Angiotensin-converting enzyme 2 regulates renal atrial natriuretic peptide through angiotensin-(1-7).

Deficiency of ACE2 (angiotensin-converting enzyme 2), which degrades Ang (angiotensin) II, promotes the development of glomerular lesions. However, the mechanisms explaining why the reduction in ACE2 is associated with the development of glomerular lesions have still to be fully clarified. We hypothesized that ACE2 may regulate the renoprotective actions of ANP (atrial natriuretic peptide). The aim of the present study was to investigate the effect of ACE2 deficiency on the renal production of ANP. We evaluated molecular and structural abnormalities, as well as the expression of ANP in the kidneys of ACE2-deficient mice and C57BL/6 mice. We also exposed renal tubular cells to AngII and Ang-(1-7) in the presence and absence of inhibitors and agonists of RAS (renin-angiotensin system) signalling. ACE2 deficiency resulted in increased oxidative stress, as well as pro-inflammatory and profibrotic changes. This was associated with a down-regulation of the gene and protein expression on the renal production of ANP. Consistent with a role for the ACE2 pathway in modulating ANP, exposing cells to either Ang-(1-7) or ACE2 or the Mas receptor agonist up-regulated ANP gene expression. This work demonstrates that ACE2 regulates renal ANP via the generation of Ang-(1-7). This is a new mechanism whereby ACE2 counterbalances the renal effects of AngII and which explains why targeting ACE2 may be a promising strategy against kidney diseases, including diabetic nephropathy.

miR-200a Prevents renal fibrogenesis through repression of TGF-ß2 expression.

OBJECTIVE: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-ß1 and TGF-ß2. RESEARCH DESIGN AND METHODS: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-ß1 and TGF-ß2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-ß signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy. RESULTS: Both TGF-ß1 and TGF-ß2 induced EMT and fibrogenesis in NRK52E cells. TGF-ß1 and TGF-ß2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-ß-dependent EMT. miR-200a also downregulated the expression of TGF-ß2, via direct interaction with the 3' untranslated region of TGF-ß2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. CONCLUSIONS: miR-200a and miR-141 significantly impact on the development and progression of TGF-ß-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-ß signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

Angiotensin-converting enzyme 2 is a key modulator of the renin-angiotensin system in cardiovascular and renal disease.

PURPOSE OF REVIEW: Angiotensin-converting enzyme 2 (ACE2) has recently emerged as a key regulator of the renin-angiotensin system in both health and disease. RECENT FINDINGS: ACE2 deficiency is associated with elevated tissue and circulating levels of angiotensin II and reduced levels of angiotensin 1-7. Phenotypically, this results in a modest elevation in systolic blood pressure and left ventricular hypertrophy. In atherosclerosis-prone apolipoprotein E knockout mice, ACE2 deficiency results in augmented vascular inflammation and an inflammatory response that contributes to increased atherosclerotic plaque formation. In the kidney, ACE2 deficiency is associated with progressive glomerulosclerosis. Interventions such as ACE2 replenishment or augmentation of its actions have proven successful in reducing hypertension, plaque accumulation, and renal and cardiac damage in a range of different models. Although promising, the balance of the renin-angiotensin system remains complicated, with some evidence that overexpression of ACE2 may have adverse cardiac effects, and ACE2 and its metabolic products may promote epithelial-to-mesenchymal transition. SUMMARY: Repletion of ACE2's activities offers a new strategy to complement current clinical interventions in treating hypertension, renal and cardiovascular disease. In particular conditions where ACE inhibition and angiotensin receptor blockade are partially effective, the adjunctive actions of ACE2 may not only reduce clinical escape but also augment the efficacy of interventions.

The molecular mediators of type 2 epithelial to mesenchymal transition (EMT) and their role in renal pathophysiology.

Common to all forms of chronic kidney disease is the progressive scarring of the tubulo-interstitial space, associated with the acquisition and accumulation of activated myofibroblasts. Many of these myofibroblasts are generated when tubular epithelial cells progressively lose their epithelial characteristics (cell-cell contact, microvilli, tight-junction proteins, apical-basal polarity) and acquire features of a mesenchymal lineage, including stress fibres, filopodia and augmented matrix synthesis. This process, known as epithelial to mesenchymal transition (EMT), plays an important role in progressive kidney disease. For EMT to occur in tubular cells, the transcriptional activation (and derepression) of genes required to sustain mesenchymal-type structures and functions (e.g. vimentin, alpha-smooth muscle actin) must occur alongside repression (or deactivation) of genes that act to maintain the epithelial phenotype (e.g. E-cadherin, bone morphogenic protein 7). Several factors have been suggested as potential initiators of EMT. With a few key exceptions, these triggers require the induction of transforming growth factor beta (TGF-beta) and downstream mediators, including SMADs, CTGF, ILK and SNAI1. Activation of TGF-beta receptors is also able to stimulate a range of additional pathways (so-called non-SMAD activation), including RhoA, mitogen-activated protein kinase and phosphoinositide 3-kinase signalling cascades, that also contribute to EMT and renal fibrogenesis. This review examines in detail the molecular mediators of EMT in tubular cells and its potential role as a long-lasting mediator of metabolic stress.

Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: implications for diabetic renal disease.

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

Connective tissue growth factor and cardiac fibrosis after myocardial infarction.

The temporal and spatial expression of transforming growth factor (TGF)-beta(1) and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-beta(1), CTGF, and procollagen alpha1(I) mRNA were localized by in situ hybridization, and TGF-beta(1) and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-beta(1), CTGF, and collagen after MI. Procollagen alpha1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-beta(1) mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-beta(1) or CTGF. TGF-beta(1) is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.

Accelerated nephropathy in diabetic apolipoprotein e-knockout mouse: role of advanced glycation end products.

Hyperlipidemia not only may be relevant to cardiovascular disease in diabetes but may also play a role in the development and progression of diabetic nephropathy. Furthermore, there is increasing evidence that advanced glycation end products (AGE) play an important role in diabetic renal disease. The objectives of this study were first to characterize renal injury in diabetic apolipoprotein E knockout (apo E-KO) mice and second to explore the role of AGE in the development and progression of renal disease in this model. Diabetes was induced by injection of streptozotocin in 6-wk-old apo E-KO mice. Diabetic animals received no treatment or treatment with the inhibitor of AGE formation aminoguanidine (1 g/kg per d) or the cross-link breaker [4,5-dimethyl-3-(2-oxo2-phenylethyl)-thiazolium chloride] ALT-711, which cleaves preformed AGE (20 mg/kg per d) for 20 wk. Nondiabetic apo E-KO mice as well as nondiabetic and diabetic C57BL/6 mice served as controls. Compared with nondiabetic apo E-KO mice, induction of diabetes in apo E-KO mice resulted in accelerated renal injury characterized by albuminuria and glomerular and tubulointerstitial injury. These abnormalities were associated with increased expression of collagen type I and type IV and transforming growth factor-beta1 (TGF-beta1), increased alpha-smooth muscle actin immunostaining and macrophage infiltration, and increased serum and renal AGE. The two treatments, which attenuated renal AGE accumulation in a disparate manner, were associated with less albuminuria, structural injury, macrophage infiltration, TGF-beta1, and collagen expression. The accelerated renal injury that was observed in diabetic apo E-KO mice was attenuated by approaches that inhibit renal AGE accumulation.

The role of advanced glycation in reduced organic cation transport associated with experimental diabetes.

Tubular dysfunction is an important early manifestation of diabetic nephropathy. Reduced renal expression of organic cation transporters (OCTs) potentially contributes to impaired cation clearance in diabetes. This study examines the role of advanced glycation end-products (AGEs) in mediating these changes. Experimental diabetes was induced with streptozotocin (55 mg/kg). Rats were randomly treated with the AGE inhibitor aminoguanidine for 32 weeks. In a second protocol, diabetic rats were followed with and without low-dose insulin therapy (2 U/day) for 4 weeks. Expression of OCTs was determined by real-time RT-PCR (reverse transcription-polymerase chain reaction) and Western blotting. As a marker of cation transport, the fractional clearance of endogenous N-methylnicotinamide (NMN) was determined by high-performance liquid chromatography. Both short- and long-term diabetes was associated with reduced gene and protein expression of the three renal OCT isotypes. This was associated with a reduction in the fractional clearance of NMN compared with control animals by over 50%. These changes correlated with the accumulation of renal and plasma AGEs. Treatment with the AGE inhibitor aminoguanidine restored the expression of OCT-2 and OCT-3 in diabetic animals and normalized renal NMN clearance. NMN clearance was also improved in diabetic animals receiving low-dose insulin, correlating with a reduction in AGEs and improvement in effective renal plasma flow. These studies demonstrate an early impairment of expression of OCTs and cation clearance associated with diabetes. These changes correlate with the accumulation of AGEs and may be partly attenuated by an AGE inhibitor, implicating a role for AGEs in organic cation transport.

Advanced glycation end product interventions reduce diabetes-accelerated atherosclerosis.

Advanced glycation end product (AGE) formation may contribute to the progression of atherosclerosis, particularly in diabetes. The present study explored atherosclerosis in streptozotocin-induced diabetic apolipoprotein E-deficient (apoE-/-) mice that were randomized (n = 20) to receive for 20 weeks no treatment, the AGE cross-link breaker ALT-711, or the inhibitor of AGE formation aminoguanidine (AG). A sixfold increase in plaque area with diabetes was attenuated by 30% with ALT-711 and by 40% in AG-treated mice. Regional distribution of plaque demonstrated no reduction in plaque area or complexity within the aortic arch with treatment, in contrast to the thoracic and abdominal aortas, where significant attenuation was seen. Diabetes-associated accumulation of AGEs in aortas and plasma and decreases in skin collagen solubility were ameliorated by both treatments, in addition to reductions in the vascular receptor for AGE. Collagen-associated reductions in the AGEs carboxymethyllysine and carboxyethyllysine were identified with both treatments. Diabetes was also accompanied by aortic accumulation of total collagen, specifically collagens I, III, and IV, as well as increases in the profibrotic cytokines transforming growth factor-beta and connective tissue growth factor and in cellular alpha-smooth muscle actin. Attenuation of these changes was seen in both treated diabetic groups. ALT-711 and AG demonstrated the ability to reduce vascular AGE accumulation in addition to attenuating atherosclerosis in these diabetic mice.

Connective tissue growth factor is up-regulated in the diabetic retina: amelioration by angiotensin-converting enzyme inhibition.

Connective tissue growth factor (CTGF) has been postulated to have prosclerotic and angiogenic properties. The aim of this present study was to characterize retinal CTGF expression in the absence and presence of diabetes and in the context of treatment with the angiotensin-converting enzyme (ACE) inhibitor, perindopril. Retinas were obtained from control, diabetic, and diabetic plus perindopril-treated (3 mg/d) rats. CTGF gene expression was quantitated by RT-PCR and localized by in situ hybridization. CTGF protein expression was analyzed by Western blotting and localized by immunohistochemistry. Diabetes was associated with a greater than 2-fold increase in CTGF mRNA levels, which was attenuated by perindopril treatment. CTGF immunoreactivity was increased almost 2-fold in diabetes and was ameliorated by the ACE inhibitor perindopril. By in situ hybridization and immunohistochemistry, the major site of CTGF gene expression in the retina of diabetic rats was the ganglion cell layer. Based on the known in vivo effects of CTGF, it is postulated that this growth factor plays a pivotal role in mediating diabetes-associated retinal pathology. Furthermore, the protective effects of ACE inhibitors on retinal pathology may partly be mediated via effects on retinal CTGF expression.

The breakdown of preexisting advanced glycation end products is associated with reduced renal fibrosis in experimental diabetes.

Renal accumulation of advanced glycation end products (AGEs) has been linked to the progression of diabetic nephropathy. Cleavage of pre-formed AGEs within the kidney by a cross-link breaker, such as ALT-711, may confer renoprotection in diabetes. STZ diabetic rats were randomized into a) no treatment (D); b) treatment with the AGE cross-link breaker, ALT-711, weeks 16-32 (DALT early); and c) ALT-711, weeks 24-32 (DALT late). Treatment with ALT-711 resulted in a significant reduction in diabetes-induced serum and renal AGE peptide fluorescence, associated with decreases in renal carboxymethyllysine and RAGE immunostaining. Cross-linking of tail tendon collagen seen in diabetic groups was attenuated only by 16 weeks of ALT-711 treatment. ALT-711, independent of treatment duration, retarded albumin excretion rate (AER), reduced blood pressure, and renal hypertrophy. It also reduced diabetes-induced increases in gene expression of transforming growth factor beta1 (TGF-beta1), connective tissue growth factor (CTGF), and collagen IV. However, glomerulosclerotic index, tubulointerstitial area, total renal collagen, nitrotyrosine, protein expression of collagen IV, and TGF-beta1 only showed improvement with early ALT treatment alone. This study demonstrates the utility of a cross-link breaker as a treatment for diabetic nephropathy and describes effects not only on renal AGEs but on putative mediators of renal injury, such as prosclerotic cytokines and oxidative stress.

Reduced tubular cation transport in diabetes: prevented by ACE inhibition.

BACKGROUND: The renal clearance of organic cations is important for the homeostasis of a number of exogenous and endogenous compounds. The organic cation transporters (OCTs) situated on the basolateral surface of proximal tubular cells mediate active cation excretion. Alterations of cation transport may occur in diabetes, although the role of the OCTs has not been previously assessed. METHODS: Experimental diabetes was induced in rats with streptozotocin (55 mg/kg) and animals were randomly assigned to receive ramipril (3 mg/mL) in drinking water for 24 weeks. In a second protocol, rats were infused with angiotensin II (Ang II) at a dose of 58.3 ng/kg/min for 2 weeks via an implanted osmotic pump. Expression of the OCTs and renal clearance of the endogenous cation N-methyl-nicotinamide (NMN) was assessed. RESULTS: Diabetes was associated with a reduction in gene and protein expression of both OCT-1 and OCT-2 and a reduction in NMN clearance. These effects were prevented by ramipril, associated with the prevention of albuminuria and tubular injury as demonstrated by the expression of osteopontin and glutathione peroxidase 3 (GPX-3). An infusion of Ang II also reduced NMN clearance but without altering the renal expression of OCTs. CONCLUSION: We hypothesize that reduced expression of OCTs in diabetes may be a marker of tubular injury. However, Ang II may also directly augment renal cation clearance independent of changes in transporter expression. Together these effects may provide additional mechanism to explain treatment-related improvements in creatinine clearance and renoprotection in diabetes following blockade of the renin-angiotensin system (RAS).

A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes.

The formation of advanced glycation end products (AGEs) on extracellular matrix components leads to accelerated increases in collagen cross linking that contributes to myocardial stiffness in diabetes. This study determined the effect of the crosslink breaker, ALT-711 on diabetes-induced cardiac disease. Streptozotocin diabetes was induced in Sprague-Dawley rats for 32 weeks. Treatment with ALT-711 (10 mg/kg) was initiated at week 16. Diabetic hearts were characterized by increased left ventricular (LV) mass and brain natriuretic peptide (BNP) expression, decreased LV collagen solubility, and increased collagen III gene and protein expression. Diabetic hearts had significant increases in AGEs and increased expression of the AGE receptors, RAGE and AGE-R3, in association with increases in gene and protein expression of connective tissue growth factor (CTGF). ALT-711 treatment restored LV collagen solubility and cardiac BNP in association with reduced cardiac AGE levels and abrogated the increase in RAGE, AGE-R3, CTGF, and collagen III expression. The present study suggests that AGEs play a central role in many of the alterations observed in the diabetic heart and that cleavage of preformed AGE crosslinks with ALT-711 leads to attenuation of diabetes-associated cardiac abnormalities in rats. This provides a potential new therapeutic approach for cardiovascular disease in human diabetes.

Characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy.

ACE2, initially cloned from a human heart, is a recently described homologue of angiotensin-converting enzyme (ACE) but contains only a single enzymatic site that catalyzes the cleavage of angiotensin I to angiotensin 1-9 [Ang(1-9)] and is not inhibited by classic ACE inhibitors. It also converts angiotensin II to Ang(1-7). Although the role of ACE2 in the regulation of the renin-angiotensin system is not known, the renin-angiotensin system has been implicated in the pathogenesis of diabetic complications and in particular in diabetic nephropathy. Therefore, the aim of this study was to assess the possible involvement of this new enzyme in the kidney from diabetic Sprague-Dawley rats to compare and contrast it to ACE. ACE2 and ACE gene and protein expression were measured in the kidney after 24 weeks of streptozocin diabetes. ACE2 and ACE mRNA levels were decreased in diabetic renal tubules by approximately 50% and were not influenced by ACE inhibitor treatment with ramipril. By immunostaining, both ACE2 and ACE protein were localized predominantly to renal tubules. In the diabetic kidney, there was reduced ACE2 protein expression that was prevented by ACE inhibitor therapy. The identification of ACE2 in the kidney, its modulation in diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of various angiotensin peptides provides a rationale to further explore the role of this enzyme in various pathophysiological states including diabetic complications.

Renal connective tissue growth factor induction in experimental diabetes is prevented by aminoguanidine.

The aim of this study was to determine whether aminoguanidine (AG), an inhibitor of advanced glycation, prevents expression of the profibrotic cytokine, connective tissue growth factor (CTGF), as well as accumulation of the previously reported CTGF-dependent matrix protein, fibronectin, in a model of experimental diabetic nephropathy. Diabetic animals were randomly allocated into groups receiving 32 wk of AG or vehicle. Diabetic rats showed increases in CTGF mRNA and protein expression as well as in advanced glycation end-product (AGE) and fibronectin immunostaining, compared with nondiabetic rats. In the diabetic kidney, the increase in CTGF gene and protein expression as well as expression of the extracellular matrix protein fibronectin were prevented by AG. To further explore the relationship between AGEs and mesangial CTGF and fibronectin production, cultured human mesangial cells were exposed in vitro to soluble AGE-BSA and carboxymethyl lysine-BSA, and this led to induction of both CTGF and fibronectin. On the basis of our in vitro findings in mesangial cells linking AGEs to CTGF expression, the known prosclerotic effects of CTGF, and the ability of AG to attenuate mesangial expansion, it is postulated that the antifibrotic effects of AG in this animal model may be partially mediated by CTGF.

Reduction of the accumulation of advanced glycation end products by ACE inhibition in experimental diabetic nephropathy.

The effect of ACE inhibition on the formation of advanced glycation end products (AGEs) and oxidative stress was explored. Streptozocin-induced diabetic animals were randomized to no treatment, the ACE inhibitor ramipril (3 mg/l), or the AGE formation inhibitor aminoguanidine (1 g/l) and followed for 12 weeks. Control groups were followed concurrently. Renal AGE accumulation, as determined by immunohistochemistry and both serum and renal fluorescence, were increased in diabetic animals. This was attenuated by both ramipril and aminoguanidine to a similar degree. Nitrotyrosine, a marker of protein oxidation, also followed a similar pattern. The receptor for AGEs, gene expression of the membrane-bound NADPH oxidase subunit gp91phox, and nuclear transcription factor-kappaB were all increased by diabetes but remained unaffected by either treatment regimen. Two other AGE receptors, AGE R2 and AGE R3, remained unchanged for the duration of the study. The present study has identified a relationship between the renin-angiotensin system and the accumulation of AGEs in experimental diabetic nephropathy that may be linked through oxidative stress

Prevention of diabetes-induced albuminuria in transgenic rats overexpressing human aldose reductase.

Studies using pharmacologic inhibitors have implicated the enzyme aldose reductase in the pathogenesis of albuminuria and diabetic renal disease. However, a clear conclusion is not easily drawn from such studies since these pharmacologic inhibitors have nonspecific properties. To examine further the role of aldose reductase, we have overexpressed the human enzyme in a transgenic rat model. Transgene expression in the kidney was predominantly localized to the outer stripe of the outer medulla, compatible with the histotopography of the straight (S3) proximal tubule. The effect of enzyme overexpression on diabetes-induced renal function and structure was then investigated. Contrary to what may have been anticipated from the previous enzyme inhibition studies, diabetes-induced albuminuria was completely prevented by the overexpression of aldose reductase. No effect of overexpression of aldose reductase on renal structure nor on urinary excretion of beta2-microglobulin and N-acetyl-beta-D-glucosaminidase was observed in this transgenic rat model. In conclusion, our study strongly suggests that multiple roles for aldose reductase may give it a more complex place in diabetic nephropathy than is currently recognized.

Nephrin expression in the post-natal developing kidney in normotensive and hypertensive rats.

Nephrin is a slit diaphragm protein and its expression in the developing kidney is largely unknown. In this study, we explored the expression of nephrin in the developmental kidney in spontaneously hypertensive (SHR) and in Wistar-Kyoto (WKY) rats at different time points, from day 5 after birth to adulthood. Real time RT-PCR, in situ hybridization and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin in the kidney. SHR had hypertension at week 10 and albuminuria at week 20. Nephrin expression in both SHR and WKY increased from day 5 to adulthood. Furthermore, both gene and protein expression of nephrin were significantly lower in SHR after birth when compared to WKY at the same age. These findings suggest that both in normotensive and hypertensive rats, nephrin expression increased from birth to the adult age and that down-regulation of nephrin in SHR evident from the early developmental kidney to adulthood may contribute to the development of albuminuria in adult SHR.

Prevention of accelerated atherosclerosis by angiotensin-converting enzyme inhibition in diabetic apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis is a major complication of diabetes, but the mechanisms by which diabetes promotes macrovascular disease have not been fully delineated. Although several animal studies have demonstrated that inhibition of ACE results in a decrease in the development of atherosclerotic lesions, information about the potential benefits of these agents on complex and advanced atherosclerotic lesions as observed in long-term diabetes is lacking. The aim of this study was to evaluate whether treatment with the ACE inhibitor perindopril affects diabetes-induced plaque formation in the apolipoprotein E (apoE)-deficient mouse. METHODS AND RESULTS: Diabetes was induced by injection of streptozotocin in 6-week-old apoE-deficient mice. Diabetic animals received treatment with perindopril (4 mg x kg(-1) x d(-1)) or no treatment for 20 weeks. Nondiabetic apoE-deficient mice were used as controls. Induction of diabetes was associated with a 4-fold increase in plaque area compared with nondiabetic animals. This accelerated atherosclerosis was associated with a significant increase in aortic ACE expression and activity and connective tissue growth factor and vascular cell adhesion molecule-1 expression. Perindopril treatment inhibited the development of atherosclerotic lesions and diabetes-induced ACE, connective tissue growth factor, and vascular cell adhesion molecule-1 overexpression in the aorta. CONCLUSIONS: The activation of the local renin-angiotensin system in the diabetic aorta and the reduction in atherosclerosis with ACE inhibitor treatment provides further evidence that the renin-angiotensin system plays a pivotal role in the development and acceleration of atherosclerosis in diabetes.

Angiotensin type 2 receptor antagonism confers renal protection in a rat model of progressive renal injury.

The role of the angiotensin type 2 (AT(2)) receptor in the pathogenesis of progressive renal injury has not been previously elucidated. The renal expression of the AT(1) and AT(2) receptors in subtotally nephrectomized rats (STNx) and the effects of AT(2) receptor blockade on renal injury were explored. Reduced renal expression of the AT(1) but not the AT(2) receptor was observed in STNx by reverse transcription-PCR, by in vitro autoradiography, and by immunohistochemical staining. The STNx rats were randomly assigned to AT(1) receptor antagonist valsartan, AT(2) receptor antagonist PD123319, or the combination of both for 4 wk. Increased proteinuria in STNx rats was reduced by PD123319 but to a lesser degree when compared with valsartan. Reduced gene and protein expression of the slit diaphragm protein nephrin was prevented by either valsartan or PD123319. Expression of osteopontin, proliferating cell nuclear antigen, and monocyte/macrophage infiltration was increased in STNx rats and was reduced by both AT(1) and AT(2) receptor antagonists. These effects of AT(2) receptor antagonism were observed in the presence of increased BP in STNx rats. These findings suggest that blockade of the AT(2) receptor alone confers a degree of renal protection; in particular, it seems that the combination of the AT(1) and AT(2) receptor antagonists may confer additive renal effects than either receptor antagonist as monotherapy.