Angiotensinogen as a Therapeutic Target for Cardiovascular and Metabolic Diseases.

AGT (angiotensinogen) is the unique precursor for the generation of all the peptides of the renin-angiotensin system, but it has received relatively scant attention compared to many other renin-angiotensin system components. Focus on AGT has increased recently, particularly with the evolution of drugs to target the synthesis of the protein. AGT is a noninhibitory serpin that has several conserved domains in addition to the angiotensin II sequences at the N terminus. Increased study is needed on the structure-function relationship to resolve many unknowns regarding AGT metabolism. Constitutive whole-body genetic deletion of Agt in mice leads to multiple developmental defects creating a challenge to use these mice for mechanistic studies. This has been overcome by creating Agt-floxed mice to enable the development of cell-specific deficiencies that have provided considerable insight into a range of cardiovascular and associated diseases. This has been augmented by the recent development of pharmacological approaches targeting hepatocytes in humans to promote protracted inhibition of AGT synthesis. Genetic deletion or pharmacological inhibition of Agt has been demonstrated to be beneficial in a spectrum of diseases experimentally, including hypertension, atherosclerosis, aortic and superior mesenteric artery aneurysms, myocardial dysfunction, and hepatic steatosis. This review summarizes the findings of recent studies utilizing AGT manipulation as a therapeutic approach.

Association of biomarkers of endothelial function, coagulation activation and kidney injury with persistent albuminuria in sickle cell anaemia.

Persistent albuminuria (PA) is common in sickle cell anaemia (SCA). With the association of chronic kidney disease (CKD) with increased mortality, biomarkers that predict its development or progression are needed. We evaluated the association of select biomarkers with PA in adults with SCA using Kruskal-Wallis rank-sum test and logistic regression models, with adjustment for multiple testing. Of 280 subjects, 100 (35.7%) had PA. Median plasma levels of soluble vascular cell adhesion molecule-1 (VCAM-1) (1176.3 vs. 953.4 ng/mL, false discovery rate [FDR] q-value <0.003), thrombin-antithrombin complex (5.5 vs. 4.7 ng/mL, FDR q-value = 0.04), and urinary angiotensinogen (AGT) (12.2 vs. 5.3 ng/mg, FDR q-value <0.003), urinary nephrin (30.6 vs. 27.2 ng/mg, FDR q-value = 0.04), and urinary kidney injury molecule-1 (KIM-1) (0.8 vs. 0.5 ng/mg, FDR q-value <0.003), normalized to urine creatinine, were significantly higher in subjects with PA. In multivariable analysis, only urinary AGT (odds ratio = 1.058, FDR q-value <0.0001) remained a significant predictor of PA. In addition, soluble VCAM-1 (FDR q-value <0.0001), D-dimer (FDR q-value <0.0001), urinary AGT (FDR q-value <0.0001), KIM-1 (FDR q-value <0.0001), and nephrin (FDR q-value <0.0001) were significantly associated with urine albumin-creatinine ratio in multivariable analyses. Longitudinal studies to evaluate the predictive capacity of biomarkers for the development and progression of CKD in SCA are warranted.

Anxiolytic effect of alamandine in male transgenic rats with low brain angiotensinogen is dependent on activation of MrgD receptors.

Alamandine is a peptide hormone belonging to the renin-angiotensin system (RAS). It acts through the Mas-related G-protein coupled receptor type D, MrgD, which is expressed in different tissues, including the brain. In the present study, we hypothesize that a lack of alamandine, through MrgD, could cause the anxiety-like behavior in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)680]. Adult male transgenic rats exhibited a significant increase in the latency to feeding time in the novelty suppressed feeding test and a decrease in the percentage of time and entries in the open arms in the elevated plus maze. These effects were reversed by intracerebroventricular infusion of alamandine. Pretreatment with D-Pro7-Ang-(1-7), a Mas and MrgD receptor antagonist, prevented the anxiolytic effects induced by this peptide. However, its effects were not altered by the selective Mas receptor antagonist, A779. In conclusion, our data indicates that alamandine, through MrgD, attenuates anxiety-like behavior in male TGR(ASrAOGEN)680, which reinforces the importance of the counter-regulatory RAS axis as promising target for the treatment of neuropsychiatric disorders.

Targeting Angiotensinogen With N-Acetylgalactosamine-Conjugated Small Interfering RNA to Reduce Blood Pressure.

Blood pressure management involves antihypertensive therapies blocking the renin-angiotensin system (RAS). Yet, it might be inadequate due to poor patient adherence or the so-called RAS escape phenomenon, elicited by the compensatory renin elevation upon RAS blockade. Recently, evidence points toward targeting hepatic AGT (angiotensinogen) as a novel approach to block the RAS pathway that could circumvent the RAS escape phenomenon. Removing AGT, from which all angiotensins originate, should prevent further angiotensin generation, even when renin rises. Furthermore, by making use of a trivalent N-acetylgalactosamine ligand-conjugated small interfering RNA that specifically targets the degradation of hepatocyte-produced mRNAs in a highly potent and specific manner, it may be possible in the future to manage hypertension with therapy that is administered 1 to 2× per year, thereby supporting medication adherence. This review summarizes all current findings on AGT small interfering RNA in preclinical models, making a comparison versus classical RAS blockade with either ACE (angiotensin-converting enzyme) inhibitors or AT1 (angiotensin II type 1) receptor antagonists and AGT suppression with antisense oligonucleotides. It ends with discussing the first-in-human study with AGT small interfering RNA.

Stronger association of intact angiotensinogen with mortality than lactate or renin in critical illness: post-hoc analysis from the VICTAS trial.

Sepsis and septic shock remain global healthcare problems associated with high mortality rates despite best therapy efforts. Circulating biomarkers may identify those patients at risk for poor outcomes, however, current biomarkers, most prominently lactate, are non-specific and have an inconsistent impact on prognosis and/or disease management. Activation of the renin-angiotensin- system (RAS) is an early event in sepsis patients and elevated levels of circulating renin are more predictive of worse outcomes than lactate. The precursor protein Angiotensinogen is another key component of the circulating RAS; it is the only known substrate for renin and the ultimate source of the vasopressor Angiotensin II (Ang II). We postulate that lower Angiotensinogen concentrations may reflect a dysfunctional RAS characterized by high renin concentrations but attenuated Ang II generation, which is disproportionate to the high renin response and may compromise adequate support of blood pressure and tissue perfusion in septic patients. The current study compared the association between serum Angiotensinogen with mortality to that of lactate and renin in the VICTAS cohort of sepsis patients at baseline (day 0) by receiver operating characteristic (ROC) and Kaplan-Meier curve analyses. Serum concentration of Angiotensinogen was more strongly associated with 30-day mortality than either the serum concentrations of renin or lactate in sepsis patients. Moreover, the clinical assessment of Angiotensinogen may have distinct advantages over the typical measures of renin. The assessment of intact Angiotensinogen may potentially facilitate more precise therapeutic approaches (including exogenous angiotensin II) to restore a dysfunctional RAS and improve patient outcomes. Additional prospective validation studies are clearly required for this biomarker in the future.

Correlation of single nucleotide polymorphisms in the AGT gene with susceptibility to systemic lupus erythematosus in Northeast China.

To investigate the correlation between susceptibility to systemic lupus erythematosus (SLE) and single nucleotide polymorphisms (SNPs) rs699, rs4762 and rs1926723 in the AGT gene in the population of Northeast China, while also introducing a new method for early detection of SLE. A total of 856 cases of SLE patients and healthy volunteers who attended the First Affiliated Hospital of Harbin Medical University from January 2020 to December 2022 were recruited. Clinical information and biood samples were collected from particpants in this study. SNaPshot sequencing technology was used to sequence the bases of the rs699, rs4762 and rs1926723 in the AGT gene. The genetic stability of SNPs was analysed by means of Hardy-Weinberg (HWE) genetic equilibrium. The study examined the correlation between genetically stable SNPs and susceptibility to SLE using logistic regression analysis. Rs699 did not adhere to the principles of the HWE genetic equilibrium (p < .01). Conversely, both rs4762 and rs1926723 conformed to the HWE genetic equilibrium (p > .05). However, no significant differences in genotypes and alleles frequencies of the rs4762 were observed between the two groups (p > .05). Furthermore, there was a significant difference in the distribution of AG, GG genotypes frequency and G allele frequency at the rs1926723 between the two groups (p < .001). Individuals with AG and GG genotypes and the G allele had a significantly lower frequency of SLE, indicating a potential genetic protective factor against susceptibility to the SLE. The SNPs rs1926723 may be linked to the susceptibility to SLE, and the AG, GG genotypes and the G allele may be important protective factors for the development of SLE in Northeast China.

Genetic Variations Related to Angiotensin II Production and Risk for Basal Cell Carcinoma.

Basal cell carcinoma (BCC) is the most prevalent human neoplasm, with constantly increasing annual incidence. Despite its slow growth, BCC is locally invasive and, if left untreated, can cause severe complications, including metastasis and death. The renin-angiotensin system (RAS) plays a key role in electrolyte balance, atrial pressure, tissue development, homeostasis, and inflammation, but also in cancer development. After binding to its type 1 receptor (AT1R), angiotensin II (ANGII), the system's principal hormonal effector, regulates cancer pathways spanning from the formation of the initial cancer cell to the construction and nutrition of the tumor microenvironment, angiogenesis, proliferation, and metastasis. Although the role of RAS in the development of skin pathologies has not been widely researched, RAS-targeting antihypertensive medications have been shown to have a chemoprotective effect against BCC. Based on those findings, our group conducted a series of genetic association studies to investigate the association between common functional variations in key genes related to ANGII production (AGT, ACE, ACE2, AT1R, AT2R, and CMA1) and the risk of BCC occurrence. This review provides a summary of the current understanding of the ANGII involvement in BCC development. The reliable and easily assessed pool of genetic biomarkers may be used for predictive testing and prevention purposes in high-risk individuals.

[Polymorphism of RAAS genes in patients with COVID-19: comparison with frequency in population and relationship with severity of course].

AIM: Evaluation of genes polymorphisms frequencies of angiotensinogen (AGT), angiotensin converting enzyme type 1 (ACE1) and angiotensin II receptors type 1 (AGTR1) and type 2 (AGTR2) in patients admitted with coronavirus disease (COVID-19) and its association the severity of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2). MATERIALS AND METHODS: The study included 100 patients admitted to the hospital with a laboratory-confirmed diagnosis of COVID-19. All patients were identified with alleles and genotypes of polymorphic markers rs4762 of the AGT gene, rs1799752 of the ACE1 gene, rs5186 of the AGTR1 gene and rs1403543 of the AGTR2 gene. The frequencies of each polymorphisms were compared with population. Statistical processing was performed using the Statistica 8.0 software package. RESULTS: In evaluated cohort there was higher frequency of D-allele ACE1 rs1799752 compared to population. Depending on the availability of criteria for the severity of coronavirus infection, 44 (44%) patients were diagnosed with severe, 56 (56%) with moderate course. The groups did not significantly differ in age, gender, cardiovascular risk factors and comorbid pathology. In the groups with severe and moderate course, the same distribution of genotypes and alleles of AGT rs4762, AGTR2 rs1403543 and ACE1 rs1799752 was revealed. For the I/D alleles of the ACE1 rs1799752 gene, a significant deviation from the papulation was found in both the group of severe and moderate COVID-19. In the group with a severe course of the disease, a higher frequency of the mutant C-allele of the AGTR1 rs5186 gene was detected. In the same group, a deviation in the frequency ratio of A and C of the AGTR1 rs5186 alleles from Hardy-Weinberg Equilibrium was found. When calculating the risk of severe COVID-19 in the presence of the C-allele compared with the A-allele, an odds ratio 2.092 (95% confidence interval 1.066-4.108) was obtained. CONCLUSION: The data obtained suggest that the genes polymorphisms of the components of renin-angiotensin-aldosterone system, namely D-allele of ACE1 rs1799752 and C-allele of AGTR1 rs5186, may make it possible to identify groups of patients predisposed to the development of more severe COVID-19.

Urinary angiotensinogen is associated with albuminuria in adults with sickle cell anaemia.

We explored the association of novel urinary biomarkers with albumin-creatinine ratio (ACR) in adults with sickle cell anaemia. Of 37 participants, 13 (35.2%) had persistent albuminuria (PA). Urinary levels of clusterin (p = 0.002), retinol-binding protein 4 (p = 0.008), alpha-1 microglobulin (p = 0.002) and angiotensinogen (p = 0.006) were significantly higher in participants with PA than in those without PA. Although univariate analysis showed significant associations between both alpha-1 microglobulin (p = 0.035) and angiotensinogen (p = 0.0021) with ACR, only angiotensinogen was associated with ACR in multivariable analysis (p = 0.04). Our results suggest that urinary angiotensinogen may identify sickle cell anaemia patients at risk for kidney disease.

Loss of Hepatic Angiotensinogen Attenuates Sepsis-Induced Myocardial Dysfunction.

[Figure: see text].

Inhibition of the Renin-Angiotensin System Fails to Suppress ß-Aminopropionitrile-Induced Thoracic Aortopathy in Mice-Brief Report.

BACKGROUND: Cross-linking of lysine residues in elastic and collagen fibers is a vital process in aortic development. Inhibition of lysyl oxidase by BAPN (ß-aminopropionitrile) leads to thoracic aortopathies in mice. Although the renin-angiotensin system contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the renin-angiotensin system protects against aortopathy caused by the impairment of elastic fiber/collagen crosslinking. METHODS: BAPN (0.5% wt/vol) was started in drinking water to induce aortopathies in male C57BL/6J mice at 4 weeks of age for 4 weeks. Five approaches were used to investigate the impact of the renin-angiotensin system. Bulk RNA sequencing was performed to explore potential molecular mechanisms of BAPN-induced thoracic aortopathies. RESULTS: Losartan increased plasma renin concentrations significantly, compared with vehicle-infused mice, indicating effective angiotensin II type 1 receptor inhibition. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor of the renin-angiotensin system, irbesartan, an insurmountable inhibitor, was also tested. Although increased plasma renin concentrations indicated effective inhibition, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin II type 1 receptor blockade. Next, we inhibited angiotensin II production by pharmacological or genetic depletion of AGT (angiotensinogen), the unique precursor of angiotensin II. However, neither suppressed BAPN-induced thoracic aortic rupture and dilatation. Aortic RNA sequencing revealed molecular changes during BAPN administration that were distinct from other types of aortopathies in which angiotensin II type 1 receptor inhibition protects against aneurysm formation. CONCLUSIONS: Inhibition of either angiotensin II action or production of the renin-angiotensin system does not attenuate BAPN-induced thoracic aortopathies in mice.

Intrarenal renin-angiotensin system activation and macrophage infiltrations in pediatric chronic glomerulonephritis.

BACKGROUND: The current study tested the hypothesis that urinary angiotensinogen (UAGT) and urinary monocyte chemoattractant protein-1 (UMCP-1) levels provide a specific index of intrarenal renin-angiotensin system (RAS) status and the degree of infiltration of macrophages associated with RAS blockade and immunosuppressant treatment in pediatric patients with chronic glomerulonephritis. METHODS: We measured baseline UAGT and UMCP-1 levels to examine the correlation between glomerular injury in 48 pediatric chronic glomerulonephritis patients before treatment. Furthermore, we performed immunohistochemical analysis of angiotensinogen (AGT) and CD68 in 27 pediatric chronic glomerulonephritis patients treated with RAS blockades and immunosuppressants for 2 years. Finally, we examined the effects of angiotensin II (Ang II) on monocyte chemoattractant protein-1 (MCP-1) expression in cultured human mesangial cells (MCs). RESULTS: Baseline UAGT and UMCP-1 levels positively correlated with urinary protein levels, scores for mesangial hypercellularity, rate of crescentic formation, and expression levels of AGT and CD68 in renal tissues (p < 0.05). UAGT and UMCP-1 levels were significantly decreased after RAS blockade and immunosuppressant treatment (p < 0.01), which was accompanied by AGT and CD68 (p < 0.01), as well as the magnitude of glomerular injury. Cultured human MCs showed increased MCP-1 messenger ribonucleic acid and protein levels after Ang II treatment (p < 0.01). CONCLUSIONS: The data indicates that UAGT and UMCP-1 are useful biomarkers of the degree of glomerular injury during RAS blockade and immunosuppressant treatment in pediatric patients with chronic glomerulonephritis.

Fetal growth restriction followed by very preterm birth is associated with smaller kidneys but preserved kidney function in adolescence.

BACKGROUND: Preterm birth and fetal growth restriction (FGR) are associated with structural and functional kidney changes, increasing long-term risk for chronic kidney disease and hypertension. However, recent studies in preterm children are conflicting, indicating structural changes but normal kidney function. This study therefore assessed kidney structure and function in a cohort of adolescents born very preterm with and without verified FGR. METHODS: Adolescents born very preterm with FGR and two groups with appropriate birthweight (AGA) were included; one matched for gestational week at birth and one born at term. Cortical and medullary kidney volumes and T1 and T2* mapping values were assessed by magnetic resonance imaging. Biochemical markers of kidney function and renin-angiotensin-aldosterone system (RAAS) activation were analyzed. RESULTS: Sixty-four adolescents were included (13-16 years; 48% girls). Very preterm birth with FGR showed smaller total (66 vs. 75 ml/m2; p = 0.01) and medullary volume (19 vs. 24 ml/m2; p < 0.0001) compared to term AGA. Corticomedullary volume ratio decreased from preterm FGR (2.4) to preterm AGA (2.2) to term AGA (1.9; p = 0.004). There were no differences in T1 or T2* values (all p ≥ 0.34) or in biochemical markers (all p ≥ 0.12) between groups. CONCLUSIONS: FGR with abnormal fetal blood flow followed by very preterm birth is associated with smaller total kidney and medullary kidney volumes, but not with markers of kidney dysfunction or RAAS activation in adolescence. Decreased total kidney and medullary volumes may still precede a long-term decrease in kidney function, and potentially be used as a prognostic marker. A higher resolution version of the Graphical abstract is available as Supplementary information.

Anomalies in neurovascular coupling during early diabetes: A review.

Diabetic retinopathy is the most feared complication for those with diabetes. Although visible vascular pathology traditionally defines the management of this condition, it is now recognised that a range of cellular changes occur in the retina from an early stage of diabetes. One of the most significant functional changes that occurs in those with diabetes is a loss of vasoregulation in response to changes in neural activity. There are several retinal cell types that are critical for mediating so-called neurovascular coupling, including Müller cells, microglia and pericytes. Although there is a great deal of evidence that suggests that Müller cells are integral to regulating the vasculature, they only modulate part of the vascular tree, highlighting the complexity of vasoregulation within the retina. Recent studies suggest that retinal immune cells, microglia, play an important role in mediating vasoconstriction. Importantly, retinal microglia contact both the vasculature and neural synapses and induce vasoconstriction in response to neurally expressed chemokines such as fractalkine. This microglial-dependent regulation occurs via the vasomediator angiotensinogen. Diabetes alters the way microglia regulate the retinal vasculature, by increasing angiotensinogen expression, causing capillary vasoconstriction and contributing to a loss of vascular reactivity to physiological signals. This article summarises recent studies showing changes in vascular regulation during diabetes, the potential mechanisms by which this occurs and the significance of these early changes to the progression of diabetic retinopathy.

Hepatic and proximal tubule angiotensinogen play distinct roles in kidney dysfunction, glomerular and tubular injury, and fibrosis progression.

Components of the renin-angiotensin system, including angiotensinogen (AGT), are critical contributors to chronic kidney disease (CKD) development and progression. However, the specific role of tissue-derived AGTs in CKD has not been fully understood. To define the contribution of liver versus kidney AGT in the CKD development, we performed 5/6 nephrectomy (Nx), an established CKD model, in wild-type (WT), proximal tubule (PT)- or liver-specific AGT knockout (KO) mice. Nx significantly elevated intrarenal AGT expression and elevated blood pressure (BP) in WT mice. The increase of intrarenal AGT protein was completely blocked in liver-specific AGT KO mice with BP reduction, suggesting a crucial role for liver AGT in BP regulation during CKD. Nx-induced glomerular and kidney injury and dysfunction, as well as fibrosis, were all attenuated to a greater extent in liver-specific AGT KO mice compared with PT-specific AGT KO and WT mice. However, the suppression of interstitial fibrosis in PT- and liver-specific AGT KO mouse kidneys was comparable. Our findings demonstrate that liver AGT acts as a critical contributor in driving glomerular and tubular injury, renal dysfunction, and fibrosis progression, whereas the role of PT AGT was limited to interstitial fibrosis progression in chronic renal insufficiency. Our results provide new insights for the development of tissue-targeted renin-angiotensin system intervention in the treatment of CKD.NEW & NOTEWORTHY Chronic kidney disease (CKD) is a major unmet medical need with no effective treatment. Current findings demonstrate that hepatic and proximal tubule angiotensinogen have distinct roles in tubular and glomerular injury, fibrogenesis, and renal dysfunction during CKD development. As renin-angiotensin system components, including angiotensinogen, are important targets for treating CKD in the clinic, the results from our study may be applied to developing better tissue-targeted treatment strategies for CKD and other fibroproliferative diseases.

Renal Angiotensinogen Is Predominantly Liver Derived in Nonhuman Primates.

[Figure: see text].

Deletion of AT1a (Angiotensin II Type 1a) Receptor or Inhibition of Angiotensinogen Synthesis Attenuates Thoracic Aortopathies in Fibrillin1C1041G/+ Mice.

Objective: A cardinal feature of Marfan syndrome is thoracic aortic aneurysm. The contribution of the renin-angiotensin system via AT1aR (Ang II [angiotensin II] receptor type 1a) to thoracic aortic aneurysm progression remains controversial because the beneficial effects of angiotensin receptor blockers have been ascribed to off-target effects. This study used genetic and pharmacological modes of attenuating angiotensin receptor and ligand, respectively, to determine their roles on thoracic aortic aneurysm in mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+). Approach and Results: Thoracic aortic aneurysm in Fbn1C1041G/+ mice was found to be strikingly sexual dimorphic. Males displayed aortic dilation over 12 months while aortic dilation in Fbn1C1041G/+ females did not differ significantly from wild-type mice. To determine the role of AT1aR, Fbn1C1041G/+ mice that were either +/+ or -/- for AT1aR were generated. AT1aR deletion reduced expansion of ascending aorta and aortic root diameter from 1 to 12 months of age in males. Medial thickening and elastin fragmentation were attenuated. An antisense oligonucleotide against angiotensinogen was administered to male Fbn1C1041G/+ mice to determine the effects of Ang II depletion. Antisense oligonucleotide against angiotensinogen administration attenuated dilation of the ascending aorta and aortic root and reduced extracellular remodeling. Aortic transcriptome analyses identified potential targets by which inhibition of the renin-angiotensin system reduced aortic dilation in Fbn1C1041G/+ mice. Conclusions: Deletion of AT1aR or inhibition of Ang II production exerted similar effects in attenuating pathologies in the proximal thoracic aorta of male Fbn1C1041G/+ mice. Inhibition of the renin-angiotensin system attenuated dysregulation of genes within the aorta related to pathology of Fbn1C1041G/+ mice.

Comparative diagnostic utility of different urinary biomarkers during pre-albuminuric stages of non-hypertensive type 2 diabetic nephropathy.

Background & objectives: Activation of renin-angiotensin system and tubulointerstitial damage might be seen in pre-albuminuria stage of diabetic nephropathy (DN). Here, diagnostic utility of four urinary biomarkers [Angiotensinogen (Angio), Interleukin (IL)-18, Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Cystatin] during pre-albuminuria stages of non-hypertensive type 2 diabetes patients was studied. Methods: A total of 952 type 2 diabetes mellitus (T2DM) patients were screened for nephropathy [estimated glomerular filtration rate (eGFR) ≥120 ml/min and albumin-creatinine ratio (ACR) ≥30], and 120 patients were followed up for one year. At one year, they were classified into hyperfiltration (43), normoalbuminuria (29) and microalbuminuria (48) groups. Another 63 T2DM patients without nephropathy were included as controls. Hypertension, patients on angiotensin-converting enzyme inhibitor/angiotensin receptor blocker, eGFR <60 ml/min/1.73 m2 and all proteinuric conditions were excluded. All were subjected to testing for urine protein, ACR, HbA1C, eGFR, along with urinary biomarkers (IL-18, cystatin-C, NGAL and AGT). Comparative analysis of all the diagnostic tests among different subgroups, correlation and logistic regression was done. Results: Urinary IL-18/Cr, cystatin/creatinine (Cr) and AGT/Cr levels were higher in groups of hyperfiltration (13.47, 12.11 and 8.43 mg/g), normoalbuminuria (9.24, 11.74 and 9.15 mg/g) and microalbuminuria (11.59, 14.48 and 10.24 mg/g) than controls (7.38, 8.39 and 1.26 mg/g), but NGAL/Cr was comparable. The area under receiver operating characteristic curve (AUC) and sensitivity of AGT to detect early CKD were higher than ACR and eGFR (0.91 and 90.4%, 0.6 and 40% and 0.6 and 37%, respectively). AUC values of other biomarkers, namely IL-18/Cr, cystatin/Cr and NGAL/Cr, were 0.65, 0.64 and 0.51, respectively. Angio/Cr and IL-18/Cr showed correlation with log albuminuria (r=0.3, P=0.00, and r=0.28, P=0.00, respectively). NGAL showed correlation with log eGFR (r=0.28 P=0.00). Multivariate logistic analysis showed that odds ratio of developing nephropathy was 7.5 times with higher values of log Angio/Cr. Interpretation & conclusions: Urinary AGT showed a higher diagnostic value than ACR and eGFR followed by IL-18 and cystatin to diagnose DN during pre-albuminuric stages.

Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

Immunosuppression by Mycophenolate Mofetil Mitigates Intrarenal Angiotensinogen Augmentation in Angiotensin II-Dependent Hypertension.

Augmentation of intrarenal angiotensinogen (AGT) leads to further formation of intrarenal angiotensin II (Ang II) and the development of hypertensive kidney injury. Recent studies demonstrated that macrophages and the enhanced production of pro-inflammatory cytokines can be crucial mediators of renal AGT augmentation in hypertension. Accordingly, this study investigated the effects of immunosuppression by mycophenolate mofetil (MMF) on intrarenal AGT augmentation. Ang II (80 ng/min) was infused with or without daily administration of MMF (50 mg/kg) to Sprague-Dawley rats for 2 weeks. Mean arterial pressure (MAP) in Ang II infused rats was slightly higher (169.7 ± 6.1 mmHg) than the Ang II + MMF group (154.7 ± 2.0 mmHg), but was not statistically different from the Ang II + MMF group. MMF treatment suppressed Ang II-induced renal macrophages and IL-6 elevation. Augmentation of urinary AGT by Ang II infusion was attenuated by MMF treatment (control: 89.3 ± 25.2, Ang II: 1194 ± 305.1, and Ang II + MMF: 389 ± 192.0 ng/day). The augmentation of urinary AGT by Ang II infusion was observed before the onset of proteinuria. Elevated intrarenal AGT mRNA and protein levels in Ang II infused rats were also normalized by the MMF treatment (AGT mRNA, Ang II: 2.5 ± 0.2 and Ang II + MMF: 1.5 ± 0.1, ratio to control). Ang II-induced proteinuria, mesangial expansion and renal tubulointerstitial fibrosis were attenuated by MMF. Furthermore, MMF treatment attenuated the augmentation of intrarenal NLRP3 mRNA, a component of inflammasome. These results indicate that stimulated cytokine production in macrophages contributes to intrarenal AGT augmentation in Ang II-dependent hypertension, which leads to the development of kidney injury.