Search

Regulation of systemic and local angiotensinogen (AGT) levels is a key determinant of tissue angiotensin II (Ang II) levels and inappropriate AGT augmentation promotes the development of hypertension and tissue injury. Kidney and urinary AGT levels are increased in Ang II-mediated hypertension. Recent studies have demonstrated that circulating hepatocyte-derived AGT (hAGT) enters kidneys sustaining kidney and urinary AGT levels. However, roles of hAGT in blood pressure elevation and kidney injury in Ang II-mediated hypertension have not been delineated. This study tested if hAGT contributes to the development of the pathophysiological events in Ang II-infused mice. A low dose of Ang II (400 ng/kg/min) was infused to male wild type (WT) and hAGT gene knockout (KO) mice (N=9 and 13) for 4 weeks. The control group in each genotype received vehicle (Veh) infusion (N=5 and 6). Western blot confirmed non-detectable levels of hAGT in KO mice. hAGT KO markedly decreased plasma AGT levels (WT+Veh:12.2±0.6 vs. hAGT KO+Veh: 0.8±0.1 μg/ml). Ang II infusion did not elevate plasma AGT levels in either WT and hAGT KO mice. Although hAGT KO mice exhibited a lower baseline of systolic blood pressure (SBP) than WT mice, Ang II-mediated increases in SBP was not attenuated in hAGT KO mice (ΔSBP in WT+Ang II: 30.1±4.4 vs. hAGT KO+Ang II: 26.0±4.2 mmHg). Kidney AGT mRNA levels were increased by Ang II infusion to the same extent in both WT and hAGT mice (WT+Ang II: 1.30±0.04 vs. hAGT KO+Ang II: 1.34±0.06, ratio to control). Likewise, Ang II infusion increased IL-6 mRNA to the same magnitude in both WT and hAGT KO mice. Urinary AGT was sustained in hAGT KO+Veh mice (66±9%) compared to WT+Veh mice. Ang II infusion did not alter urinary AGT levels in both groups. Glomerular mesangial expansion and fibrosis by Ang II infusion were not observed. Ang II infusion developed tubulointerstitial fibrosis in renal cortex and medulla. hAGT KO prevented the fibrosis only in the medulla. These outcomes demonstrate that elevation of SBP, augmentation of intrarenal AGT and IL-6 expression, and the development of renal cortical fibrosis in Ang II-mediated hypertension do not require hAGT. In contrast, hAGT contributes to renal medullary fibrosis which may be due to the lower absolute levels of blood pressure.

The development of Angiotensin II (Ang II)-induced hypertension is associated with mitochondrial dysfunction and kidney injury. Sirtuin 3 (SIRT3), a key mitochondrial protein, plays an important role in maintaining mitochondrial homeostasis. However, it remains unknown whether deletion of SIRT3 in the proximal tubules will alter the pressor and renal responses to Ang II in sex-different manners. In the present study, adult male, and female wild-type (WT) and mutant mice with proximal tubule-specific knockout of SIRT3, PT- Sirt3 -/- , were infused with or without a slow pressor dose of Ang II via an osmotic minipump (0.5 mg/kg/day, i.p.), supplemented with a 2% NaCI diet or losartan, 20 mg/kg/day, for 2 weeks. Systolic (SBP), diastolic (DBP), and mean arterial blood (MAP) pressure were determined using the tail-cuff method, whereas 24 hr. urinary sodium and potassium excretion were determined using a metabolic cage. Serum and urine creatinine were measured using colorimetric assays, whereas glomerular and tubulointerstitial injury was evaluated by Masson’s Trichrome staining. Basal SBP levels were lower in PT- Sirt3 -/- than in WT mice (SBP-WT: 112 ± 2 vs. SBP-PT- Sirt3 -/- : 93 ± 2 mmHg, P Sirt3 -/- mice with or without losartan treatment. Serum creatinine levels and urinary creatinine excretion were higher in PT- Sirt3 -/- mice than in WT mice ( P Sirt3 -/- mice with lower 24 hr. urine and urinary creatinine excretion in response to Ang II infusion or losartan. Losartan significantly increased 24 hr. urinary potassium and chloride excretion in Ang II-infused male and female PT- Sirt3 -/- mice ( P Sirt3 -/- mice showed significant renal cortical tubulointerstitial fibrotic responses ( P Sirt3 -/- mice and that Ang II induces similar hypertensive and renal fibrotic responses in male and female PT- Sirt3 -/- mice without significant sex differences.

The activation of the renin-angiotensin system (RAS) in the clipped kidney plays a critical role in the development of two-kidney, one-clip Goldblatt hypertension (2K1C), but the roles of angiotensin II (Ang II) and AT 1a receptors in the proximal tubules has not been determined previously. The present study tested the hypothesis that genetic deletion of AT 1a receptors selectively in the proximal tubules attenuates the development of 2K1C Goldblatt hypertension via AT 1a receptor-mediated, Na + /H + exchanger 3 (NHE3)-dependent mechanisms. To test the hypothesis, 2K1C Goldblatt hypertension was induced by placing a silver clip, 0.2 mm internal diameter, on the left renal artery for 4 weeks in adult male wild-type (WT), global AT 1a receptor knockout ( Agtr1a -/- ), proximal tubule (PT)-specific Agtr1a -/- (PT- Agtr1a -/- ), or PT- Nhe3 -/- mice, respectively. In WT mice, systolic blood pressure increased in a time-dependent manner reaching a maximal response by Week 3 (Basal: 112 ± 2 vs. 2K1C: 149 ± 4 mmHg, n=12, P P P P P Agtr1a -/- (Basal: 88 ± 4 vs. 2K1C: 92 ± 2 mmHg, n=9, n.s .), PT- Agtr1a -/- mice (Basal: 101 ± 2 vs. 2K1C: 104 ± 3 mmHg, n=12, n.s .) and PT- Nhe3 -/- mice (Basal: 103 ± 3 vs. 109 ± 5 mmHg, n=12, n.s .). Renin mRNA expression was not different in clipped and nonclipped kidney of Agtr1a -/- mice, but it was decreased in the nonclipped kidney of PT- Agtr1a -/- mice ( P 1a receptors selectively in the proximal tubules attenuates the development of 2K1C Goldblatt hypertension via AT 1a receptor-mediated, Na + /H + exchanger 3 (NHE3)-dependent mechanisms.

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. Although NLRP3 inflammasome is activated by ATP-P2Y7 axis and reactive oxygen species, the expression of pro-NLRP3 is promoted by NF-κB activated by cytokines or PAMPs/DAMPs. Thus, we hypothesized that mycophenolate mofetil (MMF), an immunosuppressant, attenuates augmentation of intrarenal NLRP3 and consequent progression of kidney injury in angiotensin II (Ang II)-dependent hypertension. Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to Sprague-Dawley 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, kidney injury was evaluated. MMF treatment mitigated Ang II-induced macrophage infiltration into kidneys, suggesting immunosuppression by the drug. Ang II infusion significantly increased intrarenal NLRP3 mRNA levels (normotensive control group: 4.12±1.1 copies/ng RNA vs. Ang II-infused group: 9.96±1.8 copies, N=5). The elevated NLRP3 expression in kidneys of Ang II-infused rats was attenuated by MMF treatment (6.24±1.4 copies). In contrast, intrarenal AIM2 levels were lower than NLRP3 in the control group and the levels were not altered by Ang II infusion or MMF treatment (normotensive control group: 0.42±0.1 copies, Ang II-infused group: 0.35±0.06 copies and Ang II+MMF group: 0.35±0.08 copies). Urinary protein and angiotensinogen levels were elevated in Ang II-infused rats and MMF treatment suppressed the augmentations. Histological analyses also showed the development of kidney injury including mesangial expansion and tubulointerstitial fibrosis observed in the hypertensive rats, but these injury markers were mitigated by MMF. 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.

Inappropriately activated intrarenal renin-angiotensin system, including proximal tubular angiotensinogen (AGT) augmentation, contributes to the progression of hypertension and associated kidney injury. It has been demonstrated that interleukin 6 (IL-6) is required for angiotensin II (Ang II)-induced AGT augmentation in renal proximal tubular cells (PTC). However, molecular mechanisms remain unelucidated. Recent studies suggest that Ang II downregulates cardiac class II histone deacetylases (HDAC), which exhibit protective effects against hypertension related tissue injury. Furthermore, HDAC9 (a class II HDAC) has been shown to act as an epigenetic repressor of proximal tubular AGT. Accordingly, the present study investigated the hypothesis that Ang II decreases HDAC9 levels, thereby enabling IL-6 to promote AGT expression in PTC. Ang II (400 ng/kg/min) was chronically perfused to male mice (N=7) for 14 days to investigate the regulation of intrarenal HDAC9. Moreover, cultured human PTC (hPTC) were used to create wild-type, HDAC9 overexpression, and HDAC9 knockdown by shRNA models and were treated with 100 nM Ang II, 10 ng/ml IL-6, or both for 24 hours. Chronic Ang II infusion elevated blood pressure and suppressed renal cortical HDAC9 levels. A decrease in HDAC9 was also observed in Ang II-treated hPTC. Treatment of hPTC with both Ang II and IL-6 increased AGT mRNA levels (1.50±0.10, ratio to the control, N=6), whereas individual treatments with Ang II or IL-6 did not alter AGT expression levels as previously demonstrated. This upregulation of AGT expression by Ang II and IL-6 co-stimulation was attenuated by the overexpression of HDAC9 in hPTC (1.10±0.08, ratio to the control). Importantly, AGT expression was enhanced by IL-6 without Ang II (2.31±0.08 by IL-6 and 2.27±0.15 by Ang II+IL-6, ratio to the control) in cells receiving the HDAC9 gene knockdown, suggesting that HDAC9 down-regulation mediates IL-6-induced AGT upregulation in hPTC. These results indicate that Ang II-induced epigenetic modification, such as the suppression of intrarenal HDAC9, results in increased accessibility of IL-6-derived transcription factors to the AGT promoter and consequent augmentation of intrarenal AGT expression in Ang II-dependent hypertension.

SGLT2 inhibitors lower blood glucose (BG) levels and have the potential to reduce cardiovascular and renal complications of type 2 diabetes mellitus (T2DM). Since BG levels can vary significantly during a treatment regime, hemoglobin A1c is often utilized as a stable indicator of BG status but dynamic measurements of BG may provide greater insights. A blood glucose telemetry systems was used to investigate BG dynamics in canagliflozin (CANA, an SGLT inhibitor)-treated T2DM mice. Male New Zealand Obese mice were fed a high fat diet to induce diabetes. When the mice exhibited a BG of ~350 mg/dl, the mice were treated with vehicle for 5 days followed by 10 mg/kg/day CANA for 5 days by daily oral gavage (single dosing/day). BG levels were monitored continuously via implanted telemetry devices. Chronic treatment (6 weeks) was also performed to investigate blood pressure monitored by a telemetry system and to assess kidney injury. During vehicle treatment for 5 days, BG levels in the mice averaged 336.7 mg/dl. Lower BG levels during early morning compared to night time (active time) were observed during vehicle treatment. BG levels demonstrated a large variation over 24 hours during the vehicle treatment (maximum minus minimum BG: 368.5 mg/dl on day 5). CANA rapidly reduced BG levels within 3 hours following treatment (214.8±25.4 mg/dl), and the lowered BG was sustained until the next dosing. The average 24-hour BG was gradually suppressed during the CANA treatment reaching 170.1 mg/dl on the last day of CANA treatment. Moreover, CANA reduced the variation of BG (maximum minus minimum BG: 214.8 mg/dl on day 5 of CANA treatment). In mice receiving chronic treatment, CANA started lowering systolic blood pressure on week 2 and significant suppression was observed on week 6 (vehicle: 157.9±2.2 vs. CANA: 124.7±7.6 mmHg). The development of kidney injury, especially renal tubular fibrosis and inflammation, was attenuated by CANA. These findings demonstrate that CANA treatment results in rapid and sustained reductions of both BG levels and BG variability which precede the reduction of blood pressure. The temporal dissociation between the lowering of BG and of arterial pressure levels by CANA suggests hyperglycemia-induced factors mediate the development of hypertension in T2DM.

Intrarenal renin-angiotensin system (RAS), including proximal tubular angiotensinogen (AGT), plays crucial roles in the progression of hypertension, kidney injury, and kidney development and has been investigated in vivo using animal models. For translational relevance, we sought to further our investigations in human tissue. This study investigated RAS expression and AGT regulation by histone deacetylase 9 (HDAC9), an epigenetic repressor of AGT, in human iPSC-derived kidney organoids. After pre-treatment of human iPSC with CHIR99201, a glycogen synthase kinase inhibitor, and fibroblast growth factor 9, cells were moved to transwell membranes. Cells were harvested on day 0, 5, 12 or 18 to determine mRNA copy numbers of developmental markers and RAS genes by digital PCR. Marker genes of renal structures were induced during the culture with concomitant decrease in progenitor markers including Cited1. Immunostaining revealed that the organoids contain podocytes, proximal tubules expressing AGT and distal tubules. Angiotensin II type 1 receptor (AT1R) levels were higher than other RAS components on day 0 and the expression was downregulated on day 5. AT2R induction peaked on day 5 and reduced until day 18. Renin expression was strongly induced on day 5 and sustained until day 18. Angiotensin-converting enzyme levels were moderately augmented during the culture. AGT levels were elevated on day 12 and remained until day 18 (7.6-fold, ratio to day 0). Conversely, HDAC9 levels decreased by day 18. On day 18, AGT and HDAC9 levels were inversely correlated in a CHIR99201 concentration-dependent manner. Moreover, an HDAC9 inhibitor increased AGT expression (2.25±0.21-fold, ratio to control). These results suggest that all RAS components are expressed and independently regulated during the development of iPSC-derived human kidney organoids. The existence of all RAS components including high renin expression, their regulation, and epigenetic regulation of AGT in the organoids support previous findings in rodent models. Concerted effort, including this study, to overcome technical challenges to generate complete nephrons will provide human kidney organoids to study development, pathophysiological mechanisms, novel drugs and clinical therapies.

Intrarenal angiotensinogen (AGT) is mainly expressed in proximal tubular cells (PTC). AGT is increased by hyperglycemia (HG) in type 1 and 2 diabetes mellitus, which causes elevated intrarenal angiotensin formation contributing to the development of hypertension and kidney injury. Sodium glucose co-transporter 2 (SGLT2) is abundantly expressed in early PTC and may promote increased intrarenal AGT by increasing intracellular glucose levels. This study tested the effects of canagliflozin (CANA), an SGLT2 inhibitor, on HG-induced AGT elevation in cultured PTC. Mouse PTC were treated with 5, 10 or 25 mM glucose. 0-10 μM CANA was applied one hour before glucose treatment. AGT mRNA and protein levels were measured by digital PCR and western blot analysis. Levels of intracellular reactive oxygen species (ROS) were determined with H 2 DCF-DA. Tempol, an antioxidant, was used to test if elevated ROS is involved in HG-induced AGT upregulation. 10 mM glucose increased AGT protein levels at 12 hours (3.06±0.48-fold compared with 5 mM glucose) and treatment with 10 μM CANA attenuated the AGT augmentation (1.68±0.05-fold). AGT protein levels were also increased by 25 mM glucose; but CANA did not suppress the AGT levels caused by this glucose concentration. In PTC treated with 10 mM glucose for 12 hours, the suppressing effect on AGT upregulation was observed with 1 and 10 μM CANA. Lower concentrations of CANA (0.01 and 0.1 μM) did not lower AGT protein levels significantly. Elevated AGT mRNA expression by glucose was also attenuated by CANA. Treatment of PTC with 1 mM pyruvate also increased AGT expression levels, indicating that glycolysis is involved in HG-induced AGT upregulation. After incubation of PTC with 10 mM glucose for 12 hours, intracellular ROS levels were elevated compared to baseline (4.24±0.23-fold) and these were also inhibited by CANA (0.2±0.08-fold). Furthermore, tempol attenuated AGT upregulation in HG-treated PTC. These results indicate that enhanced glucose entry via SGLT2 into PTC elevates intracellular ROS generation by stimulation of glycolysis and consequent AGT augmentation. Thus, SGLT2 inhibition by CANA may limit HG-induced AGT stimulation which suppress intrarenal angiotensin formation and reduce kidney injury in diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a complex disease where hyperglycemia occurs as a result of the development of insulin resistance. T2DM leads to complications, including renal and cardiovascular injury. Renal proximal tubular angiotensinogen (AGT) is stimulated by hyperglycemia via elevated oxidative stress, and is associated with activation of the intrarenal renin-angiotensin system which contributes to development of high blood pressure and diabetic nephropathy in diabetes mellitus. Sodium glucose co-transporter 2 (SGLT2) is mainly expressed in early renal proximal tubules and is responsible for most of the glucose reabsorption by the tubules. This study tested the effects of canagliflozin (CANA), an SGLT2 inhibitor, on intrarenal AGT regulation in T2DM mice. Male New Zealand Obese mice were fed a regular fat diet (RFD, 4% fat) or a high fat diet (HFD, 40% fat) to induce diabetes. When the mice fed with HFD exhibited >350 mg/dl blood glucose levels (week 0), both RFD and HFD fed mice were treated with 10 mg/kg/day CANA or vehicle for 6 weeks by daily oral gavage. Systolic blood pressure (SBP) levels were measured by a tail cuff system and 24-hour urine samples were collected using metabolic cages. Intrarenal AGT mRNA and protein levels were determined by digital PCR and western blot analysis. CANA treatment decreased blood glucose levels in HFD mice, which remained suppressed for duration of the study. SBP in HFD groups were higher than in the RFD and RFD+CANA groups (134.7±3.6, 118.7±10.8 and 108.3±7.6 mmHg) at week 6. The elevated SBP was normalized by CANA (110.0±6.0 mmHg). The HFD group exhibited greater renal cortical AGT mRNA levels than the RFD group (3.7±0.4 vs. 7.4±1.0 copies/reaction). Likewise, intrarenal AGT mRNA expression was lower in CANA treated group (4.5±0.8 copies/reaction). Western blot analysis also showed lower AGT protein levels in CANA treated group (1.02±0.08-fold compared with RFD) than in HFD group (1.49±0.08-fold). In the HFD group, CANA treatment also suppressed elevated urinary 8-isoprostane levels, a marker of renal oxidative stress. These results demonstrate that CANA attenuates intrarenal AGT augmentation that occurs in T2DM, which may mitigate the development of diabetic nephropathy and progression of high blood pressure.