Heterogeneous nuclear ribonucleoprotein F deficiency in mouse podocyte promotes podocytopathy mediated by methyltransferase-like 14 nuclear translocation resulting in Sirtuin 1 gene inhibition.

Heterogeneous nuclear ribonucleoprotein F (HnRNP F) is a key regulator for nucleic acid metabolism; however, whether HnRNP F expression is important in maintaining podocyte integrity is unclear. Nephroseq analysis from a registry of human kidney biopsies was performed. Age- and sex-matched podocyte-specific HnRNP F knockout (HnRNP FPOD KO) mice and control (HnRNP Ffl/fl) were studied. Podocytopathy was induced in male mice (more susceptible) either by adriamycin (ADR)- or low-dose streptozotocin treatment for 2 or 8 weeks. The mouse podocyte cell line (mPODs) was used in vitro. Nephroseq data in three human cohorts were varied greatly. Both sexes of HnRNP FPOD KO mice were fertile and appeared grossly normal. However, male 20-week-old HnRNP FPOD KO than HnRNP Ffl/fl mice had increased urinary albumin/creatinine ratio, and lower expression of podocyte markers. ADR- or diabetic- HnRNP FPOD KO (vs. HnRNP Ffl/fl) mice had more severe podocytopathy. Moreover, methyltransferase-like 14 (Mettl14) gene expression was increased in podocytes from HnRNP FPOD KO mice, further enhanced in ADR- or diabetic-treated HnRNP FPOD KO mice. Consequently, this elevated Mettl14 expression led to sirtuin1 (Sirt1) inhibition, associated with podocyte loss. In mPODs, knock-down of HnRNP F promoted Mettl14 nuclear translocation, which was associated with podocyte dysmorphology and Sirt1 inhibition-mediated podocyte loss. This process was more severe in ADR- or high glucose- treated mPODs. Conclusion: HnRNP F deficiency in podocytes promotes podocytopathy through activation of Mettl14 expression and its nuclear translocation to inhibit Sirt1 expression, underscoring the protective role of HnRNP F against podocyte injury.

NRF2 Deficiency Attenuates Diabetic Kidney Disease in Db/Db Mice via Down-Regulation of Angiotensinogen, SGLT2, CD36, and FABP4 Expression and Lipid Accumulation in Renal Proximal Tubular Cells.

The role(s) of nuclear factor erythroid 2-related factor 2 (NRF2) in diabetic kidney disease (DKD) is/are controversial. We hypothesized that Nrf2 deficiency in type 2 diabetes (T2D) db/db mice (db/dbNrf2 knockout (KO)) attenuates DKD progression through the down-regulation of angiotensinogen (AGT), sodium-glucose cotransporter-2 (SGLT2), scavenger receptor CD36, and fatty -acid-binding protein 4 (FABP4), and lipid accumulation in renal proximal tubular cells (RPTCs). Db/dbNrf2 KO mice were studied at 16 weeks of age. Human RPTCs (HK2) with NRF2 KO via CRISPR-Cas9 genome editing and kidneys from patients with or without T2D were examined. Compared with db/db mice, db/dbNrf2 KO mice had lower systolic blood pressure, fasting blood glucose, kidney hypertrophy, glomerular filtration rate, urinary albumin/creatinine ratio, tubular lipid droplet accumulation, and decreased expression of AGT, SGLT2, CD36, and FABP4 in RPTCs. Male and female mice had similar results. NRF2 KO attenuated the stimulatory effect of the Nrf2 activator, oltipraz, on AGT, SGLT2, and CD36 expression and high-glucose/free fatty acid (FFA)-stimulated lipid accumulation in HK2. Kidneys from T2D patients exhibited markedly higher levels of CD36 and FABP4 in RPTCs than kidneys from non-diabetic patients. These data suggest that NRF2 exacerbates DKD through the stimulation of AGT, SGLT2, CD36, and FABP4 expression and lipid accumulation in RPTCs of T2D.

Angiotensin II type-2-receptor stimulation ameliorates focal and segmental glomerulosclerosis in mice.

Podocyte damage and loss are the early event in the development of focal segmental glomerulosclerosis (FSGS). Podocytes express angiotensin II type-2-receptor (AT2R), which may play a key role in maintaining kidney integrity and function. Here, we examined the effects of AT2R deletion and AT2R agonist compound 21 (C21) on the evolution of FSGS. FSGS was induced by adriamycin (ADR) injection in both male wild-type (WT) and AT2R knockout (KO) mice. C21 was administered to WT-FSGS mice either one day before or 7 days after ADR (Pre-C21 or Post-C21), using two doses of C21 at either 0.3 (low dose, LD) or 1.0 (high dose, HD) mg/kg/day. ADR-induced FSGS was more severe in AT2RKO mice compared with WT-FSGS mice, and included profound podocyte loss, glomerular fibrosis, and albuminuria. Glomerular cathepsin L expression increased more in AT2RKO-FSGS than in WT-FSGS mice. C21 treatment ameliorated podocyte injury, most significantly in the Pre C21-HD group, and inhibited glomerular cathepsin L expression. In vitro, Agtr2 knock-down in mouse podocyte cell line given ADR confirmed the in vivo data. Mechanistically, C21 inhibited cathepsin L expression, which protected synaptopodin from destruction and stabilized actin cytoskeleton. C21 also prevented podocyte apoptosis. In conclusion, AT2R activation by C21 ameliorated ADR-induced podocyte injury in mice by the inhibition of glomerular cathepsin L leading to the maintenance of podocyte integrity and prevention of podocyte apoptosis.

Deletion of heterogeneous nuclear ribonucleoprotein F in renal tubules downregulates SGLT2 expression and attenuates hyperfiltration and kidney injury in a mouse model of diabetes.

AIMS/HYPOTHESIS: We previously reported that renal tubule-specific deletion of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) results in upregulation of renal angiotensinogen (Agt) and downregulation of sodium-glucose co-transporter 2 (Sglt2) in HnrnpfRT knockout (KO) mice. Non-diabetic HnrnpfRT KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated Sglt2 expression. Here, we investigated the effect of renal tubular Hnrnpf deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes. METHODS: Akita HnrnpfRT KO mice were generated through crossbreeding tubule-specific (Pax8)-Cre mice with Akita floxed-Hnrnpf mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita HnrnpfRT KO mice were studied up to the age of 24 weeks (n = 8/group). RESULTS: Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita HnrnpfRT KO mice than Akita mice. Compared with Akita mice, Akita HnrnpfRT KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita HnrnpfRT KO mice. Treatment of Akita HnrnpfRT KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal Agt expression was elevated in Akita mice and further increased in Akita HnrnpfRT KO mice. In contrast, Sglt2 expression was increased in Akita and decreased in Akita HnrnpfRT KO mice. CONCLUSIONS/INTERPRETATION: The renoprotective effect of Sglt2 downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.

Overexpression of Nrf2 in Renal Proximal Tubular Cells Stimulates Sodium-Glucose Cotransporter 2 Expression and Exacerbates Dysglycemia and Kidney Injury in Diabetic Mice.

We investigated the impact of nuclear factor erythroid 2-related factor 2 (Nrf2) overexpression in renal proximal tubular cells (RPTCs) on blood glucose, kidney injury, and sodium-glucose cotransporter 2 (Sglt2) expression in diabetic Akita Nrf2 -/-/Nrf2RPTC transgenic (Tg) mice. Immortalized human RPTCs (HK2) stably transfected with plasmid containing the SGLT2 promoter and human kidneys from patients with diabetes were also studied. Nrf2 overexpression was associated with increased blood glucose, glomerular filtration rate, urinary albumin-to-creatinine ratio, tubulointerstitial fibrosis, and Sglt2 expression in Akita Nrf2 -/-/Nrf2RPTC Tg mice compared with their Akita Nrf2 -/- littermates. In vitro, oltipraz or transfection of NRF2 cDNA stimulated SGLT2 expression and SGLT2 promoter activity in HK2, and these effects were inhibited by trigonelline or NRF2 siRNA. The deletion of the NRF2-responsive element (NRF2-RE) in the SGLT2 promoter abolished the stimulatory effect of oltipraz on SGLT2 promoter activity. NRF2 binding to the NRF2-RE of the SGLT2 promoter was confirmed by gel mobility shift assay and chromatin immunoprecipitation assays. Kidneys from patients with diabetes exhibited higher levels of NRF2 and SGLT2 in the RPTCs than kidneys from patients without diabetes. These results suggest a link by which NRF2 mediates hyperglycemia stimulation of SGLT2 expression and exacerbates blood glucose and kidney injury in diabetes.

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.

Comparison of the effects of insulin and SGLT2 inhibitor on the Renal Renin-Angiotensin system in type 1 diabetes mice.

AIMS: SGLT2 inhibitors have been proposed as an adjunct to insulin therapy for glycemic control in type 1 diabetes (T1D) patients. However, concern has been raised due to an increase in renin-angiotensin-system (RAS) activity reported in a clinical trial in which an SGLT2 inhibitor was added while insulin dose was reduced in T1D patients. We previously reported that insulin inhibits intrarenal angiotensinogen (Agt) gene transcription and RAS activation. We hypothesized that insulin, rather than SGLT2 inhibition might regulate the intrarenal RAS. METHODS: We compared RAS activity in non-diabetic wild type mice, Akita mice (T1D model) and Akita mice treated with insulin or the SGLT2 inhibitor canagliflozin. RESULTS: Treatment of Akita mice with insulin or canagliflozin produced similar reductions in blood glucose, whereas insulin, but not canagliflozin, reduced elevated systolic blood pressure. Akita mice exhibited increased renal Agt mRNA/protein expression, which was attenuated by insulin, but not by canagliflozin. Furthermore, insulin was more effective than canagliflozin in lowering kidney weight and albuminuria. CONCLUSIONS: Insulin, but not canagliflozin, lowers intrarenal RAS activity in Akita mice. Our findings can be of potential clinical importance, especially for T1D patients who are not on RAS inhibitors at the time of adding SGLT2 inhibitors.

Increased urinary excretion of hedgehog interacting protein (uHhip) in early diabetic kidney disease.

Glomerular endothelial cell (GEC) dysfunction occurs in diabetic kidney disease (DKD) and generally precedes albuminuria. We recently reported that hedgehog interacting protein (Hhip), highly expressed in GECs, contributes to DKD development in diabetic mice. Here, we hypothesized that urinary Hhip (uHhip) could identify early DKD; we tested uHhip in mice and humans with diabetes (DM). In both type 1 (Akita) and type 2 (db/db) DM mice, uHhip is elevated prior to the development of albuminuria, while non-DM controls excrete minimal amount of uHhip. In 87 type 2 DM patients and 39 healthy controls, the uHhip/creatinine (Cr) ratio provides a significant discrimination between non-DM and DM groups; 0 [0-69.5] in non-DM, 9.9 [1.7-39.5] in normoalbuminuric DM, 167.7 [95.7-558.7] in microalbuminuric DM, and 207.9 [0-957.2] in macroalbuminuric DM (median [IQR] ng/mmol, P < 0.0001). The log-uHhip/Cr is positively correlated with urine albumin/Cr ratio (UACR) (spearman correlation coefficient 0.47, P < 0.0001). The log-uHhip/Cr is also associated with eGFR, pulse pressure, and urinary cytokines (IL-1ß, IL-6, IL-8, and TGFß1) independent of UACR. By immunostaining, Hhip is localized in glomeruli and tubules, and is increased in human DM kidneys compared with non-DM kidneys. TGFß1 shares the similar staining pattern as Hhip in human DM kidneys. Thus, uHhip appears to be a novel indicator of diabetic GEC injury and is elevated in early DKD before the development of microalbuminuria in mice and humans. Clinical value for detecting early DKD warrants further investigation.

Tubular Deficiency of Heterogeneous Nuclear Ribonucleoprotein F Elevates Systolic Blood Pressure and Induces Glycosuria in Mice.

We reported previously that overexpression of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in renal proximal tubular cells (RPTCs) suppresses angiotensinogen (Agt) expression, and attenuates systemic hypertension and renal injury in diabetic Hnrnpf-transgenic (Tg) mice. We thus hypothesized that deletion of Hnrnpf in the renal proximal tubules (RPT) of mice would worsen systemic hypertension and kidney injury, perhaps revealing novel mechanism(s). Tubule-specific Hnrnpf knockout (KO) mice were generated by crossbreeding Pax8-Cre mice with floxed Hnrnpf mice on a C57BL/6 background. Both male and female KO mice exhibited elevated systolic blood pressure, increased urinary albumin/creatinine ratio, tubulo-interstitial fibrosis and glycosuria without changes in blood glucose or glomerular filtration rate compared with control littermates. However, glycosuria disappeared in male KO mice at the age of 12 weeks, while female KO mice had persistent glycosuria. Agt expression was elevated, whereas sodium-glucose co-transporter 2 (Sglt2) expression was down-regulated in RPTs of both male and female KO mice as compared to control littermates. In vitro, KO of HNRNPF in human RPTCs (HK-2) by CRISPR gRNA up-regulated AGT and down-regulated SGLT2 expression. The Sglt2 inhibitor canagliflozin treatment had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice but induced glycosuria. Our results demonstrate that Hnrnpf plays a role in the development of hypertension and glycosuria through modulation of renal Agt and Sglt2 expression in mice, respectively.

Hedgehog Interacting Protein (Hhip) Regulates Insulin Secretion in Mice Fed High Fat Diets.

Hedgehog interacting protein (Hhip) is essential for islet formation and beta-cell proliferation during pancreatic development; abnormally elevated Hhip expression has been linked to human pancreatitis. Here, we investigate the role of Hhip in modulating insulin secretion in adult Hhip mice (Hhip +/- vs. Hhip+/+) fed high fat diets (HFD). Both sexes of HFD-Hhip +/+ mice developed impaired glucose intolerance, that was only ameliorated in male HFD-Hhip +/- mice that had high levels of circulating plasma insulin, but not in female HFD-Hhip +/- mice. HFD stimulated Hhip gene expression, mainly in beta cells. Male HFD-Hhip +/+ mice had more large islets in which insulin content was reduced; islet architecture was disordered; and markers of oxidative stress (8-OHdG and Nox 2) were increased. In contrast, male HFD-Hhip +/- mice had more small islets with increased beta cell proliferation, enhanced GSIS, less oxidative stress and preserved islet integrity. In vitro, recombinant Hhip increased Nox2 and NADPH activity and decreased insulin-positive beta cells. siRNA-Hhip increased GSIS and abolished the stimulation of sodium palmitate (PA)-BSA on Nox2 gene expression. We conclude that pancreatic Hhip gene inhibits insulin secretion by altering islet integrity and promoting Nox2 gene expression in beta cells in response to HDF-mediated beta cell dysfunction, a novel finding.

Heterogeneous Nuclear Ribonucleoprotein F Mediates Insulin Inhibition of Bcl2-Modifying Factor Expression and Tubulopathy in Diabetic Kidney.

We investigated the molecular mechanism(s) by which insulin prevents Bcl2-modifying factor (Bmf)-induced renal proximal tubular cell (RPTC) apoptosis and loss in diabetic mice. Transgenic mice (Tg) mice specifically overexpressing human BMF in RPTCs and non-Tg littermates were studied at 10 to 20 weeks of age. Non-diabetic littermates, diabetic Akita mice +/- insulin implant, Akita Tg mice specifically overexpressing heterogeneous nuclear ribonucleoprotein F (hnRNP F) in their RPTCs and immortalized rat renal proximal tubular cells (IRPTCs) were also studied. BMF-Tg mice exhibited higher systolic blood pressure, urinary albumin/creatinine ratio, RPTC apoptosis and urinary RPTCs than non-Tg mice. Insulin treatment in Akita mice and Akita mice overexpressing hnRNP F suppressed Bmf expression and RPTC apoptosis. In hyperinsulinemic-euglycemic wild type mice, renal Bmf expression was down-regulated with up-regulation of hnRNP F. In vitro, insulin inhibited high glucose-stimulation of Bmf expression, predominantly via p44/42 mitogen-activated protein kinase (MAPK) signaling. Transfection of p44/42 MAPK or hnRNP F small interfering RNA (siRNA) prevented insulin inhibition of Bmf expression. HnRNP F inhibited Bmf transcription via hnRNP F-responsive element in the Bmf promoter. Our results demonstrate that hnRNP F suppression of Bmf transcription is an important mechanism by which insulin protects RPTCs from apoptosis in diabetes.

Hedgehog Interacting Protein Promotes Fibrosis and Apoptosis in Glomerular Endothelial Cells in Murine Diabetes.

We investigated whether renal hedgehog interacting protein (Hhip) expression contributes to the progression of diabetic nephropathy (DN) and studied its related mechanism(s) in vivo and in vitro. Here, we show that Hhip expression is highly elevated in glomerular endothelial cells of adult type 1 diabetic (T1D) Akita and T2D db/db mouse kidneys as compared to non-diabetic control littermates. Hyperglycemia enhances reactive oxygen species (ROS) generation via NADPH oxidase 4 (Nox4) activation and stimulates renal Hhip gene expression, and that elevated renal Hhip gene expression subsequently activates the TGFß1- Smad2/3 cascade and promotes endothelial to mesenchymal transition associated with endothelial cell fibrosis/apoptosis in vivo and in vitro. Furthermore, kidneys of low-dose streptozotocin-induced diabetic heterozygous Hhip deficient (Hhip+/-) mice displayed a normal albumin/creatinine ratio with fewer features of DN (glomerulosclerosis/fibrosis and podocyte apoptosis/loss) and less evidence of renal compensation (glomerular hypertrophy and hyperfiltration) as compared to diabetic wild type controls (Hhip+/+). Thus, our studies demonstrated that renal Hhip expression is associated with nephropathy development in diabetes and that hyperglycemia-induced renal Hhip expression may mediate glomerular endothelial fibrosis and apoptosis in diabetes, a novel finding.

Nrf2 Deficiency Upregulates Intrarenal Angiotensin-Converting Enzyme-2 and Angiotensin 1-7 Receptor Expression and Attenuates Hypertension and Nephropathy in Diabetic Mice.

We investigated the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in renin-angiotensin system (RAS) gene expression in renal proximal tubule cells (RPTCs) and in the development of systemic hypertension and kidney injury in diabetic Akita mice. We used adult male Akita Nrf2 knockout mice and Akita mice treated with trigonelline (an Nrf2 inhibitor) or oltipraz (an Nrf2 activator). We also examined rat immortalized RPTCs (IRPTCs) stably transfected with control plasmids or plasmids containing rat angiotensinogen (Agt), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme-2 (Ace2), or angiotensin 1-7 (Ang 1-7) receptor (MasR) gene promoters. Genetic deletion of Nrf2 or pharmacological inhibition of Nrf2 in Akita mice attenuated hypertension, renal injury, tubulointerstitial fibrosis, and the urinary albumin/creatinine ratio. Furthermore, loss of Nrf2 upregulated RPTC Ace2 and MasR expression, increased urinary Ang 1-7 levels, and downregulated expression of Agt, ACE, and profibrotic genes in Akita mice. In cultured IRPTCs, Nrf2 small interfering RNA transfection or trigonelline treatment prevented high glucose stimulation of Nrf2 nuclear translocation, Agt, and ACE transcription with augmentation of Ace2 and MasR transcription, which was reversed by oltipraz. These data identify a mechanism, Nrf2-mediated stimulation of intrarenal RAS gene expression, by which chronic hyperglycemia induces hypertension and renal injury in diabetes.

AT2 R deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein (Hhip) gene expression.

Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFß1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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/dbhnRNP 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/dbhnRNP 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.

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.

Overexpression of angiotensinogen downregulates aquaporin 1 expression via modulation of Nrf2-HO-1 pathway in renal proximal tubular cells of transgenic mice.

INTRODUCTION: We aimed to examine the regulation of aquaporin 1 expression in an angiotensinogen transgenic mouse model, focusing on underlying mechanisms. METHODS: Male transgenic mice overexpressing rat angiotensinogen in their renal proximal tubular cells (RPTCs) and rat immortalised RPTCs stably transfected with rat angiotensinogen cDNA were used. RESULTS: Angiotensinogen-transgenic mice developed hypertension and nephropathy, changes that were either partially or completely attenuated by treatment with losartan or dual renin-angiotensin system blockade (losartan and perindopril), respectively, while hydralazine prevented hypertension but not nephropathy. Decreased expression of aquaporin 1 and heme oxygenase-1 and increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and sodium-hydrogen exchanger 3 were observed in RPTCs of angiotensinogen-transgenic mice and in angiotensinogen-transfected immortalised RPTCs. These parameters were normalised by dual renin-angiotensin system blockade. Both in vivo and in vitro studies identified a novel mechanism in which angiotensinogen overexpression in RPTCs enhances the cytosolic accumulation of Nrf2 via the phosphorylation of pGSK3ß Y216. Consequently, lower intranuclear Nrf2 levels are less efficient to trigger heme oxygenase-1 expression as a defence mechanism, which subsequently diminishes aquaporin 1 expression in RPTCs. CONCLUSIONS: Angiotensinogen-mediated downregulation of aquaporin 1 and Nrf2 signalling may play an important role in intrarenal renin-angiotensin system-induced hypertension and kidney injury.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-ß1-induced kidney injury in a mouse model of diabetes.

AIMS/HYPOTHESIS: We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-ß1 signalling, TGF-ß1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. METHODS: Adult 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. RESULTS: Overexpression 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-ß1, TGF-ß receptor II [Tgf-ßrII]) 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-ß1 and Tgf-ßrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-ß1 signalling and TGF-ß1 inhibition of Ace-2 gene expression. CONCLUSIONS/INTERPRETATION: These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-ß1 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.

Angiotensin-(1-7) prevents systemic hypertension, attenuates oxidative stress and tubulointerstitial fibrosis, and normalizes renal angiotensin-converting enzyme 2 and Mas receptor expression in diabetic mice.

We investigated the relationship between Ang-(1-7) [angiotensin-(1-7)] action, sHTN (systolic hypertension), oxidative stress, kidney injury, ACE2 (angiotensin-converting enzyme-2) and MasR [Ang-(1-7) receptor] expression in Type 1 diabetic Akita mice. Ang-(1-7) was administered daily [500 µg/kg of BW (body weight) per day, subcutaneously] to male Akita mice from 14 weeks of age with or without co-administration of an antagonist of the MasR, A779 (10 mg/kg of BW per day). The animals were killed at 20 weeks of age. Age-matched WT (wild-type) mice served as controls. Ang-(1-7) administration prevented sHTN and attenuated kidney injury (reduced urinary albumin/creatinine ratio, glomerular hyperfiltration, renal hypertrophy and fibrosis, and tubular apoptosis) without affecting blood glucose levels in Akita mice. Ang-(1-7) also attenuated renal oxidative stress and the expression of oxidative stress-inducible proteins (NADPH oxidase 4, nuclear factor erythroid 2-related factor 2, haem oxygenase 1), pro-hypertensive proteins (angiotensinogen, angiotensin-converting enzyme, sodium/hydrogen exchanger 3) and profibrotic proteins (transforming growth factor-ß1 and collagen IV), and increased the expression of anti-hypertensive proteins (ACE2 and MasR) in Akita mouse kidneys. These effects were reversed by A779. Our data suggest that Ang-(1-7) plays a protective role in sHTN and RPTC (renal proximal tubular cell) injury in diabetes, at least in part, through decreasing renal oxidative stress-mediated signalling and normalizing ACE2 and MasR expression.

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.