Intrarenal Renin-angiotensin System Research Articles

POS-195 ROLE OF S1P-DERIVED SOLUBLE (PRO)RENIN RECEPTOR IN ALBUMIN OVERLOAD INDUCED NEPROPATHY IN 129/SV MICE

In albumin overload (AO) nephropathy model, PRR activation mediated activation of intrarenal renin-angiotensin system (RAS) and thus elevated inflammatory and fibrosis factors of the kidney. In the present study, our goal was to determine whether such action of PRR was mediated by PRR soluble form (sPRR), cleaved by site-1 protease (S1P), a serine protease in the Golgi.

Characterization of the intrarenal renin-angiotensin system in cats with naturally occurring chronic kidney disease.

BackgroundThe role of the renin‐angiotensin‐aldosterone system in cats with chronic kidney disease (CKD) is incompletely understood.ObjectiveTo characterize components of the intrarenal renin‐angiotensin system (RAS) in cats with CKD.AnimalsEleven cats with naturally occurring CKD (CKD group) and 8 healthy control cats.MethodsRenal tissue samples were evaluated by reverse‐transcription polymerase chain reaction for renin, angiotensinogen, angiotensin‐converting enzyme (ACE), and angiotensin II type 1 receptor transcript levels, and by liquid chromatography‐mass spectrometry for quantification of angiotensin I, II, III, and IV concentrations. Linear mixed models were used to compare gene transcript levels and concentrations of angiotensin peptides between groups.ResultsCats of the CKD group were significantly older (P < .001) and more likely to be neutered (P = .007) than healthy control cats. Kidneys from cats with CKD had significantly higher transcript levels of angiotensinogen (P < .001) and lower transcript levels of ACE (P < .001) than those from control cats. Renal angiotensin I concentrations were increased in CKD compared with control kidneys (P = .001). No other significant differences in renal transcript levels or angiotensin peptide concentrations were noted between groups.Conclusion and Clinical ImportanceThe intrarenal RAS might be activated in cats with CKD. Small sample size and differences in age, neuter status, and dietary sodium intake between groups might have limited the ability to identify a significant difference in concentration of renal angiotensin II.

Biomarkers for early diagnosis of diabetic kidney disease: still a long way to go.

Biomarkers for early diagnosis of diabetic kidney disease: still a long way to go.

Pathophysiological mechanisms of hypertension development induced by fructose consumption.

During the past several decades, there has been a dramatic increase in fructose consumption worldwide in parallel with epidemics of metabolic diseases. Accumulating evidence has suggested that excessive fructose consumption is extensively linked to an increase in blood pressure. A combined intake of high fructose and high salt induced salt-sensitive hypertension and maternal high-fructose consumption induced programmed hypertension in adult offspring. The underlying mechanisms of these two events are similar and complex. These mainly include activation of the intrarenal renin-angiotensin system, gut dysbiosis, enhanced oxidative stress, activation of nephron ion transporters, and dysregulation of T-lymphocytes. The major objective of this article is to review recent advances in these fields and suggest novel therapies targeting these mechanisms that have potentially beneficial effects on diet-associated hypertension.

IgA Nephropathy with Macroproteinuria and a GFR of 20-30 ml/min/1.73 m2 May Still Benefit from RAS Inhibition.

There has been controversy about renin-angiotensin system (RAS) inhibition in IgAN patients with advanced (stage 4) chronic kidney disease (CKD). Therefore, we investigated the effect of RAS blockade in these patients. Renal specimens of 50 IgAN patients who underwent renal biopsy during stage 4 CKD between 2010 and 2020, were stained using immunohistochemistry to detect the expression of RAS receptors (AT1R, AT2R, MasR, and MrgD). The primary endpoint was a composite of end-stage renal disease (ESRD) and death. Main baseline information and the administration of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin II receptor blocker (ARB) were collected. During a median follow-up time of 25.5 months, 21 (42.0%) patients reached ESRD and none died. Six patients had a baseline eGFR of 15-20 ml/min/1.73m2, and reached ESRD with a median renal survival time of 7.0 (range 6.0-23.0) months. Among patients with a baseline eGFR of 20-30 ml/min/1.73m2, the percentage of patients using ACEI/ARB in progressive group was much lower than that in stable group (33.3% vs. 62.1%, P = 0.045), together with a shorter renal survival time in progressive group (26.0 vs. 30.5 months, P = 0.033). Macroproteinuria (24 h - UP ≥ 2.5 g) was also associated with a shorter renal survival time, as well as a significant decline in eGFR of stable group (24.4 vs. 26.4 ml/min/1.73 m2, P = 0.026). Lower eGFR [hazards ratio (HR), 0.829, 95% confidence interval (CI), 0.724-0.950; P = 0.007] and use of ACEI/ARB (HR, 0.356, 95% CI, 0.133-0.953; P = 0.040) were predictive of time to ESRD in this stage. No differences were found in the expression of AT1R, AT2R, MasR, and MrgD of renal tissues at the time of biopsy between stable and progressive groups. Contingent on monitoring serum creatinine and potassium levels, IgAN with macroproteinuria and a GFR of 20-30 ml/min/1.73m2 may still benefits from intrarenal RAS inhibition.

Foe and friend in the COVID-19-associated acute kidney injury: an insight on intrarenal renin-angiotensin system.

Since the first reported case in December of 2019, the coronavirus disease 2019 (COVID-19) has became an international public health emergency. So far, there are more than 228,206,384 confirmed cases including 4,687,066 deaths. Kidney with high expression of angiotensin-converting enzyme 2 (ACE2) is one of the extrapulmonary target organs affected in patients with COVID-19. Acute kidney injury (AKI) is one of the independent risk factors for the death of COVID-19 patients. The imbalance between ACE2-Ang(1-7)-MasR and ACE-Ang II-AT1R axis in the kidney may contribute to COVID-19-associated AKI. Although series of research have shown the inconsistent effects of multiple common RAS inhibitors on ACE2 expression and enzyme activity, most of the retrospective cohort studies indicated the safety and protective effects of ACEI/ARB in COVID-19 patients. This review article highlights the current knowledge on the possible involvement of intrarenal RAS in COVID-19-associated AKI with a primary focus on the opposing effects of ACE2-Ang(1-7)-MasR and ACE-Ang II-AT1R signaling in the kidney. Human recombinant soluble ACE2 or ACE2 variants with preserved ACE2-enzymatic activity may be the best options to improve COVID-19-associated AKI.

Ренин-ангиотензиновая система в регуляции экскреторной функции почек при экспериментальном сахарном диабете

Целью исследования было изучение роли ренин-ангиотензиновой системы в нарушении экскреторной функции почек в динамике аллоксан-индуцированного экспериментального сахарного диабета. Материалы и методы. Исследования выполнены на 78 нелинейных половозрелых самцах белых крыс с 11-, 26- и 46-дневным экспериментальным сахарным диабетом, вызванным внутрибрюшинным введением аллоксана (160 мг/кг), на фоне фармакологической блокады внутрипочечной ренин-ангиотензиновой системы, индуцированной внутрибрюшинным введением каптоприла (10 мг/кг). Изучение экскреторной функции почек осуществляли клиренс-методом в условиях водного индуцированного 2-часового диуреза с определением клиренса эндогенного креатинина, скорости клубочковой фильтрации, относительной реабсорбции воды, содержания белка в моче, его экскреции. Результаты. Анализ изменений функции почек после фармакологической блокады ренин-ангиотензиновой системы у крыс на 11-й день аллоксанового диабета показал достоверное возрастание диуреза, скорости клубочковой фильтрации, концентрационного индекса эндогенного креатинина, а также экскреции белка. При этом фармакологическая блокада ренин-ангиотензиновой системы практически не влияла на интенсивность относительной реабсорбции воды у аллоксан-диабетических крыс. На 26-й день развития аллоксанового диабета после введения каптоприла наблюдалось незначительное уменьшение объема конечной мочи, интенсивности гломерулярной фильтрации, относительной реабсорбции воды, концентрационного индекса эндогенного креатинина. Вместе с тем каптоприл не вызывал антипротеинурического эффекта, а экскреция белка даже продемонстрировала тенденцию к увеличению. На 46-й день аллоксан-индуцированного диабета после введения каптоприла достоверно снижались диурез крыс, клиренс эндогенного креатинина и скорость клубочковой фильтрации, а также концентрация белка в моче и его экскреция. Выводы. Результаты исследования позволяют сделать вывод о том, что начальный этап формирования ренальных нарушений при аллоксан-индуцированном экспериментальным сахарным диабетом связан с гемодинамически-гиперперфузионным характером функционирования почек при сохранном почечном функциональном резерве и структуре клубочково-канальцевого аппарата почки, авторегуляторных механизмов. Указанные компенсаторно-функциональные изменения функций почек постепенно осложняются истощением почечного функционального резерва и патологической активацией внутрипочечной ренин-ангиотензиновой системы с последующим прогрессированием гиперперфузионно-ишемического поражения почек, уменьшением массы функционирующих нефронов.

Tetrahydrocurcumin Ameliorates Kidney Injury and High Systolic Blood Pressure in High-Fat Diet-Induced Type 2 Diabetic Mice.

BackgroundActivation of the intrarenal renin-angiotensin system (RAS) is implicated in the pathogenesis of kidney injury and hypertension. We aimed to investigate the protective effect of tetrahydrocurcumin (THU) on intrarenal RAS expression, kidney injury, and systolic blood pressure (SBP) in high-fat diet (HFD)-induced type 2 diabetic mice.MethodsEight-week-old male mice were fed a regular diet (RD) or HFD for 12 weeks, and THU (50 or 100 mg/kg/day) was intragastrically administered with HFD. Physiological and metabolic changes were monitored and the expression of RAS components and markers of kidney injury were assessed.ResultsHFD-fed mice exhibited hyperglycemia, insulin resistance, and dyslipidemia compared to those in the RD group (P<0.05). Kidney injury in these mice was indicated by an increase in the ratio of albumin to creatinine, glomerular hypertrophy, and the effacement of podocyte foot processes. Expression of intrarenal angiotensin-converting enzyme, angiotensin II type I receptor, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4, and monocyte chemoattractant protein-1 was also markedly increased in HFD-fed mice. HFD-fed mice exhibited elevated SBP that was accompanied by an increase in the wall thickness and vascular cross-sectional area (P<0.05), 12 weeks post-HFD consumption. Treatment with THU (100 mg/kg/day) suppressed intrarenal RAS activation, improved insulin sensitivity, and reduced SBP, thus, attenuating kidney injury in these mice.ConclusionTHU alleviated kidney injury in mice with HFD-induced type 2 diabetes, possibly by blunting the activation of the intrarenal RAS/nicotinamide adenine dinucleotide phosphate oxidase IV (NOX4)/monocyte chemoattractant protein 1 (MCP-1) axis and by lowering the high SBP.

Urinary excretion of renin and angiotensinogen in hypertensive children and adolescents.

IntroductionIn recent years hypertension has become an emerging condition in the young population. It has been proposed that the renin–angiotensin system plays an important role in regulation of blood pressure. We assessed whether activation of the intrarenal renin-angiotensin system occurs in hypertensive children and adolescents and what better reflects its activity: urine angiotensinogen (AGT) or urine renin (REN).Material and methodsThe study was conducted on a sample of 58 subjects with primary hypertension (HT) and 29 normotensive children and adolescents. We measured urine REN and AGT excretion and assessed the values in relation to blood pressure (BP) and other clinical parameters. Both REN and AGT values were calculated by urine creatinine: REN/cr. and AGT/cr., respectively.ResultsWe observed higher urine REN/cr. values in hypertensive subjects in comparison to the reference group (6.99 vs. 2.93, p = 0.003). Hypertensive participants showed positive correlations between urine REN/cr. and diastolic 24-hour BP (r = 0.42, p = 0.002) as well as between urine REN/cr. and urine AGT/cr. (r = 0.266, p = 0.044, respectively).ConclusionsIncreased urine REN/cr. in hypertensive children and adolescents and its positive correlation with BP may indicate its important role in the pathogenesis of HT. Perhaps urine REN/cr. could be a marker of intrarenal renin-angiotensin system activity. Nevertheless, further research should be undertaken to confirm this observation.

Renal denervation does not affect hypertension or the renin-angiotensin system in a rodent model of juvenile-onset polycystic kidney disease: clinical implications

We examined the effect of total and afferent renal denervation (RDN) on hypertension and the renin-angiotensin system (RAS) in a rodent model of juvenile-onset polycystic kidney disease (PKD). Lewis Polycystic Kidney (LPK) and control rats received total, afferent or sham RDN by periaxonal application of phenol, capsaicin or normal saline, respectively, and were monitored for 4-weeks. Afferent RDN did not affect systolic blood pressure (SBP) determined by radiotelemetry in either strain (n = 19) while total RDN significantly reduced SBP in Lewis rats 4-weeks post-denervation (total vs. sham, 122 ± 1 vs. 130 ± 2 mmHg, P = 0.002, n = 25). Plasma and kidney renin content determined by radioimmunoassay were significantly lower in LPK vs. Lewis (plasma: 278.2 ± 6.7 vs. 376.5 ± 11.9 ng Ang I/ml/h; kidney: 260.1 ± 6.3 vs. 753.2 ± 37.9 ng Ang I/mg/h, P < 0.001, n = 26). These parameters were not affected by RDN. Intrarenal mRNA expression levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE)2, and angiotensin II receptor type 1a were significantly lower, whereas ACE1 expression was significantly higher in the LPK vs. Lewis (all P < 0.05, n = 26). This pattern of intrarenal RAS expression was not changed by RDN. In conclusion, RDN does not affect hypertension or the RAS in the LPK model and indicates RDN might not be a suitable antihypertensive strategy for individuals with juvenile-onset PKD.

Sex differences in soluble prorenin receptor in patients with type 2 diabetes

BackgroundThe soluble prorenin receptor (sPRR), a member of the renin-angiotensin system (RAS), is elevated in plasma of patients with preeclampsia, hypertension, chronic kidney disease (CKD), and type 2 diabetes. Our goal was to examine the relationship between sPRR and RAS activation to define whether sexual dimorphisms in sPRR might explain sex disparities in renal outcomes in patients with type 2 diabetes.MethodsTwo hundred sixty-nine participants were included in the study (mean age, 48 ± 16 years; 42% men, 58% women), including 173 controls and 96 subjects with type 2 diabetes. In plasma and urine, we measured sPRR, plasma renin activity (PRA), and prorenin. In the urine, we also measured angiotensinogen along with other biomarkers of renal dysfunction.ResultsPlasma sPRR and PRA were significantly higher in women with type 2 diabetes compared to men. In these women, plasma sPRR was positively correlated with PRA, age, and body mass index (BMI). In contrast, in men the sPRR in urine but not in plasma positively correlated with eGFR in urine, but negatively correlated with urine renin activity, plasma glucose, age, and BMI.ConclusionsIn patients with type 2 diabetes, sPRR contributes to RAS stimulation in a sex-dependent fashion. In diabetic women, increased plasma sPRR parallels the activation of systemic RAS; while in diabetic men, decreased sPRR in urine matches intrarenal RAS stimulation. sPRR might be a potential indicator of intrarenal RAS activation and renal dysfunction in men and women with type 2 diabetes.

Renal Inflammation Induces Salt Sensitivity in Male db/db Mice through Dysregulation of ENaC.

Hypertension is considered a major risk factor for the progression of diabetic kidney disease. Type 2 diabetes is associated with increased renal sodium reabsorption and salt-sensitive hypertension. Clinical studies show that men have higher risk than premenopausal women for the development of diabetic kidney disease. However, the renal mechanisms that predispose to salt sensitivity during diabetes and whether sexual dimorphism is associated with these mechanisms remains unknown. Female and male db/db mice exposed to a high-salt diet were used to analyze the progression of diabetic kidney disease and the development of hypertension. Male, 34-week-old, db/db mice display hypertension when exposed to a 4-week high-salt treatment, whereas equivalently treated female db/db mice remain normotensive. Salt-sensitive hypertension in male mice was associated with no suppression of the epithelial sodium channel (ENaC) in response to a high-salt diet, despite downregulation of several components of the intrarenal renin-angiotensin system. Male db/db mice show higher levels of proinflammatory cytokines and more immune-cell infiltration in the kidney than do female db/db mice. Blocking inflammation, with either mycophenolate mofetil or by reducing IL-6 levels with a neutralizing anti-IL-6 antibody, prevented the development of salt sensitivity in male db/db mice. The inflammatory response observed in male, but not in female, db/db mice induces salt-sensitive hypertension by impairing ENaC downregulation in response to high salt. These data provide a mechanistic explanation for the sexual dimorphism associated with the development of diabetic kidney disease and salt sensitivity.

Angiotensin II up-regulates sodium-glucose co-transporter 2 expression and SGLT2 inhibitor attenuates Ang II-induced hypertensive renal injury in mice.

Clinical trials indicate that sodium/glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) improve kidney function, yet, the molecular regulation of SGLT2 expression is incompletely understood. Here, we investigated the role of the intrarenal renin-angiotensin system (RAS) on SGLT2 expression. In adult non-diabetic participants in the Nephrotic Syndrome Study Network (NEPTUNE, n=163), multivariable linear regression analysis showed SGLT2 mRNA was significantly associated with angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE) mRNA levels (P<0.001). In vitro, angiotensin II (Ang II) dose-dependently stimulated SGLT2 expression in HK-2, human immortalized renal proximal tubular cells (RPTCs); losartan and antioxidants inhibited it. Sglt2 expression was increased in transgenic (Tg) mice specifically overexpressing Agt in their RPTCs, as well as in WT mice with a single subcutaneous injection of Ang II (1.44 mg/kg). Moreover, Ang II (1000 ng/kg/min) infusion via osmotic mini-pump in WT mice for 4 weeks increased systolic blood pressure (SBP), glomerulosclerosis, tubulointerstitial fibrosis, and albuminuria; canaglifozin (Cana, 15 mg/kg/day) reversed these changes, with the exception of SBP. Fractional glucose excretion (FeGlu) was higher in Ang II+Cana than WT+Cana, whereas Sglt2 expression was similar. Our data demonstrate a link between intrarenal RAS and SGLT2 expression and that SGLT2i ameliorates Ang II-induced renal injury independent of SBP.

The evolving complexity of the collecting duct renin-angiotensin system in hypertension.

The intrarenal renin-angiotensin system is critical for the regulation of tubule sodium reabsorption, renal haemodynamics and blood pressure. The excretion of renin in urine can result from its increased filtration, the inhibition of renin reabsorption by megalin in the proximal tubule, or its secretion by the principal cells of the collecting duct. Modest increases in circulating or intrarenal angiotensin II (ANGII) stimulate the synthesis and secretion of angiotensinogen in the proximal tubule, which provides sufficient substrate for collecting duct-derived renin to form angiotensin I (ANGI). In models of ANGII-dependent hypertension, ANGII suppresses plasma renin, suggesting that urinary renin is not likely to be the result of increased filtered load. In the collecting duct, ANGII stimulates the synthesis and secretion of prorenin and renin through the activation of ANGII type 1 receptor (AT1R) expressed primarily by principal cells. The stimulation of collecting duct-derived renin is enhanced by paracrine factors including vasopressin, prostaglandin E2 and bradykinin. Furthermore, binding of prorenin and renin to the prorenin receptor in the collecting duct evokes a number of responses, including the non-proteolytic enzymatic activation of prorenin to produce ANGI from proximal tubule-derived angiotensinogen, which is then converted into ANGII by luminal angiotensin-converting enzyme; stimulation of the epithelial sodium channel (ENaC) in principal cells; and activation of intracellular pathways linked to the upregulation of cyclooxygenase 2 and profibrotic genes. These findings suggest that dysregulation of the renin-angiotensin system in the collecting duct contributes to the development of hypertension by enhancing sodium reabsorption and the progression of kidney injury.

POS-373 Advanced oxidation protein products activate oxidative stress in vitro via soluble (pro)renin receptor mediated intrarenal renin angiotensin system and Nox4/H2O2 pathway

Advanced oxidation protein products (AOPPs) are newly identified as a novel oxidative stress biomarkers. Previous research has indicated potential associations between AOPPs and activation intrarenal local renin angiotensin system (RAS). However, no known empirical research has focused on exploring relationships between AOPPs and the new member of RAS, (pro) renin receptor (PRR). Aim of this study was to test the role of soluble PRR (sPRR) in AOPPs-induced oxidative stress responses in cultured human renal proximal tubular cell line HK-2 cells. The HK-2 cells were incubated for 24h in the presence of different concentrations of AOPPs, or incubated for different time lengths in the presence of 100 μg/ml of AOPPs. In HK-2 cells treated with PRO20, furin inhibitors, GM6001, PF-429242, sPRR-His, or transfected with PRR siRNA, site-1 protease (S1P) siRNA, Nox4 siRNA prior to exposure to AOPPs, the changes in the protein expressions of PRR/sPRR and Nox4 were examined with Western blotting, mRNA lelvels of PRR, angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R), angiotensinogen (AGT), intercellular adhesion molecule (ICAM1), tumor necrosis factor-α (TNF-α) by using real-time quantitative PCR, the cell medium for renin activity, ACE activity, sPRR, Ang II, H2O2, thiobarbituric acid reactive substances (TBARS) concentrations by using ELISA Assay kits. AOPPs showed time-dependent and dose-independent effects on PRR cleavage produces sPRR. AOPP-albumin treatment induced a >4-times increase in medium sPRR due to enhanced cleavage of PRR but unmodified albumin has no effect. Inhibition of S1P with PF-429242 or siRNA remarkably suppressed AOPPs-induced sPRR production. Surprisingly, this cleavage event was unaffected by inhibition of furin or ADAM19. AOPPs (100 μg/ml) treatment for 24 h induced renin activity and oxidative stress, both of which were attenuated by a PRR decoy inhibitor PRO20 and S1P inhibitor PF429242. What’s more, PF-429242 significantly decreased AOPPs stimulated Nox4 and H2O2 expression and administration of a recombinant sPRR, termed sPRR-His, reversed the effects of S1P inhibition. Taken together, these findings suggest that AOPPs induce oxidative stress via S1P-derived soluble (pro)renin receptor activation intrarenal renin angiotensin system and Nox4/H2O2 pathway in vitro.

ENHANCED PRODUCTIONS OF INTRARENAL NLRP-3 INFLAMMASOME AND ANGIOTENSINOGEN IN ANGIOTENSIN II-DEPENDENT HYPERTENSION ARE PREVENTED BY IMMUNOSUPPRESSANT TREATMENT

Objective: Activated inflammasomes enhance maturation of pro-inflammatory cytokines, which facilitates the development of kidney injury. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), one of major subunits in the inflammasome complex, plays a crucial role in innate immunity and inflammation. NLRP3 inflammasome is activated by ATP-P2X7 axis and reactive oxygen species. Expression of pro-NLRP3 is promoted by activated NF-κB by cytokines or PAMPs/DAMPs. In angiotensin II (Ang II)-dependent hypertension, there is augmentation of renal immune cells leading to enhanced production of cytokines and consequent activation of NF-κB. Therefore, we hypothesized that intrarenal NLRP3 levels are increased in Ang II-dependent hypertension in association with the kidney injury, and treatment with mycophenolate mofetil (MMF), an immunosuppressant, prevents the augmentation of NLRP3 and attenuates the consequent progression of kidney injury. Design and method: Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to male rats for 2 weeks. mRNA levels of intrarenal NLRP3 and AIM2, which forms another type of inflammasome complex by viral or bacterial infections, were measured by droplet digital PCR. Furthermore, levels of intrarenal angiotensinogen (AGT), a critical contributor to activation of intrarenal renin-angiotensin system leading to the development of hypertension and associated kidney injury, were evaluated. Histological analyses of kidney injury were also performed. Results: MMF prevented increases in intrarenal macrophage/monocyte and IL-6 levels induced by Ang II infusion, confirming immunosuppression by the drug. Ang II infusion significantly increased intrarenal NLRP3 levels (control group: 4.12 ± 1.1 copies/ng RNA vs. Ang II-infused group: 9.96 ± 1.8 copies). The elevated NLRP3 expression was attenuated by MMF (6.24 ± 1.4 copies). In contrast, intrarenal AIM2 levels were not altered by Ang II infusion or MMF. Urinary protein and AGT levels were elevated in Ang II-infused rats, which were prevented by MMF. Histological analyses showed mesangial expansion and tubulointerstitial fibrosis observed in Ang II-infused group, but these injury markers were in normal ranges in the group receiving Ang II + MMF. Conclusions: These results demonstrate activation of the NLRP3 inflammasome in Ang II dependent hypertension and indicate that immunosuppression by MMF mitigates the inflammasome activation, which contributes to attenuation of the kidney injury.

POS-389 PODOCYTE MATURATION IN HUMAN KIDNEY ORGANOIDS IS ACCELERATED WITH RENIN-ANGIOTENSIN SYSTEM ACTIVATION

The global prevalence of chronic kidney disease (CKD) is approximately 10% and rising. Current renal replacement modalities (dialysis and transplant) carry significant socioeconomic burden and may struggle to meet rising demands. PSC-derived kidney organoids have emerged as a promising alternative for providing functional nephrons to CKD patients. However, this nascent technology faces numerous challenges most pressing of which is maturation. Organoid differentiation protocols rely on application of morphogens in a temporal restricted fashion mimicking ureteric bud and metanephric mesenchyme development. However, organoids are poorly vascularized and resemble first and second trimester fetal kidneys. The renin-angiotensin system (RAS) is critical for normal kidney morphogenesis guiding glomerular, vascular and ureteric formation. Indeed, prenatal exposure to RAS inhibitors is associated with kidney dysgenesis and mortality. Can RAS modulators like angiotensin 2 (ANG2) and RAS inhibitors alter kidney organoid maturation? We hypothesize that ANG2 and losartan perturb developmental programs within nephron progenitor populations, alter cell fate specification and maturation of glomerular epithelial cells (i.e. podocytes). We performed single-cell RNA sequence analyses on human iPSC derived kidney organoids treated acutely (24hrs) with vehicle, ANG2 alone, losartan alone, or ANG2 and losartan on the 28th day of the Takasato protocol. Kidney organoids expressed all major RAS genes. AGT, ACE1 and ACE2 were restricted to proximal tubule clusters while REN (renin), AGTR1 and AGTR2 were preferentially expressed in stromal cells. ACE1 and REN were also found in endothelial cells. ATP6AP2 (pro-renin receptor) was ubiquitously expressed. REN expression was downregulated by ANG2 and normalized with losartan pre-treatment suggesting a functioning RAS network. Unbiased clustering identified 3 late podocyte (LP) clusters (NPHS2hiPODXLhi), 1 early podocyte (EP) cluster (OLFM3+NPHS2loPDOXLlo), 1 parietal epithelial (PEC) cluster (CLDN1+CAV2+) and 2 nephron progenitor (NPC) clusters (SIX2+CITED1+). RNA velocity analysis revealed 2 distinct differentiation trajectories: NPC – EP – LP and NPC – EP – PEC – LP. The EP and NPC transcriptional profiles were disproportionately sensitive to ANG2 as we noted significant upregulation of podocyte differentiation genes such as BMP7, FOXC2 PODXL and NPHS2. Moreover, ANG2 depleted the immature populations (NPC and EP) and expanded mature cell cell states (PEC and LP). On the other hand, losartan downregulated the podocyte differentiation programs and increased the proportion of immature cells (EP and NPC) to mature (PEC and LP) cells. The proportion of glomerular epithelial cells relative to all cells was unchanged across treatments. Human kidney organoids developed a functioning intraorganoid RAS resembling in vivo intrarenal RAS. The lineage plasticity observed between NPCs, EPs, PECs and LPs was sensitive to acute RAS perturbation. ANG2 accelerated podocyte maturity while RAS inhibition held podocytes in an immature state. Next steps include testing different ANG2 treatment durations, validate findings using immunocytochemistry, investigate the podocyte differentiation programs closely and their relationship to RAS, and reproduce this analysis on endothelial and tubule cells.

Novel roles of the renal angiotensin-converting enzyme

The observation that all components of the renin angiotensin system (RAS) are expressed in the kidney and the fact that intratubular angiotensin (Ang) II levels greatly exceed the plasma concentration suggest that the synthesis of renal Ang II occurs independently of the circulating RAS. One of the main components of this so-called intrarenal RAS is angiotensin-converting enzyme (ACE). Although the role of ACE in renal disease is demonstrated by the therapeutic effectiveness of ACE inhibitors in treating several conditions, the exact contribution of intrarenal versus systemic ACE in renal disease remains unknown. Using genetically modified mouse models, our group demonstrated that renal ACE plays a key role in the development of several forms of hypertension. Specifically, although ACE is expressed in different cell types within the kidney, its expression in renal proximal tubular cells is essential for the development of high blood pressure. Besides hypertension, ACE is involved in several other renal diseases such as diabetic kidney disease, or acute kidney injury even when blood pressure is normal. In addition, studies suggest that ACE might mediate at least part of its effect through mechanisms that are independent of the Ang I conversion into Ang II and involve other substrates such as N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), Ang-(1–7), and bradykinin, among others. In this review, we summarize the recent advances in understanding the contribution of intrarenal ACE to different pathological conditions and provide insight into the many roles of ACE besides the well-known synthesis of Ang II.

Baseline Urinary Angiotensinogen Excretion Predicts Deterioration of the Kidney Function in Patients with Chronic Kidney Disease.

Objective The intrarenal renin-angiotensin system (RAS) is activated in patients with chronic kidney disease (CKD), and urinary angiotensinogen (AGT) levels, a surrogate marker of the intrarenal RAS activation, are associated with blood pressure (BP) and urinary albumin excretion. In addition, it has been shown that changes in urinary AGT levels correlate with annual changes in the estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes and that elevated levels of urinary AGT in type 2 diabetic patients with albuminuria are a high-risk factor for worsening renal and cardiovascular complications. However, whether or not baseline urinary AGT levels predict deterioration of the kidney function in all patients with CKD is unclear. Methods We recruited 62 patients with CKD whose eGFR was ＞15 mL/min/1.73 m2. We performed 24-hour ambulatory BP monitoring at 30-min intervals and daily urinary collection to examine the urinary AGT levels and albumin excretion and measured the levels of plasma angiotensin II (Ang II), a surrogate marker of circulating RAS. In addition, annual changes in the eGFR were followed up for 3.4±1.5 years. Results Annual changes in the eGFR were significantly and negatively associated with urinary AGT levels (r=-0.31, p=0.015) as well as the age, systolic BP, and urinary albumin levels. In contrast, annual changes in the eGFR were not correlated with plasma Ang II levels. Furthermore, when dividing patients into quartiles according to urinary AGT levels, patients with the highest urinary AGT levels showed a progressive decline in the eGFR. Conclusion These results suggest that elevated baseline urinary AGT levels can predict renal dysfunction in patients with CKD.

Circadian rhythm of the intrarenal renin-angiotensin system is caused by glomerular filtration of liver-derived angiotensinogen depending on glomerular capillary pressure in adriamycin nephropathy rats.

Circadian fluctuation disorder of the intrarenal renin-angiotensin system (RAS) causes that of blood pressure (BP) and renal damage. In renal damage with an impaired glomerular filtration barrier, liver-derived angiotensinogen (AGT) filtered through damaged glomeruli regulates intrarenal RAS activity. Furthermore, glomerular permeability is more strongly affected by glomerular hypertension than by systemic hypertension. Thus, we aimed to clarify whether the circadian rhythm of intrarenal RAS activity is influenced by AGT filtered through damaged glomeruli due to glomerular capillary pressure. Rats with adriamycin nephropathy and an impaired glomerular filtration barrier were compared with control rats. In adriamycin nephropathy rats, olmesartan medoxomil (an angiotensin II type 1 receptor blocker) or hydralazine (a vasodilator) was administered, and the levels of intrarenal RAS components in the active and rest phases were evaluated. Moreover, the diameter ratio of afferent to efferent arterioles (A/E ratio), an indicator of glomerular capillary pressure, and the glomerular sieving coefficient (GSC) based on multiphoton microscopy in vivo imaging, which reflects glomerular permeability, were determined. Mild renal dysfunction was induced, and the systemic BP increased, resulting in increased A/E ratios in the adriamycin nephropathy rats compared with the control rats. Fluctuations in intrarenal RAS activity occurred in parallel with circadian fluctuations in glomerular capillary pressure, which disappeared with olmesartan treatment and were maintained with hydralazine treatment. Furthermore, the GSCs for AGT also showed similar changes. In conclusion, intrarenal RAS activity is influenced by the filtration of liver-derived AGT from damaged glomeruli due to circadian fluctuation disorder of the glomerular capillary pressure.