OBJECTIVE-The present study investigated the relationships between reactive oxygen species (ROS), interstitial fibrosis, and renal proximal tubular cell (RPTC) apoptosis in type 2 diabetic db/db mice and in db/db transgenic (Tg) mice overexpressing rat catalase (rCAT) in their RPTCs (db/db rCAT-Tg).RESEARCH DESIGN AND METHODS-Blood pressure, blood glucose, and albuminuria were monitored for up to 5 months. Kidneys were processed for histology and apoptosis studies (terminal transferase-mediated dUTP nick-end labeling or immunostaining for active caspase-3 and Bax). Real-time quantitative PCR assays were used to quantify angiotensinogen (ANG), p53, and Bax mRNA levels.RESULTS-db/db mice developed obesity, hyperglycemia, hypertension, and albuminuria. In contrast, db/db rCAT-Tg mice became obese and hyperglycemic but had normal blood pressure and attenuated albuminuria compared with db/db mice. Kidneys from db/db mice displayed progressive glomerular hypertrophy, glomerulosclerosis, interstitial fibrosis, and tubular apoptosis and increased expression of collagen type IV, Bax, and active caspase-3, as well as increased ROS production. These changes, except glomerular hypertrophy, were markedly attenuated in kidneys of db/db rCAT-Tg mice. Furthermore, ANG, p53, and Bax mRNA expression was increased in renal proximal tubules of db/db mice but not of db/db rCAT-Tg mice. D iabetes affects 5-10% of the world population. It is estimated that 30 -40% and 5-10% of patients with type 1 and type 2 diabetes, respectively, will eventually develop kidney failure or endstage renal disease (1). Diabetic nephropathy is now the most common cause of end-stage renal disease, accounting for 40 -50% (2). Diabetic nephropathy is associated with an increased risk of hypertension, myocardial infarction, stroke, and cardiovascular dysfunction (3). Multiple factors have been implicated in the pathogenesis of diabetic nephropathy, including hyperglycemia, hypertension, insulin resistance, and oxidative stress (4). However, the molecular mechanisms of action of these risk factors are incompletely understood. CONCLUSIONS-OurOxidative stress has long been implicated in the progression of diabetes complications. High glucose induces reactive oxygen species (ROS) generation, and ROS contribute to apoptosis in podocytes and mesangial and tubular cells (5-7). Ang II stimulates ROS generation via heightened NADPH oxidase activity in various renal cell types, whereas antioxidants provide renal protection in part by ameliorating oxidative stress (8 -11). Such data strongly indicate a link between ROS, renin-angiotensin system (RAS) activation, and renal cell apoptosis in diabetes.Recent studies have reported that 71% of glomeruli from proteinuric type 1 diabetic patients have glomerulo-tubular junction abnormalities, including atubular glomeruli, which may occur in 8 -17% of nephrons (12,13). Atubular glomeruli, glomeruli that are not connected to their proximal tubules as an end result of tubular atrophy, are characteristic of a variety of human kid...

The intrarenal renin-angiotensin system (RAS) plays an important role in the progression of diabetic nephropathy. We have previously reported that mice overexpressing angiotensinogen in renal proximal tubular cells (RPTC) develop hypertension, albuminuria, and renal injury. Here, we investigated whether activation of the intrarenal RAS contributes to apoptosis of RPTC in diabetes. Induction of diabetes with streptozotocin in these transgenic mice led to significant increases in BP, albuminuria, RPTC apoptosis, and proapoptotic gene expression compared with diabetic nontransgenic littermates. Insulin and/or RAS blockers markedly attenuated these changes. Hydralazine prevented hypertension but not albuminuria, RPTC apoptosis, or proapoptotic gene expression. In vitro, high-glucose medium significantly increased apoptosis and caspase-3 activity in rat immortalized RPTC overexpressing angiotensinogen compared with control cells, and these changes were prevented by insulin and/or RAS blockers. In conclusion, intrarenal RAS activation and high glucose may act in concert to increase tubular apoptosis in diabetes, independent of systemic hypertension. Diabetic nephropathy (DN) is the leading cause of all ESRD in North America, accounting for 45% to 50% of all cases. 1,2 Intensive insulin therapy and chronic treatment with renin-angiotensin system (RAS) blockers are effective in retarding DN progression but do not provide a cure. [3][4][5] The local intrarenal RAS is well accepted. 6,7 Renal proximal tubular cells (RPTC) express all components of the RAS. 8 -10 Angiotensin II (Ang II) levels and RAS genes are elevated in the kidneys of diabetic rats and humans, 11-13 implying an important role for the intrarenal RAS in DN progression.Glomerular changes in DN first consist of hypertrophy and, later, thickening of the glomerular basement membrane, followed by expansion of the mesangial matrix and glomerulosclerosis. 14 -16 However, the gradual decline of renal function in later stages of DN is invariably associated with tubular atrophy and interstitial fibrosis, hallmarks of ESRD. 17,18 In fact, tubular atrophy and interstitial fibrosis are closely associated with loss of renal function and appear to be better predictors of renal disease progression than glomerular pathology. 15-18 Tubular atrophy in DN is incompletely understood, although apoptosis is a candidate mechanism. Indeed, apoptosis has been detected in RPTC of diabetic mouse, rat, and human kidneys. 19 -24

Maternal diabetes leads to an adverse in utero environment, but whether maternal diabetes impairs nephrogenesis is unknown. Diabetes was induced with streptozotocin in pregnant Hoxb7-green fluorescence protein mice at embryonic day 13, and the offspring were examined at several time points after birth. Compared with offspring of nondiabetic controls, offspring of diabetic mice had lower body weight, body size, kidney weight, and nephron number. The observed renal dysmorphogenesis may be the result of increased apoptosis, because immunohistochemical analysis revealed significantly more apoptotic podocytes as well as increased active caspase-3 immunostaining in the renal tubules compared with control mice. Regarding potential mediators of these differences, offspring of diabetic mice had increased expression of intrarenal angiotensinogen and renin mRNA, upregulation of NF-B isoforms p50 and p65, and activation of the NF-B pathway. In conclusion, maternal diabetes impairs nephrogenesis, possibly via enhanced intrarenal activation of the renin-angiotensin system and NF-B signaling.

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.

. Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice. Am J Physiol Renal Physiol 302: F840 -F852, 2012. First published December 28, 2011 doi:10.1152/ajprenal.00340.2011.-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 Ϫ1 ·day Ϫ1 ) and angiotensinconverting enzyme (ACE) inhibitor perindopril (4 mg·kg Ϫ1 ·day Ϫ1 ) 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.HYPERTENSION AFFECTS 25% OF the adult population in North America (1), and 40% of patients with diabetes develop hypertension (32). Hypertension and diabetes account for 65-70% of all end-stage renal disease (ESRD) cases in North America (1). ESRD is a major risk factor for cardiovascular diseases, including myocardial infarction and stroke (8). While intensive insulin therapy and chronic treatment with reninangiotensin system (RAS) blockers effectively retard the progression of diabetic nephropathy, they do not provide a cure (9,14,27,28,44). Such findings, however, indicate that hyperglycemia, hypertension, and RAS activation are major risk factors in the pathogenesis of ESRD.Human and murine renal proximal tubular cells (RPTCs) express all components of the RAS (19,22,34,41). We have reported that transgenic (Tg) mice that specifically overexpress angiotensinogen (Agt), the sole precursor of angiotensins in RPTCs, develop hypertension, albuminuria, and tubular apoptosis (21, 30). Furthermore, Agt overexpression enhances tubular apoptosis in streptozotocin (STZ)-induced diabetic mice (20). Although these findings indicate that intrarenal RAS activation and hypergly...