Yixuan Shi

Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice

We investigated the effects of dual renin-angiotensin system (RAS) blockade on angiotensin-converting enzyme-2 (Ace2) expression, hypertension, and renal proximal tubular cell (RPTC) apoptosis in type 1 diabetic Akita angiotensinogen (Agt)-transgenic (Tg) mice that specifically overexpress Agt in their RPTCs. Adult (11 wk old) male Akita and Akita Agt-Tg mice were treated with two RAS blockers (ANG II receptor type 1 blocker losartan, 30 mg·kg(-1)·day(-1)) and angiotensin-converting enzyme (ACE) inhibitor perindopril (4 mg·kg(-1)·day(-1)) in drinking water. Same-age non-Akita littermates and Agt-Tg mice served as controls. Blood pressure, blood glucose, and albuminuria were monitored weekly. The animals were euthanized at age 16 wk. The left kidneys were processed for immunohistochemistry and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess gene and protein expression. Urinary ANG II and ANG 1-7 were quantified by ELISA. RAS blockade normalized renal Ace2 expression and urinary ANG 1-7 levels (both of which were low in untreated Akita and Akita Agt-Tg), prevented hypertension, albuminuria, tubulointerstitial fibrosis and tubular apoptosis, and inhibited profibrotic and proapoptotic gene expression in RPTCs of Akita and Akita Agt-Tg mice compared with non-Akita controls. Our results demonstrate the effectiveness of RAS blockade in preventing intrarenal RAS activation, hypertension, and nephropathy progression in diabetes and support the important role of intrarenal Ace2 expression in modulating hypertension and renal injury in diabetes.

Overexpression of catalase prevents hypertension and tubulointerstitial fibrosis and normalization of renal angiotensin-converting enzyme-2 expression in Akita mice

We investigated the relationship among oxidative stress, hypertension, renal injury, and angiotensin-converting enzyme-2 (ACE2) expression in type 1 diabetic Akita mice. Blood glucose, blood pressure, and albuminuria were monitored for up to 5 mo in adult male Akita and Akita catalase (Cat) transgenic (Tg) mice specifically overexpressing Cat, a key antioxidant enzyme in their renal proximal tubular cells (RPTCs). Same-age non-Akita littermates and Cat-Tg mice served as controls. In separate studies, adult male Akita mice (14 wk) were treated with ANG 1-7 (500 μg·kg⁻¹·day⁻¹ sc) ± A-779, an antagonist of the Mas receptor (10 mg·kg⁻¹·day⁻¹ sc), and euthanized at the age of 18 wk. The left kidneys were processed for histology and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess protein and gene expression. Urinary angiotensinogen (AGT), angiotensin II (ANG II), and ANG 1-7 were quantified by specific ELISAs. Overexpression of Cat attenuated renal oxidative stress; prevented hypertension; normalized RPTC ACE2 expression and urinary ANG 1-7 levels (both were low in Akita mice); ameliorated glomerular filtration rate, albuminuria, kidney hypertrophy, tubulointerstitial fibrosis, and tubular apoptosis; and suppressed profibrotic and proapoptotic gene expression in RPTCs of Akita Cat-Tg mice compared with Akita mice. Furthermore, daily administration of ANG 1-7 normalized systemic hypertension in Akita mice, which was reversed by A-779. These data demonstrate that Cat overexpression prevents hypertension and progression of nephropathy and highlight the importance of intrarenal oxidative stress and ACE2 expression contributing to hypertension and renal injury in diabetes.

Angiotensin-(1-7): A Novel Peptide to Treat Hypertension and Nephropathy in Diabetes?

The renin-angiotensin system (RAS) plays a pivotal role in mammalian homeostasis physiology. The RAS can be delineated into a classical RAS (the pressor arm) including angiotensinogen (Agt), renin, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) and angiotensin type 1 receptor (AT1R), and a counterbalancing novel RAS (the depressor arm) including Agt, renin, angiotensin-converting enzyme-2 (ACE-2), angiotensin-(1-7) (Ang 1-7) and Ang 1-7 receptor (or Mas receptor (MasR)). Hyperglycemia (diabetes) induces severe tissue oxidative stress, which stimulates the pressor arm of the renal RAS axis and leads to an increase in ACE/ACE-2 ratio, with excessive formation of Ang II. There is a growing body of evidence for beneficial effects of the depressor arm of RAS (ACE-2/Ang 1-7/MasR) axis in diabetes, hypertension and several other diseased conditions. Evidence from in vitro, in vivo and clinical studies reflects anti-oxidant, anti-fibrotic, and anti-inflammatory properties of Ang 1-7. Most of the currently available therapies only target suppression of the pressor arm of RAS with angiotensin receptor blockers (ARBs) and ACE inhibitors (ACEi). However, it is time to consider simultaneous activation of the depressor arm for more effective outcomes. This review summarizes the recent updates on the protective role of Ang 1-7 in hypertension and kidney injury in diabetes, as well as the possible underlying mechanism(s) of Ang 1-7 action, suggesting that the ACE-2/Ang 1-7/MasR axis can be developed as a therapeutic target for the treatment of diabetes-induced hypertension and renal damage.

Catalase Overexpression Prevents Nuclear Factor Erythroid 2-Related Factor 2 Stimulation of Renal Angiotensinogen Gene Expression, Hypertension and Kidney Injury in Diabetic Mice

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2–related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice.

High Glucose Up-regulates ADAM17 through HIF-1α in Mesangial Cells.

Background: ADAM17 is an important promoter of fibrosis in diabetic kidney disease. Results: ADAM17 is transcriptionally up-regulated by glucose through HIF-1α in kidney mesangial cells, associated with enhanced enzymatic activity. Conclusion: ADAM17 induces its own up-regulation, thereby augmenting its activity, in response to glucose. Significance: These studies provide a strong foundation for further evaluation of the role of ADAM17 in the profibrotic response of kidney cells to high glucose. We previously showed that ADAM17 mediates high glucose-induced matrix production by kidney mesangial cells. ADAM17 expression is increased in diabetic kidneys, suggesting that its up-regulation may augment high glucose profibrotic responses. We thus studied the effects of high glucose on ADAM17 gene regulation. Primary rat mesangial cells were treated with high glucose (30 mm) or mannitol as osmotic control. High glucose dose-dependently increased ADAM17 promoter activity, transcript, and protein levels. This correlated with augmented ADAM17 activity after 24 h versus 1 h of high glucose. We tested involvement of transcription factors shown in other settings to regulate ADAM17 transcription. Promoter activation was not affected by NF-κB or Sp1 inhibitors, but was blocked by hypoxia-inducible factor-1α (HIF-1α) inhibition or down-regulation. This also prevented ADAM17 transcript and protein increases. HIF-1α activation by high glucose was shown by its increased nuclear translocation and activation of the HIF-responsive hypoxia-response element (HRE)-luciferase reporter construct. Assessment of ADAM17 promoter deletion constructs coupled with mutation analysis and ChIP studies identified HIF-1α binding to its consensus element at −607 as critical for the high glucose response. Finally, inhibitors of epidermal growth factor receptor (EGFR) and downstream PI3K/Akt, or ADAM17 itself, prevented high glucose-induced HIF-1α activation and ADAM17 up-regulation. Thus, high glucose induces ADAM17 transcriptional up-regulation in mesangial cells, which is associated with augmentation of its activity. This is mediated by HIF-1α and requires EGFR/ADAM17 signaling, demonstrating the potentiation by ADAM17 of its own up-regulation. ADAM17 inhibition thus provides a potential novel therapeutic strategy for the treatment of diabetic nephropathy.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-β1-induced kidney injury in a mouse model of diabetes

Aims/hypothesisWe investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes.MethodsAdult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied.ResultsOverexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1–7 receptor [MasR]) expression, and normalised urinary Ang 1–7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression.Conclusions/interpretationThese data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.

Insulin Inhibits Nrf2 Gene Expression via Heterogeneous Nuclear Ribonucleoprotein F/K in Diabetic Mice

Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2–related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and angiotensinogen (Agt) gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F and hnRNP K) expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signaling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F, or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was downregulated, whereas hnRNP F/K expression was upregulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes.

Heterogeneous Nuclear Ribonucleoprotein F Stimulates Sirtuin-1 Gene Expression and Attenuates Nephropathy Progression in Diabetic Mice

We investigated the mechanism of heterogeneous nuclear ribonucleoprotein F (hnRNP F) renoprotective action in a type 2 diabetes (T2D) mouse model (db/db). Immortalized rat renal proximal tubular cells (IRPTCs) and kidneys from humans with T2D were also studied. The db/db mice developed hyperglycemia, oxidative stress, and nephropathy at age 20 weeks compared with their db/m littermates. These abnormalities, with the exception of hyperglycemia, were attenuated in db/db hnRNP F–transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs. Sirtuin-1, Foxo3α, and catalase expression were significantly decreased in RPTCs from db/db mice and normalized in db/db hnRNP F–Tg mice. In vitro, hnRNP F overexpression stimulated Sirtuin-1 and Foxo3α with downregulation of acetylated p53 expression and prevented downregulation of Sirtuin-1 and Foxo3α expression in IRPTCs by high glucose plus palmitate. Transfection of Sirtuin-1 small interfering RNA prevented hnRNP F stimulation of Foxo3α and downregulation of acetylated p53 expression. hnRNP F stimulated Sirtuin-1 transcription via hnRNP F–responsive element in the Sirtuin-1 promoter. Human T2D kidneys exhibited more RPTC apoptosis and lower expression of hnRNP F, SIRTUIN-1, and FOXO3α than nondiabetic kidneys. Our results demonstrate that hnRNP F protects kidneys against oxidative stress and nephropathy via stimulation of Sirtuin-1 expression and signaling in diabetes.

OS 25-03 OVEREXPRESSION OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN F PREVENTS SYSTEMIC HYPERTENSION AND KIDNEY INJURY AND NORMALIZES RENAL RENIN-ANGIOTENSIN SYSTEM GENES EXPRESSION IN TYPE 2 DIABETIC DB/DB TRANSGENIC MICE.

Objective: We aimed to investigate whether overexpression of heterogeneous nuclear ribonucleoprotein F (HnRNP F, a transcription factor) in renal proximal tubular cells (RPTCs) prevents systemic hypertension and kidney injury in type 2 diabetic db/db transgenic (Tg) mice and study its underlying mechanism (s) of action. Design and Method: Db/db hnRNP F-Tg mice (BKS strain) were generated by cross-breeding of db/m + mice with Tg mice specifically overexpressing HnRNP F in their RPTCs using kidney-specific androgen-regulated protein promoter (KAP2). Adult (10–20 weeks of age) male wild type (WT), db/db and db/db HnRNP F- Tg mice were studied. Kidneys were processed for immunohistochemistry. Blood glucose (BG), systolic blood pressure (SBP) and urinary albumin/creatinine ratio (ACR) were monitored bi-weekly. Immunostaining, Western blotting and real-time qPCR were employed to assess angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme-2 (ACE-2), angiotensin 1–7 receptor (MasR), transforming growth factor-beta 1 (TGF-&bgr;1), Bax and active caspase-3 (Csp-3) protein expression and their mRNA levels in RPTCs, respectively. Rat immortalizaed RPTCs stably transfected with hnRNP F cDNA were studied in vitro. Results: Db/db mice exhibited increased BG, SBP and ACR as compared to WT mice. Db/db hnRNP F-Tg mice specifically overexpressing HnRNP F in their RPTCs attenuated these abnormalities except BG. AGT, ACE, TGF-&bgr;1, Bax and active Csp-3 protein and their respective mRNA expression in RPTCs were significantly increased whereas ACE2 and MasR 2 protein and mRNA were decreased in db/db mice compared to WT. These changes were normalized in db/db HnRNP F-Tg mice except ACE. In vitro, overexpression of HnRNP F inhibited AGT, TGF-&bgr;1, Bax and active Csp-3 protein and mRNA expression, whereas it increased ACE-2 and MasR expression in rat RPTCs. Conclusions: Our data suggest that intrarenal HnRNP F overexpression attenuates SBP, tubule-interstitial fibrosis and tubular apoptosis, predominantly through decreasing AGT and TGF-&bgr;1 expression and increasing ACE-2 and MasR expression.