys. The hypothesis herein raised is that GHS-R1a would be player in the renal contribution to the neurogenic hypertension pathophysiology. To investigate GHS-R1a role on renal function and hemodynamics, we used Wistar (WT) and spontaneously hypertensive rats (SHR). First, we assessed the effect of systemically injected vehicle, ghrelin, GHS-R1a antagonist PF04628935, ghrelin plus PF04628935 or GHS-R1a synthetic agonist MK-677 in WT and SHR rats housed in metabolic cages (24 h). Blood and urine samples were also analyzed. Then, we assessed the GHS-R1a contribution to the control of renal vasomotion and hemodynamics in WT and SHR. Finally, we assessed the GHS-R1a levels in brain areas, aorta, renal artery, renal cortex and medulla of WT and SHR rats using western blot. We found that ghrelin and MK-677 changed osmolarity parameters of SHR, in a GHS-R1a-dependent manner. GHS-R1a antagonism reduced the urinary Na+ and K+ and creatinine clearance in WT but not in SHR. Ghrelin reduced arterial pressure and increased renal artery conductance in SHR. GHS-R1a protein levels were decreased in the kidney and brain areas of SHR when compared to WT. Therefore, GHS-R1a role in the control of renal function and hemodynamics during neurogenic hypertension seem to be different, and this may be related to brain and kidney GHS-R1a downregulation.

Polyprenol-Based Lipofecting Agents for In Vivo Delivery of Therapeutic DNA to Treat Hypertensive Rats

Development of efficient vectors for transfection is one of the major challenges in genetic engineering. Previous research demonstrated that cationic derivatives of polyisoprenoids (PTAI) may serve as carriers of nucleic acids. In the present study, the effectiveness of two PTAI-based formulations (PTAI-6–8 and 10–14) was investigated and compared to the commercial reagents. The purpose of applied gene therapy was to enhance the expression of vascular endothelial growth factor (VEGF-A) in the renal medulla of spontaneously hypertensive rats (SHR) and to test its potential as a novel antihypertensive intervention. In the first part of the study (in vitro), we confirmed that PTAI-based lipoplexes efficiently transfect XC rat sarcoma cells and are stable in 37 °C for 7 days. In the in vivo experiments, we administered selected lipoplexes directly to the kidneys of conscious SHR (via osmotic pumps). There were no blood pressure changes and VEGF-A level in renal medulla was significantly higher only for PTAI-10–14-based formulation. In conclusion, despite the promising results, we were not able to achieve VEGF-A expression level high enough to verify VEGF-A gene therapy usefulness in SHR. However, results of our study give important indications for the future development of PTAI-based DNA carriers and kidney-targeted gene delivery.

Enhanced Myogenic Constriction in the SHR Preglomerular Vessels Is Mediated by Thromboxane A2 Synthesis.

BackgroundSpontaneously Hypertensive Rats (SHR) have chronically elevated blood pressures at 30 weeks of age (systolic: 191.0 ± 1.0, diastolic: 128.8 ± 0.9). However, despite this chronic malignant hypertension, SHR kidneys remain relatively free of pathology due to having an augmented myogenic constriction (MC). We hypothesized that the enhanced MC in the SHR preglomerular vessels was due to increased prostaglandin and decreased nitric oxide (NO) synthesis, providing renal protection.MethodsSHR and Wistar Kyoto (WKY) arcuate and mesenteric arteries were treated with indomethacin (prostaglandin synthesis inhibitor), N omega-nitro-L-arginine (L-NNA, NO synthase inhibitor), and nifedipine (L-type calcium channel blocker); and MC was measured in these vessels. The role of endothelium in MC was examined by removing endothelium from WKY and SHR preglomerular and mesenteric arteries using human hair, and measuring MC. We also studied the source of prostaglandin in the SHR by treating endothelium-removed arcuate arteries with indomethacin and furegrelate (thromboxane synthase inhibitor).ResultsMC was enhanced in the SHR preglomerular vessels but not the mesenteric arteries. Indomethacin and LNNA removed the enhanced MC in the SHR. Nifedipine also inhibited MC in both WKY and SHR arcuate and mesenteric arteries. Removing endothelium did not change MC in either arcuate or mesenteric arteries of WKY and SHR rats; and did not remove the augmented MC in the SHR arcuate arteries. Indomethacin and furegrelate decreased MC in endothelium-removed SHR arcuate arteries and obliterated the enhanced MC in the SHR.ConclusionThe enhanced MC in the SHR arcuate arteries was due to thromboxane A2 synthesis from the tunica media and/or adventitia layers. MC was not dependent on endothelium, but was dependent on L-type calcium channels. Nevertheless, SHR arcuate arteries displayed differential intracellular calcium signaling compared to the WKYs.

P0916OLIVE LEAF EXTRACT IMPROVES RENAL ANTIOXIDANT DEFENSE WITHOUT ALTERING THE HEME OXYGENASE-1/BILIVERDIN REDUCTASE PATHWAY IN HYPERTENSIVE RATS WITH FOCAL SEGMENTAL GLOMERULOSCLEROSIS

Abstract Background and Aims Oxidative stress is implicated in the pathophysiology of chronic kidney disease. Previously, we showed that adriamycin (anticancer agent, enhances reactive oxygen species production) induced focal segmental glomerular sclerosis (FSGS) with massive proteinuria in spontaneously hypertensive rats (SHR). The heme oxygenase (HO) system plays an important role in regulating oxidative stress and is protective in chronic kidney disease. HO-1 is a cytoprotective enzyme that catalyzes the conversion of highly reactive free heme molecules into biliverdin, carbon monoxide, and iron. Biliverdin is subsequently converted to bilirubin by biliverdin reductase and has potent antioxidant effect. Olive leaf extract (OLE, Olea europaea L.) is rich in phenolic compounds that are known to possess powerful antioxidant properties. Here, we aimed to investigate the effects of OLE, focusing on its modulatory role on oxidative stress and HO-1/BVR pathway in the kidney of SHR with adriamycin-induced FSGS. Method Adult females SHR were divided into three groups. Control rats received vehicle. Two other groups, FSGS and FSGS+OLE, received adriamycin (2 mg/kg body weight i.v.) twice in 3-week-interval. After the second injection, FSGS+OLE group received OLE (80 mg/kg/day) by gavage for 6 weeks. Mean blood pressure (MAP), urine albumin-to-creatinine (Ualb/cr), renal HO-1 and biliverdin reductase protein expressions (Western Blot), protein carbonyl content (PCOs), and antioxidant capacity (ABTS) were analyzed. Results In FSGS group albuminuria was significantly increased in comparison to the level in control. Chronic consumption of OLE markedly, but not significantly decreased Ualb/cr compared to that in control. Analysis of renal PCOs revealed that significant enhancement of protein oxidation in the kidney of model group was reduced after OLE treatment to the level as in control. The ABTS level in kidney homogenates significantly decreased in FSGS group in comparison to the level in control. OLE significantly increased renal antioxidant capacity in FSGS+OLE group compared to that in model group. Western blot analysis of HO-1, and biliverdin reductase in the kidney revealed that protein expressions of both enzymes were significantly decreased in FSGS group compared to that in control. Following OLE treatment in FSGS+OLE group protein expressions of HO-1, and biliverdin reductase remained at similar level as in model group. No change in MAP values were observed between control and model groups. OLE significantly decreased MAP in FSGS+OLE group in comparison to the value of model group, and nearly significant reduction of MAP compared to the value of control. Conclusion Collectively, our results showed that OLE expressed its antioxidant property and improved oxidative status in the kidney of SHR with ADR-induced FSGS, independently of the HO-1/BVR pathway.