AGTR1 mRNA Research Articles

Renin-angiotensin system blockade: Effect on renal mRNA expression in 5/6 nephrectomized rats

The aim of this study was to determinate the gene expression levels of angiotensinogen, angiotensin converting enzyme, renin, (pro)renin receptor, and the final rennin-angiotensin system (RAS) products Angiotensin (Ang) II and Ang 1-7 inthe remnant kidney of 5/6 nephrectomized rats and its response to RAS pharmacological blockade. Male Wistar rats were divided into five groups: sham operated (SO), 5/6 nephrectomized (NFX), NFX + captopril (50 mg/ kg/day), NFX + losartan (10 mg/kg/day), and NFX + aliskiren (10 mg/kg/day). Animals were followed up for 60 days and protein urine excretion was measured. Systolic blood pressure, renal tissue RAS mRNA expression levels, plasma Ang II, and plasma Ang 1-7 were evaluated at day 60 after nephrectomy. Blood pressure and urine protein were increased after 5/6 nephrectomy. Ang II levels were increased 9.4 fold, whereas Ang 1-7 decreased 72.9% in NFX animals compared with SO rats. 5/6 nephrectomy increased renal angiotensinogen and (pro)renin receptor mRNA expression but down-regulated renin mRNA expression. RAS blockade restored the systolic blood pressure to normal values and slowed down urinary protein excretion, and also prevented changes in Ang II and Ang 1-7. RAS blockade reduced (pro)renin receptor, ACE, and AGT mRNA expression in the remnant kidney. However, renin mRNA expression increased compared with NFX rats. In conclusion these results suggest that inhibition of Ang II synthesis by RAS blockade is associated with renal regulation of RAS mRNA expression and this may be through a mechanism related with the Ang II/Ang 1-7 balance.

Heterogeneous Nuclear Ribonucleoprotein F Suppresses Angiotensinogen Gene Expression and Attenuates Hypertension and Kidney Injury in Diabetic Mice

We investigated the impact of heterogeneous nuclear ribonucleoprotein F (hnRNP F) overexpression on angiotensinogen (Agt) gene expression, hypertension, and renal proximal tubular cell (RPTC) injury in high-glucose milieu both in vivo and in vitro. Diabetic Akita transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs were created, and the effects on systemic hypertension, Agt gene expression, renal hypertrophy, and interstitial fibrosis were studied. We also examined immortalized rat RPTCs stably transfected with control plasmid or plasmid containing hnRNP F cDNA in vitro. The results showed that hnRNP F overexpression attenuated systemic hypertension, suppressed Agt and transforming growth factor-β1 (TGF-β1) gene expression, and reduced urinary Agt and angiotensin II levels, renal hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg mice. In vitro, hnRNP F overexpression prevented the high-glucose stimulation of Agt and TGF-β1 mRNA expression and cellular hypertrophy in RPTCs. These data suggest that hnRNP F plays a modulatory role and can ameliorate hypertension, renal hypertrophy, and interstitial fibrosis in diabetes. The underlying mechanism is mediated, at least in part, via the suppression of intrarenal Agt gene expression in vivo. hnRNP F may be a potential target in the treatment of hypertension and kidney injury in diabetes.

Abstract 627: Antisense Inhibition Of Angiotensinogen Reduces BP In Normotensive Sprague-Dawley Rats And Effectively Eliminates RAS-dependent BP Control In Hypertensive SHR Rats

Uncontrolled hypertension is an important contributor to stroke, myocardial infarction, heart failure and renal failure. Despite the armamentarium of antihypertensive therapeutics, there still exists a need for a novel agent effective in individuals with Resistant Hypertension (RHTN). Second generation antisense oligonucleotides (ASOs) are a novel therapeutic class of inhibitors that cause RNase H-mediated target mRNA degradation in organs such as the liver and kidneys. Experiments were conducted to determine the efficacy of ASO treatment targeting angiotensinogen (Agt). Normotensive Sprague-Dawley rats were given 10 - 80 mg/kg/wk of an Agt ASO for 4 weeks via subcutaneous injections. Dose-responsive reductions of liver Agt mRNA (20 - 96%), kidney Agt mRNA (40 - 75%) and plasma Agt (25 - 90%) were observed. BP, measured by tail-cuff volume-pressure recordings, was dose-dependently reduced by Agt ASO treatments as well. At the maximal ASO dose SBP, MAP and DBP were reduced by 39, 43 and 45 mm Hg, respectively. Studies comparing Agt ASO to captopril treatments were conducted in SHR rats. Agt ASO administered at 50, 75 or 100 mg/kg/wk for 2 weeks resulted in dose-dependent reductions of BP of 35 - 57 mm Hg (SBP), 32 - 52 mm Hg (MAP) and 31 - 49 mm Hg (DBP). Addition of 50 mg/kg/day of captopril did not result in further BP reductions in rats receiving 100 mg/kg/wk Agt ASO, suggesting maximal inhibition of RAS signaling was already achieved via Agt ASO treatment in this group. However, captopril treatment in the 50 and 75 mg/kg/wk Agt ASO treated groups did result in further reductions of 17 & 27 mm Hg (SBP), 17 & 25 mm Hg (MAP) and 17 & 24 mm Hg (DBP). These data demonstrate robust and predictive reductions of BP with long term Agt ASO treatment in normo- or hypertensive rats. Additionally, complete inhibition of RAS-dependent BP control could be achieved within 2 wks of ASO treatment. Finally, additive BP lowering was achieved with the combination treatment of Agt ASO and captopril, suggesting improvements in efficacy are possible when ASOs are added to existing RAS inhibitor therapeutics. Such improvements could be desirable in RHTN individuals and/or individuals not at their BP goal with existing RAS inhibitors.

Abstract 331: Angiotensinogen Inhibition by Antisense Oligonucleotides Decreases Atherosclerosis and Obesity in a Dose-Dependent Manner

Background and Objective: The renin-angiotensin system (RAS) exerts profound effects on experimental atherosclerosis. While there is evolving evidence of the complexity of the RAS, angiotensinogen (Agt) remains the single precursor for all known angiotensin peptides. The aim of this study was to determine the dose-dependent effects of inhibiting Agt synthesis on atherosclerosis. Methods and results: Male LDL receptor -/- mice were fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.2% wt/wt cholesterol) for 3 months. Mice were injected weekly i.p. with either an antisense oligonucleotide (ASO) to Agt (ISIS #261333 at 50 mg/kg/week or #487022 at 12.5, 25 and 50 mg/kg/week) or a control ASO (ISIS #141923 at 50 mg/kg/week; n=10 mice per group). Administration of Agt ASO led to ∼90% reduction in plasma Agt concentrations (P<0.001) and a marked reduction in hepatic Agt mRNA abundance (P<0.05). There was also an expected dose-dependent increase in plasma renin concentrations (P<0.001). Agt ASO administration also decreased systolic blood pressure (SBP) in a dose-dependent manner (P<0.05). Inhibition of Agt significantly decreased total plasma cholesterol concentrations in the two highest dose Agt ASO groups. Atherosclerotic lesion areas were markedly decreased by Agt ASO administration in highest dose groups when compared to the PBS group (16.9 ± 1.0 versus 6.0 ± 1.3 and 6.2 ± 1.1 % of intimal area; P<0.001). Unexpectedly, Agt ASO administration also markedly attenuated body weight gain. MRI analyses demonstrated a dose-dependent reduction in fat mass (P<0.001) with no effect on lean mass. Adipose (retroperitoneal, inguinal and subcutaneous) tissue weights were decreased in a dose-dependent manner. Conclusions: Inhibition of Agt synthesis markedly decreases SBP, development of atherosclerosis and body weight gain in a dose-dependent manner.

N-3 and n-6 polyunsaturated fatty acids differentially regulate adipose angiotensinogen and other inflammatory adipokines in part via NF-κB-dependent mechanisms

Excessive secretion of proinflammatory adipokines has been linked to metabolic disorders. We have previously documented anti-inflammatory effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) in adipose tissue; however, the mechanisms by which these fatty acids regulate adipokine secretion remain unclear. Here, we determined differential effects of eicosapentaenoic acid (EPA, n-3 PUFA) vs. arachidonic acid (AA, n-6 PUFA) on expression and secretion of angiotensinogen (Agt), interleukin 6 (IL-6) and monocyte chemotactic protein (MCP-1) in 3T3-L1 adipocytes. While both PUFAs increased intracellular Agt protein and mRNA expression, Agt secretion into culture media was increased only by AA treatment, which in turn was prevented by co-treatment with EPA. At various AA/EPA ratios, increasing AA concentrations significantly increased secretion of the above three adipokines, whereas increasing EPA dose-dependently, while lowering AA, decreased their secretion. Moreover, IL-6 and MCP-1 were more significantly reduced by EPA treatment compared to Agt (IL-6>MCP>Agt). Next, we tested whether nuclear factor-κB (NF-κB), a major proinflammatory transcription factor, was involved in regulation of these adipokines by PUFAs. EPA significantly inhibited NF-κB activation compared to control or AA treatments. Moreover, EPA attenuated tumor necrosis factor-α-induced MCP-1 and further reduced its secretion in the presence of an NF-κB inhibitor. Taken together, we reported here novel beneficial effects of EPA in adipocytes. We demonstrated direct anti-inflammatory effects of EPA, which are at least in part due to the inhibitory effects of this n-3 PUFA on the NF-κB pathway in adipocytes. In conclusion, these studies further support beneficial effects of n-3 PUFAs in adipocyte inflammation and metabolic disorders.

Association of left ventricular mass with the AGTR1 A1166C polymorphism

The A1166C polymorphism is located within the microRNA-155 binding site of the human angiotensin II (Ang II) type-1 receptor (AGTR1) gene. The C allele interferes with the base-pairing complementariness between AGTR1 mRNA and microRNA-155 and thereby increases AGTR1 protein expression in vitro. We hypothesized that left ventricular (LV) mass is associated with the AGTR1 A1166C polymorphism. Among 708 individuals (mean age, 49.4 years; 51.8% women) randomly recruited in a white European population, we measured LV structure by two-dimensional guided M-mode echocardiography, the AGTR1 A1166C polymorphism and the 24-h urinary aldosterone. We applied a mixed model to assess phenotype-genotype associations while adjusting for covariables and accounting for relatedness. The AA (49.1%), AC (42.8%), and CC (8.1%) genotypes were in Hardy-Weinberg equilibrium. Using a recessive model, CC homozygotes compared to A-allele carriers showed significant increases (P < 0.021) in LV mass index (+5.78 ± 2.25 g/m(2)), mean wall thickness (MWT) (+0.48 ± 0.15 mm), interventricular septum (IVS) (+0.60 ± 0.18 mm) and posterior wall thickness (PWT) (+0.34 ± 0.15 mm), but lower 24-h urinary aldosterone excretion (geometric mean, 22.4 vs. 19.0 nmol; P = 0.050). Sensitivity analyses in 552 participants untreated for hypertension were confirmatory. LV mass index is associated with the AGTR1 A1166C polymorphism. Further research should clarify to what extent this association might be mediated via different expression of AGTR1 as modulated by microRNA-155.

The expression and localization of the human placental prorenin/renin-angiotensin system throughout pregnancy: Roles in trophoblast invasion and angiogenesis?

The renin-angiotensin system (RAS) is thought to regulate placentation, however, the expression and localization of RAS pathways in early gestation human placenta is not known. Here we describe the expression of prorenin (REN), (pro)renin receptor (ATP6AP2), angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE; ACE2), angiotensin II type 1 and 2 receptors (AGTR1; AGTR2) and angiotensin 1–7 receptor (MAS1), as well as the angiogenic factor, vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), in early gestation (6–16 weeks) and term (>37 weeks) human placentae. We also describe the location of all of the key RAS proteins in the early gestation placentae. The highest levels of REN, ATP6AP2, AGT, AGTR1 and ACE2 mRNAs were found in early gestation, whereas ACE1 mRNA was highest at term. AGTR2 and MAS1 mRNA expression were low to undetectable in all samples. REN, ATP6AP2 and AGTR1 mRNA levels were correlated with VEGF expression, but not with TGF-β1 mRNA. In early gestation placentae, prorenin, (pro)renin receptor and the angiotensin II type 1 receptor (AT1R) were localized to extravillous trophoblast cells, suggesting they play a key role in trophoblast migration. ACE2 in syncytiotrophoblasts could regulate release of Ang 1-7 into the maternal circulation contributing to the vasodilation of the maternal vasculature. ACE was only found in fetal vascular endothelium and may specifically target the growing fetal placental vessels. Because REN, ATP6AP2 and AGTR1 show strong correlations with expression of VEGF this pathway is likely to be important in placental angiogenesis.

Common genetic variations of the renin–angiotensin–aldosterone system and response to acute angiotensin I-converting enzyme inhibition in essential hypertension

In order to get insight into possible genetic determinants of antihypertensive drug action, we analysed the relations between polymorphisms of the genes of the renin-angiotensin-aldosterone system and acute effects of ACE inhibition on blood pressure as well as circulating renin and aldosterone levels in hypertensive patients. A total of 315 hypertensive patients referred for problems in drug treatment were given a single 50 mg dose of captopril. Plasma renin and aldosterone were measured before and 60 min after the drug administration. Four DNA variants, including angiotensin type I receptor (AGTR1) 1166 A/C, angiotensin-converting enzyme (ACE) I/D, angiotensinogen (AGT) M235T and AGT -217 G/A, were genotyped in the patients and normotensive men (n = 175). A replication study on the relation between AGTR1 1166 A/C and plasma renin and aldosterone levels was carried out in the 244 hypertensive men of the pharmacogenetic GENRES Study. Referred hypertensive patients with the AGTR1 CC genotype had higher aldosterone at baseline (P = 0.02) and after 60 min of captopril administration (P = 0.01) compared with the AA genotype. Replicate analysis in the GENRES patients showed a similar trend. When the two studies were combined (315 and 244 patients, respectively), plasma aldosterone level (P = 0.007) as well as aldosterone/renin ratio (P = 0.04) were significantly higher in the CC genotype (n = 13) than in the AA genotype (n = 370). Transfection studies in cultured HEK293 cells indicated that the 1166C allele was associated with higher mRNA levels than the 1166A allele. The AGTR1 1166C allele when present in homozygous form may be associated with a form of essential hypertension characterized by high plasma aldosterone and low plasma renin levels, possibly due to increased AGTR1 mRNA levels and augmented angiotensin II action.

147. MOLECULAR CHARACTERIZATION OF RENIN - ANGIOTENSIN SYSTEM COMPONENTS IN HUMAN INTRAUTERINE TISSUES AND FETAL MEMBRANES FROM VAGINAL DELIVERY AND CAESAREAN SECTION

The expression of the (pro)renin receptor (ATP6AP2) in late gestational human tissues suggests that the prorenin-angiotensin system (RAS) might influence pregnancy outcome. Here w e characterized the RAS in term fetal membranes (amnion and chorion), decidua and placenta (n = 38) from women undergoing elective cesarean section (non-labouring) or following spontaneous delivery (after labour), and myometrium (n = 16) from elective or emergency cesarean (labouring) deliveries. RT-qPCR was used to quantify prorenin (REN), AGT, ACE, ACE2, AGTR1, AGTR2, ATP6AP2 and MAS1 mRNAs, and immunohistochemistry was used to localize prorenin, AGT, ACE, ACE2 and AGTR1 proteins. In myometrium, mRNAs for downstream signalling proteins (ZBTB16, TGFB1 and PTGS2) were also measured. ACE and AGT mRNA levels were higher in labouring myometrium (P &lt; 0.05), consistent with elevated production of angiotensin II (Ang II), which, by the upregulation of PTGS2 occurring in labour (P = 0.022), could influence labour. In amnion, expression of all RAS component mRNAs, except ATP6AP2, was low. After labour amnion showed lower ACE (P = 0.014) and higher AGTR2 (P = 0.01) mRNA levels. In decidua, RAS components other than AGTR1 and AGTR2 were abundant. Amnion and chorion exhibited higher immunostaining of AGT and prorenin than expected from their low mRNA levels, suggesting that these proteins could have been originated from decidua, where the cognate genes are more active. In placenta, prorenin and AGT were localized to syncytiotrophoblasts and ACE was localized to fetal capillary endothelial cells, while ACE2 distribution was diffuse. AGTR1 mRNA and protein expression was high in the placenta. We propose that ACE in fetal vessels could contribute Ang II to the fetus, while ACE2 in syncytiotrophoblasts might convert placental or maternal circulating Ang II to angiotensin-(1–7), which might then be supplied to the maternal bloodstream. In conclusion, the abundance and distribution of intrauterine RAS components suggest diverse roles for this local RAS in pregnancy.

311. REGULATION OF THE RENIN ANGIOTENSIN SYSTEM (RAS) IN A TROPHOBLAST CELL LINE BY CYCLIC ADENOSINE MONOPHOSPHATE (cAMP) AND 5'-AZA-2'-DEOXYCYTIDINE (AZA)

Renin and renin-like activities have been detected in early gestation placentae1,2. To explore what regulates RAS expression in trophoblasts we studied the effects of cAMP and AZA (DNA demethylating agent) on the expression of mRNAs for prorenin receptor (ATP6AP2), prorenin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), ACE2, angiotensin II type 1 receptor (AGTR1), and a downstream target of the prorenin receptor, cyclooxygenase-2 (PTGS2) in the early human trophoblast cell line HTR-8/SVneo. Cells were cultured for 24 or 48 h with vehicle, 0.5 mM cAMP or 15 mM AZA. Messenger RNA abundances were measured relative to Alien RNA using real-time qRT-PCR. All mRNAs were detected except for ACE and ACE2. REN mRNA abundance was increased by cAMP and AZA (P &lt; 0.0001). However, cAMP was more effective than AZA at both 24 and 48 h (P &lt; 0.0001; P &lt; 0.05) at the concentrations employed. AZA significantly increased AGT mRNA levels at 24 h (P = 0.001) and AGTR1 mRNA levels at 48 h (P &lt; 0.0001), while cAMP had no effect. P TGS2 mRNA expression was increased by cAMP at 24 h (P &lt; 0.0001) and by AZA at 48 h (P &lt; 0.0001). cAMP and AZA had no effect on ATP6AP2 abundance. Thus, prorenin may function directly through the prorenin receptor, since cAMP upregulated both REN and its downstream target PTGS2 in HTR-8/SVneo cells, especially considering that ACE and ACE2 were not expressed. Our findings also suggest that expression of the RAS in trophoblast is regulated by DNA methylation as there was sustained over expression of REN, AGT, AGTR1 and PTGS2 with AZA. The ATP6AP2 promoter possesses CpG islands; however, its expression was not affected by AZA. This may suggest that either DNA methylation is not involved in the regulation of this gene, or that the over expressed prorenin binding to its receptor is causing suppression of ATP6AP2 abundance via a previously identified negative feedback pathway. (1) Pringle et al., Perinatal Society of Australia &amp; New Zealand (PSANZ) 2010, Wellington, New Zealand (Abstract).(2) Itskovitz et al., Journal of Clinical Endocrinology and Metabolism, 1992, 75: 906–10.

Effect of valsartan on the expression of angiotensin II receptors in the lung of chronic antigen exposure rats

Many studies have suggested that angiotensin II (Ang II) and its receptors may be involved in the development of asthma. However, the expression of angiotensin II receptors (AGTR) is not clear in the lung tissue of chronic asthmatics. This study was designed to determine the relationship between airway remodeling, dysfunction and the expression of AGTRs in a rat model of asthma. Rats were sensitized with ovalbumin (OVA) for 2 weeks. Sixty minutes before an inhalation challenge, the rats were pretreated either with valsartan (15, 30, 50 mg x kg(-1) x d(-1)) or saline intragastrically. Then the rats received an OVA challenge for 30 alternative days. Acetylcholine (Ach)-induced bronchoconstriction was measured after the final antigen challenge. White cell counts in bronchoalveolar lavage fluid (BALF) and morphological changes in the airways were then assessed. The levels of transforming growth factor-beta 1 (TGF-beta(1)) and platelet-derived growth factor (PDGF) in BALF were detected by ELISA. The levels of AGTR1 and AGTR2 mRNA and protein in lung tissues were measured by RT-PCR and Western blotting. AGTR1 mRNA and protein levels in repeatedly OVA-challenged rats were significantly increased as compared with negative controls. The AGTR1 mRNA expression versus white cell counts of BALF and airway wall thickness (mainly in small airways) in lungs of chronic antigen-exposed rats were positively correlated. Valsartan decreased the level of AGTR1 in repeatedly OVA-challenged rats. However, AGTR2 mRNA and protein levels in the OVA-challenged rats and high-dose valsartan-treated rats (50 mg x kg(-1) x d(-1)) were also increased. Valsartan significantly decreased inflammatory cell accumulation and attenuated Ach-evoked bronchoconstriction in repeatedly antigen-challenged rats. Valsartan also decreased allergen-induced structural changes in rat airway (including total airway wall thickness and smooth muscle area) and the levels of TGF-beta(1) and PDGF in BALF. AGTR1 expression is potentially associated with airway remodeling and dysfunction in asthma. Ang II and AGTR1 may participate in airway inflammation and airway remodeling of chronic antigen-exposed rats. Valsartan, a AGTR1 antagonist, could inhibit AGTR1 expression and partially inhibits structural airway changes as well as airway inflammation in chronic OVA-exposed rats.

P034 Bone marrow renin-angiotensin system expression in polycythaemia vera and essential thrombocythaemia depends on JAK2 mutational status

Discovery of V617F mutation in JAK2 tyrosine kinase gene in myeloid progenitors provided new insight into the pathogenesis and clinical understanding of CMPD. There are several lines of evidence suggesting the existence of local hematopoietic bone marrow renin-angiotensin system (RAS) which contributes to the regulation of normal and disturbed hematopoiesis. Recently, it was shown that Jak2 kinase is important upstream component in the regulation of the AGT mRNA transcription: activated Jak2 kinase stimulates AGT gene transcription. These observations suggest possibility for constitutively active, mutated Jak2 to modulate transcriptional activation of AGT gene as well as the expression of other RAS genes in CMPD. Aim: We analyzed the expression of RAS genes (ACE, AGT, AT2R1 and REN) in normal BM and that of PV and ET patients with the respect to the presence of activating V617F JAK2 mutation. Patients and methods: Fourteen PV-JAK2V617Fpos patients (7 male, 7 female, median age 68), thirteen ET-JAKV617Fpos patients (6 male, 7 female, median age 72) and seven ETJAK2V617Fneg patients (3 male, 4 female, median age 66) were included in the study. Samples of normal bone marrow were obtained from fi ve healthy donors. Real-time PCR analysis for ACE, AGT, AT1R1, REN genes and internal housekeeping gene GAPDH was performed using Real Time PCR System. To compensate for inter-PCR variations, normalisation of target genes (ACE, AGT, AT2R1, REN) with an endogenous control (GAPDH) was performed. JAK2 V617F mutational status was determined by the allele-specifi c PCR. Results: ACE expression in BM of PV and ET patients was down-regulated to 2-20% of the donor BM values with no statistically signifi cant difference in expression between patients. AGT expression was signifi cantly higher in PV and in ETJAK2V617Fpos comparing to ET JAK2V617Fneg patients. Renin has shown similar pattern of expression as AGT: it was signifi cantly higher in PV and in ET JAK2V617Fpos patients comparing to ET JAK2V617Fneg patients. AT1R gene was signifi cantly higher expressed in PV patients in comparison to both ET subgroups, JAK2V617F positive or negative patients. ET patients did not differ for AT2R1 expression by their JAK2 mutational status. Conclusion: Our fi ndings indicate up-regulation of AGT, AT2R1 and REN genes and down-regulation of ACE gene expression in clonal hematopoiesis of PV and ET. Different expression pattern of major RAS components in BM of PV and ET compared to normal BM is clearly related to the existence of JAK2V617F mutation and less to the PV or ET disease phenotype. However, interesting exception is excessive expression of AT2R1 in PV that was not observed in ET irrespective of JAK2 mutation. This latest observation provides ground for the future experimental and clinical studies for the use of AT2R1 blockers in improving clinical management of JAK2V617Fpos PV.

Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy.

Evaluation of Gene Panel mRNAs in Urine Samples of Kidney Transplant Recipients as a Non-invasive Tool of Graft Function

Non-invasive monitoring may be useful after kidney transplantation (KT), particularly for predicting acute rejection (AR). It is less clear whether chronic allograft nephropathy (CAN) is also associated with changes in urine cells. To identify non-invasive markers of allograft function in kidney transplant patients (KTP), mRNA levels of AGT, TGF-β1, EGFR, IFN-γ, TSP-1, and IL-10 in urine (Ur) samples were studied using QRT-PCR. Ninety-five KTP and 111 Ur samples were evaluated. Patients (Pts) were divided as, within six months (N = 31), and with more than six months post-KT (N = 64). KTP with more than six months post-KT were classified as KTP with stable kidney function (SKF) (N = 32), KTP with SKF (creatinine < 2 mg/dL) and proteinuria > 500 mg/24 h (N = 18), and KTP with biopsy proven CAN (N = 14). F-test was used to test for equality of variances between groups. IL-10 mRNA was decreased in Ur samples from KTP with less than six months post-KT (P = 0.005). For KTR groups with more than six months post-KT, AGT and EGFR mRNA were statistically different among KTP with SKF, KTP with SKF and proteinuria, and CAN Pts (P= 0.003, and P = 0.01), with KTP with SKF having higher mean expression. TSP-1 mRNA levels also were significantly different among these three groups (P = 0.04), with higher expression observed in CAN Pts. Using the random forest algorithm, AGT, EGFR, and TGF-β1 were identified as predictors of CAN, SKF, SKF with proteinuria. A characteristic pattern of mRNA levels in the different KTP groups was observed indicating that the mRNA levels in Ur cells might reflect allograft function.

Quantitated transcript haplotypes (QTH) ofAGTR1, reduced abundance of mRNA haplotypes containing 1166C (rs5186:A&gt;C), and relevance to metabolic syndrome traits

The angiotensin II type 1 receptor (AGTR1) is the main target through which angiotensin II influences cardiovascular tone, cell growth, and fluid and electrolyte balance. AGTR1 polymorphism has been reported to associate with hypertension, myocardial infarction (MI), and metabolic traits. Here we describe a novel approach to quantitation of transcript haplotypes (QTH) of AGTR1. To determine relative allelic expression from haplotypes, within-individual-between-allele ratiometric analyses in placental cDNA were developed for the transcribed SNPs rs5182:C>T (encoding p.L191) and rs5186:A>C (3'-noncoding "A1166C"). Additionally, between-individual comparisons were made using TaqMan assays applied to both homozygous and heterozygous genotypes and haplotypes. In conjunction, linkage disequilibrium (LD) and genomic haplotype associations with metabolic syndrome were examined. There was no significant difference of mRNA level for alleles of rs5182:C>T, but allele and mRNA haplotypes carrying 1166C exhibited reduced abundance. The effect was much greater in CC homozygotes than in heterozygotes. The promoter region was confirmed to be in a separate haplotype block from the AGTR1 3' region containing rs5182:C>T and rs5186:A>C. Metabolic syndrome trait associations were strongest for the 3' block generally and for the C allele of rs5186:A>C specifically. All effects were much more prominent in homozygotes, possibly reflecting interallelic interaction through feedback loops of mRNA regulation. Differential abundance of AGTR1 mRNA haplotypes may mediate clinical phenotypic observations of the AGTR1 genotype.

L-arginine reverses renin, hypertension, and electrolyte excretion changes in pregnancy induced hypertensive rats

Pregnancy-induced hypertensive (PIH) disorders are a leading cause of maternal mortality and fetal morbidity. Spontaneous sustained hypertension and intrauterine growth restriction often characterize these disorders. The SHHF/Mcc-facp (SHHF) has been used as an animal model of the human disorders because it has the above features plus abnormal gene expression in the placental and kidney. Although the mechanisms responsible for PIH are unknown, altered function of the renin-angiotensin system (RAS) has been implicated. To gain a molecular understanding of the role of the RAS in spontaneous PIH in SHHF rats, kidney mRNA expression profile of RAS components at gestation day 20 were compared with normotensive pregnant WKY controls. By gestation day 10, systolic blood pressure was 40 ± 5 mm Hg higher than pre-pregnant values compared to hypotension in pregnant WKY and remained elevated for the duration of pregnancy. Na and Ca excretion were lower than WKY controls despite higher blood pressure. PRA was significantly lower in SHHF rats (2.1 ± 0.1 ng/ml.hr vs 9.7 ± 1.3 ng/ml.hr, p<0.05) and renin mRNA was downregulated 66± 7% compared to pregnant WKY. Although AGT mRNA was undetectable in SHHF rat kidney, ACE RnBP, and AT1 receptor were all highly expressed. L-arginine administration beginning on day 1 reversed changes in blood pressure, Na and Ca excretion, as well as renin mRNA expression, suggesting that there may be an association between Na, Ca, and renin in PIH. L-arginine did not alter abundance of AGT, RnBP, ACE, or AT1receptor mRNA, suggesting that regulation of renin expression and Na and Ca expression in PIH rats may be mediated by the NO pathway. We conclude that the impaired RAS in PIH rats is at the level of renin gene expression and that this impairment may be reversed by L-arginine.

Functional Analysis of a Mutation Occurring between the Two In-frame AUG Codons of Human Angiotensinogen

Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin system, a major participant in blood pressure control. We have identified a natural mutation at the -30 amino acid position of the angiotensinogen signal peptide, in which an arginine is replaced by a proline (R-30P). Heterozygous individuals with R-30P showed a tendency to lowered plasma angiotensinogen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal individuals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiotensinogen mRNA has two in-phase translation initiation codons (AUG) starting upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a cluster of basic residues adjacent to the first AUG codon that may affect intracellular sorting of the nascent protein. Pulse-chase experiments in transiently transfected cultured cells revealed that the R-30P mutation was associated with reduced amounts of both intra- and extracellular protein. In a cell-free system, we found that two forms of native angiotensinogen were generated by alternative initiation of translation at either AUG codon. Alteration of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively. Furthermore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fluorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytoplasm, to the secretory pathway. Although the R-30P mutation may not affect intracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen through decreased translocation or increased residence time in the endoplasmic reticulum.

Role of the Angiotensin Type 2 Receptor Gene in Congenital Anomalies of the Kidney and Urinary Tract, CAKUT, of Mice and Men

Angiotensin type 2 receptor gene null mutant mice display congenital anomalies of the kidney and urinary tract (CAKUT). Various features of mouse CAKUT impressively mimic human CAKUT. Studies of the human type 2 receptor ( AGTR2) gene in two independent cohorts found that a significant association exists between CAKUT and a nucleotide transition within the lariat branchpoint motif of intron 1, which perturbs AGTR2 mRNA splicing efficiency. AGTR2, therefore, has a significant ontogenic role for the kidney and urinary tract system. Studies revealed that the establishment of CAKUT is preceded by delayed apoptosis of undifferentiated mesenchymal cells surrounding the urinary tract during key ontogenic events, from the ureteral budding to the expansive growth of the kidney and ureter.