Renal Rats Research Articles

Abstract P104: Kidney arteriolar responses to liver-targeted small interference RNA (siRNA) targeting angiotensinogen in diabetic rats: comparison with other renin-angiotensin system blockers

Objective: Inhibition of the renin-angiotensin system (RAS) with ACE inhibitors (ACEi) or angiotensin receptor blockers (ARB) is associated with concentric thickening of renal arteries and arterioles and parenchymal disease. Here we evaluated to what degree this also occurs during RAS inhibition with siRNA targeting hepatic angiotensinogen (AGT). Methods: Male hypertensive heterozygous transgenic rats overexpressing mouse renin (TGR(mRen2)27 rats), made diabetic with streptozotocin, were treated with the ARB valsartan (4 mg/kg/day), the ACEi captopril (6 mg/kg/day), AGT siRNA (30 mg/kg every 2 weeks), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks, starting at 15 weeks after induction of diabetes. After 3 weeks of treatment, kidneys were removed, and immunocytochemistry was performed on Bouin’s fixed kidney sections using antibodies against renin or α-smooth muscle actin. Juxtaglomerular index and wall thickness were assessed using tiling pictures of both renin and α-smooth muscle actin sections from the slides. Tubular degeneration was scored by summing up scores for tubular atrophy, interstitial fibrosis, and tubulointerstitial inflammation. Concentric vascular hypertrophy was assessed in a blinded manner. Results: All treatments increased juxtaglomerular index, and the largest increases were observed in dual treatment groups, consistent with the highest degree of RAS blockade in these groups. Tubular scores increased after induction of diabetes, and the highest scores were seen in dual treatment groups, significance versus vehicle being reached in the valsartan + captopril group only (P<0.05). Afferent arteriole wall thickness increased in all treatment groups, and the greatest increases were observed during dual treatment. Finally, concentric vascular hypertrophy was observed in 1 of 7 (14%) diabetic Ren2 rats; this percentage increased during RAS blocker treatment of these rats, numerically reaching the highest values during dual treatment. Conclusions: Our data suggest that, in the hypertensive diabetic rat model, high levels of RAS blockade, irrespective of the type of blockade, may result in an increase in afferent arteriole thickness and concentric vascular hypertrophy. AGT siRNA in combination with RAS inhibitors should be further explored in clinical studies.

O52 KIDNEY ARTERIOLAR RESPONSES TO LIVER-TARGETED SMALL INTERFERENCE RNA (SIRNA) TARGETING ANGIOTENSINOGEN IN DIABETIC RATS: COMPARISON WITH OTHER RENIN-ANGIOTENSIN SYSTEM BLOCKERS

Objective: Inhibition of the renin-angiotensin system (RAS) with ACE inhibitors (ACEi) or angiotensin receptor blockers (ARB) is associated with concentric thickening of renal arteries and arterioles and parenchymal disease. Here we evaluated to what degree this also occurs during RAS inhibition with siRNA targeting hepatic angiotensinogen (AGT). Methods: Male hypertensive heterozygous transgenic rats overexpressing mouse renin (TGR(mRen2)27 rats), made diabetic with streptozotocin, were treated with the ARB valsartan (4 mg/kg/day), the ACEi captopril (6 mg/kg/day), AGT siRNA (30 mg/kg every 2 weeks), AGT siRNA + valsartan, or captopril+valsartan for 3 weeks, starting at 15 weeks after induction of diabetes. After 3 weeks of treatment, kidneys were removed, and immunocytochemistry was performed on Bouin's fixed kidney sections using antibodies against renin or α-smooth muscle actin. Juxtaglomerular index and wall thickness were assessed using tiling pictures of both renin and α-smooth muscle actin sections from the slides. Tubular degeneration was scored by summing up scores for tubular atrophy, interstitial fibrosis, and tubulointerstitial inflammation. Concentric vascular hypertrophy was assessed in a blinded manner. Results: All treatments increased juxtaglomerular index, and the largest increases were observed in dual treatment groups, consistent with the highest degree of RAS blockade in these groups. Tubular scores increased after induction of diabetes, and the highest scores were seen in dual treatment groups, significance versus vehicle being reached in the valsartan+captopril group only (P<0.05). Afferent arteriole wall thickness increased in all treatment groups, and the greatest increases were observed during dual treatment. Finally, concentric vascular hypertrophy was observed in 1 of 7 diabetic Ren2 rats; this percentage increased during RAS blocker treatment of these rats, numerically reaching the highest values during dual treatment. Conclusions: Our data suggest that, in the hypertensive diabetic rat model, high levels of RAS blockade, irrespective of the type of blockade, may result in an increase in afferent arteriole thickness and concentric vascular hypertrophy. AGT siRNA in combination with RAS inhibitors should be further explored in clinical studies.

Phosphate Toxicity and Vascular Calcification in Chronic Kidney Disease: A Closer Look Utilizing Transmission Electron Microscopy.

Hyperphosphatemia is independently linked with vascular calcification, cardiovascular disease, bone-mineral disease, progression of renal insufficiency, and all-cause mortality in chronic kidney disease (CKD) and end-stage renal disease (ESRD). The emerging importance of fibroblast growth factor-23 (FGF-23) and its co-factor Klotho play very important roles as phosphaturic hormones; however, phosphate levels rise due to a loss of renal Klotho production and the phosphaturic effects of the FGF-23/Klotho axis. Hyperphosphatemia is also associated with calciphylaxis, acceleration of renal tubulointerstitial disease, renal osteodystrophy, and uremic cardiomyopathy. This review incorporates ultrastructural remodeling of the thoracic aorta to provide a different perspective on vascular calcification. Nine-week-old male heterozygous (mRen2) 27 (Ren2) rat models of hypertension, insulin resistance, vascular oxidative stress and albuminuria are utilized to demonstrate aortic remodeling associated with vascular calcification. Nine-week-old male Zucker obese (fa/fa) rat models are utilized to better understand nephrolith formation. Phosphate homeostasis, toxicity, multiple metabolic and uremic toxicities, renal osteodystrophy, and vascular calcification are also discussed. Additionally, the role of the endothelium, vascular smooth muscle cells, inflammatory monocytes/macrophages and mast cells, pericytes, oxidative stress, hydrogen sulfide, and extraosseous calcification in the kidney are discussed as they relate to CKD, ESRD and calciphylaxis.

PS-BPB06-8: THE NON-STEROIDAL MINERALOCORTICOID RECEPTOR ANTAGONIST, FINERENONE, REDUCES VASCULAR PATHOLOGY AND INFLAMMATION IN PRE-CLINICAL DIABETIC RETINOPATHY

Objective: Diabetic retinopathy is the leading cause of vision loss in the working population across the globe. Diabetes has devastating effects on the retinal vasculature, and can lead to vision-threatening vascular leakage, odema, and neovascularization. Current treatments such as anti-vascular endothelial growth factor (VEGF) are administered directly into the eye late in the disease process but are ineffective in about 50% of patients. Hypertension is a risk factor for the development of diabetic retinopathy and the renin-angiotensin aldosterone system contributes to disease pathogenesis. Here we aimed to determine if finerenone, a non-steroidal mineralocorticoid receptor (MR) antagonist, reduced vascular pathology and inflammation in animal models of diabetic retinopathy. Methods: To study diabetic retinopathy, streptozotocin diabetes was induced in female hypertensive transgenic Ren-2 rats overexpressing renin in extra-renal tissues. Diabetic Ren-2 rats were randomised to receive finerenone (10 mg/kg/day) by oral gavage or the angiotensin converting enzyme inhibitor perindopril (10 mg/kg/day) in drinking water for 12 weeks. As retinal neovascularization does not develop in diabetic rodents, the oxygen-induced retinopathy (OIR) model was studied. Litters of C57BL/6J mice were exposed to high oxygen (75%) between postnatal days 7 to 12 and then room air until postnatal day 18. Mouse pups were randomised to be OIR controls, or treated with finerenone (5 mg/kg/day) by intraperitoneal injection between postnatal days 7 to 18. Comparisons were made to mice exposed to room air. Damage to the blood-retinal barrier was evaluated by measuring the gliosis of macroglial Müller cells which confer barrier properties, vascular leakage (albumin ELISA), neovascularization, and the expression of VEGF. Inflammation was evaluated by the quantitation of retinal microglia and the expression of pro-inflammatory mediators. Results: Systolic blood pressure was similar between untreated non-diabetic (174.2 ± 6.3 mmHG) and diabetic (184.2 ± 8.5 mmHG) Ren-2 rats. Finerenone (138.5 ± 5.01 mmHG, P < 0.0001) and perindopril (106 ± 2.9 mmHG, P < 0.0001) reduced systolic blood pressure compared to untreated diabetic rats. In diabetic rats, finerenone reduced the gliosis of retinal Müller cells, vascular leakage, microglial density and the expression of VEGF, ICAM-1 and IL-1β;. In diabetic rats, perindopril did not reduce retinal VEGF levels, microglial density and the expression of pro-inflammatory mediators. In OIR mice, finerenone reduced retinal neovascularization, vascular leakage and microglial density. Conclusion: Our findings placed in the context of the FIDELIO-DKD trial reporting the benefits of finerenone on renal and cardiovascular outcomes in diabetic kidney disease, indicate the potential of finerenone to be an effective orally administered treatment for diabetic retinopathy.

The Mighty Mitochondria Are Unifying Organelles and Metabolic Hubs in Multiple Organs of Obesity, Insulin Resistance, Metabolic Syndrome, and Type 2 Diabetes: An Observational Ultrastructure Study.

Mitochondria (Mt) are essential cellular organelles for the production of energy and thermogenesis. Mt also serve a host of functions in addition to energy production, which include cell signaling, metabolism, cell death, and aging. Due to the central role of Mt in metabolism as metabolic hubs, there has been renewed interest in how Mt impact metabolic pathways and multiple pathologies. This review shares multiple observational ultrastructural findings in multiple cells and organs to depict aberrant mitochondrial (aMt) remodeling in pre-clinical rodent models. Further, it is intended to show how remodeling of Mt are associated with obesity, insulin resistance, metabolic syndrome (MetS), and type 2 diabetes mellitus (T2DM). Specifically, Mt remodeling in hypertensive and insulin-resistant lean models (Ren2 rat models), lean mice with streptozotocin-induced diabetes, obesity models including diet-induced obesity, genetic leptin-deficient ob/ob, and leptin receptor-deficient db/db diabetic mice are examined. Indeed, aMt dysfunction and damage have been implicated in multiple pathogenic diseases. Manipulation of Mt such as the induction of Mt biogenesis coupled with improvement of mitophagy machinery may be helpful to remove leaky damaged aMt in order to prevent the complications associated with the generation of superoxide-derived reactive oxygen species and the subsequent reactive species interactome. A better understanding of Mt remodeling may help to unlock many of the mysteries in obesity, insulin resistance, MetS, T2DM, and the associated complications of diabetic end-organ disease.

Angiotensin II and aldosterone activate retinal microglia

Microglial cells are important contributors to the neuroinflammation and blood vessel damage that occurs in ischemic retinopathies. We hypothesized that key effectors of the renin-angiotensin aldosterone system, angiotensin II (Ang II) and aldosterone, increase the density of microglia in the retina and stimulate their production of reactive oxygen species (ROS) as well as pro-angiogenic and pro-inflammatory factors. Two animal models were studied that featured up-regulation of Ang II or aldosterone and included transgenic Ren-2 rats which overexpress renin and Ang II in tissues including the retina, and Sprague Dawley rats with ischemic retinopathy and infused with aldosterone. Complementary studies were performed in primary cultures of retinal microglia from neonatal Sprague Dawley rats exposed to hypoxia (0.5% O2) and inhibitors of the angiotensin type 1 receptor (valsartan), the mineralocorticoid receptor (spironolactone) or aldosterone synthase (FAD286). In both in vivo models, the density of ionized calcium-binding adaptor protein-1 labelled microglia/macrophages was increased in retina compared to genetic or vehicle controls. In primary cultures of retinal microglia, hypoxia increased ROS (superoxide) levels as well as the expression of the NADPH oxidase (NOX) isoforms, NOX1, NOX2 and NOX4. The elevated levels of ROS as well as NOX2 and NOX4 were reduced by all of the treatments, and valsartan and FAD286 also reduced NOX1 mRNA levels. A protein cytokine array of retinal microglia revealed that valsartan, spironolactone and FAD286 reduced the hypoxia-induced increase in the potent pro-angiogenic and pro-inflammatory agent, vascular endothelial growth factor as well as the inflammatory factors, CCL5 and interferon γ. Valsartan also reduced the hypoxia-induced increase in IL-6 and TIMP-1 as well as the chemoattractants, CXCL2, CXCL3, CXCL5 and CXCL10. Spironolactone and FAD286 reduced the levels of CXCL2 and CXCL10, respectively. In conclusion, our findings that both Ang II and aldosterone influence the activation of retinal microglia implicates the renin-angiotensin aldosterone system in the pathogenesis of ischemic retinopathies.

Ultrastructure study of the transgenic REN2 rat aorta – part 2: media, external elastic lamina, and adventitia

Background The renin-angiotensin-aldosterone system (RAAS) plays an important role in the development and progression of vascular stiffness, hypertension and accelerated atherosclerosis, which are associated with the metabolic syndrome (MetS) and type 2 diabetes mellitus. In addition to the intima, RAAS plays an important role in vascular media and adventitial remodeling. Methods Descending thoracic aortas of young male transgenic heterozygous (mRen2) 27 (Ren2) rats were utilized for ultrastructural study. This lean model of hypertension, insulin resistance, and oxidative stress harbors the mouse renin gene and is known to have increased aortic tissue levels of angiotensin II, angiotensin type 1 receptors, and elevated plasma aldosterone levels. Results Ultrastructural observations substantiate known and novel findings in the tunica media, internal and external elastic lamina, and tunica adventitia, which includes: increased media collagen - proteoglycan matrix expansion, increased secretory and proliferative activity and migration of vascular smooth muscle cells (VSMCs) into a newly developing subendothelial neointima, increased VSMC caveolae, mitochondria degeneration, apoptosis; and lipid retention at the elastin lamellar interface. Openings in the external elastic lamina allow pericyte-to-VSMC contacts. The tunica adventitia exhibits stromal pericyte hyperplasia with actively synthetic phenotype and pericyte-pericyte connections. Conclusion While these studies only represent a single snapshot in time, they provide an evaluation of early abnormal ultrastructural vascular remodeling in Ren-2 models of the conduit-elastic thoracic aorta.

Exaggerated blood pressure response to fasudil or nifedipine in hypertensive Ren-2 transgenic rats: role of altered baroreflex.

Basal calcium sensitization is decreased in spontaneously hypertensive rats, although their blood pressure (BP) response to acute Rho-kinase inhibition is enhanced. Using fasudil (Rho-kinase inhibitor) or nifedipine (L-VDCC blocker), we evaluated the contribution of calcium sensitization and calcium entry to BP maintenance in hypertensive transgenic Ren-2 rats (TGR) focusing on the influence ofmajor vasoactive systems and/or baroreflex efficiency on BP responses to these two drugs. Homozygous TGR and normotensive Hannover Sprague-Dawley (HanSD) control rats aged 5, 11, or 22 weeks were used. The acute BP-lowering effects of fasudil or nifedipine were studied in intact rats, nitric oxide-deficient L-NAME-pretreated rats and rats subjected to combined blockade of the renin-angiotensin system (RAS), sympathetic nervous system (SNS) and nitric oxide synthase (NOS). Fasudil- or nifedipine-induced BP reduction increased during hypertension development in TGR. By contrast, the nifedipine-induced BP response decreased, whereas the fasudil-induced BP response increased with age inHanSD controls. Our data indicated a major contribution of nifedipine-sensitive calcium entry and relative attenuation of calcium sensitization in hypertensive rats compared with normotensive controls. The BP responses to fasudil or nifedipine were enhanced by NOS inhibition and combined blockade in normotensive HanSD rats but not in hypertensive TGR. In conclusion, calcium sensitization is attenuated by endogenous nitric oxide in normotensive HanSD rats but not in hypertensive TGR. Moreover, BP reduction elicited by acute Rho-kinase inhibition is partially compensated by enhanced sympathetic vasoconstriction. The decreased compensation in hypertensive rats with impaired baroreflex efficiency explains their greater BP response to fasudil than in normotensive animals.

Breeding Characteristics and Dose-dependent Blood Pressure Responses of Transgenic Cyp1a1-Ren2 Rats.

Hypertension is a leading risk factor for cardiovascular and chronic kidney disease. A new rodent model (transgenic male Cyp1a1-Ren2 rats) provides reversible induction of hypertension through the addition of indole-3-carbinol (I3C) to the diet, without the need for surgical intervention, thus giving researchers control over both the onset of hypertension and its magnitude (I3C dose-dependency). We here report the breeding performance and productivity of Cyp1a1-Ren2 rats. Despite being transgenic, these animals proved to be efficient breeders. In addition to confirming inducible and reversible dose-dependent hypertension (by using I3C doses of 0.125%, 0.167%, and 0.25% [w/w] in the diet for 14 d, followed by normal chow for 4 d), we demonstrated that hypertension can be sustained chronically (14 wk) by continuous dosing with I3C (0.167% [w/w]) in the diet. In chronically dosed male rats, systolic blood pressure continued to rise, from 173 ± 11 mm Hg after 1 mo to 196 ± 19 mm Hg after 3 mo, with no adverse phenotypic features observed. In conclusion, Cyp1a1-Ren2 rats are a useful animal model to investigate hypertension-induced end-organ damage and potential new therapeutic targets to manage hypertension.

Neprilysin inhibition and endothelin-1 elevation: Focus on the kidney

Increasing the degree of renin-angiotensin system (RAS) blockade by combining ≥2 RAS blockers marginally increases efficacy, but results in more side effects. Hence, interference with other systems is currently being investigated, like potentiation of natriuretic peptides with neprilysin inhibitors. However, the neprilysin inhibitor thiorphan was recently found to increase endothelin-1 when administered to TGR(mREN2)27 (Ren2) rats on top of RAS blockade. Here we investigated whether this effect is thiorphan-specific, by comparing the neprilysin inhibitors thiorphan and sacubitril, administered by osmotic minipumps at a low or high dose for 7 days, in Ren2 rats. Plasma and urinary levels of endothelin-1, atrial and brain natriuretic peptide (ANP, BNP) and their second messenger cyclic guanosine 3′5′ monophosphate (cGMP) were monitored. No significant differences were found in the plasma concentrations of endothelin-1, cGMP, ANP and BNP after treatment, although plasma ANP tended to be higher in the high-dose thiorphan treatment group and the low- and high-dose sacubitril treatment groups, compared with vehicle. Urinary endothelin-1 increased in the low-dose thiorphan and high-dose sacubitril groups, compared with baseline, although significance was reached for the former only. Urinary cGMP rose significantly in the high-dose sacubitril treatment group compared with baseline. Both urinary endothelin-1 and cGMP were significantly higher in the high-dose sacubitril group compared with the low-dose sacubitril group. In conclusion, endothelin-1 upregulation occurs with both thiorphan and sacubitril, and is particularly apparent in neprilysin-rich organs like the kidney. High renal neprilysin levels most likely also explain why sacubitril increased cGMP in urine only.

FT011, a Novel Cardiorenal Protective Drug, Reduces Inflammation, Gliosis and Vascular Injury in Rats with Diabetic Retinopathy.

Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.

Chronic bilateral renal denervation reduces cardiac hypertrophic remodelling but not β-adrenergic responsiveness in hypertensive type 1 diabetic rats.

What is the central question of this study? Can bilateral renal denervation, an effective antihypertensive treatment in clinical and experimental studies, improve cardiac β-adrenoceptor responsiveness in a diabetic model with underlying hypertension? What is the main finding and its importance? Bilateral renal denervation did not affect β-adrenergic responsiveness in the diabetic hypertensive rat heart, but denervation reduced the hypertension-induced concentric hypertrophic remodelling. This suggests that the positive haemodynamic changes induced by renal denervation are most likely to reflect an attenuation of sympathetic effects on the systemic vasculature and/or the renal function rather than direct sympathetic modulation of the heart. Bilateral renal denervation (BRD) has been shown to normalise blood pressure in clinical and experimental studies of hypertension by reducing systemic sympathetic output. This study determined the effect of BRD on cardiac β-adrenoceptor (AR) responsiveness in a diabetic model with underlying hypertension using the transgenic (mRen-2)27 rats. Bilateral renal denervation or sham surgeries were conducted repeatedly at 3, 6 and 9weeks in Ren-2 rats with or without streptozotocin (STZ)-induced diabetes (4×n=7); Sprague-Dawley rats (n=6) served as control animals. Cardiac function was determined in isolated hearts at 18weeks of age. Normalised left ventricular developed pressure and relaxation was recorded in response to incremental concentrations of the β-AR agonist isoprenaline (from 10-10 to 10-7m) or the β3 -AR agonist BRL37344 (from 10(-13) to 10(-6) m). Expression levels of β1 -AR were determined by Western blot. Both inotropic and lusitropic β-AR responsiveness was reduced in the hypertensive diabetic hearts, but these responses were unaltered after BRD. Expression levels of β1 -AR were increased after BRD (Sham, 0.85±0.11versus 1.01±0.05a.u.; BRD, 1.45±0.11versus 1.46±0.07a.u.; Ren-2versus Ren-2 STZ, P<0.05versus Sham). No effect of β3 -AR agonist stimulation with BRL37344 was observed. Interestingly, BRD increased left ventricular diastolic volume in both the Ren-2 and the Ren-2 STZ groups. Bilateral renal denervation did not restore the attenuated cardiac β-AR responsiveness in the diabetic hypertensive rats, but it reduced the extent of hypertension-induced concentric hypertrophic remodelling. Thus, the haemodynamic protection offered by renal denervation appears to reflect an attenuated sympathetic innervation of the systemic vasculature and/or kidney rather than a direct cardiac effect.

Pharmacological inhibition of galectin-3 protects against hypertensive nephropathy.

Galectin-3 activation is involved in the pathogenesis of renal damage and fibrogenesis. Limited data are available to suggest that galectin-3-targeted intervention is a potential therapeutic candidate for the prevention of chronic kidney disease. Homozygous TGR(mREN)27 (REN2) rats develop severe high blood pressure (BP) and hypertensive end-organ damage, including nephropathy and heart failure. Male REN2 rats were treated with N-acetyllactosamine [galectin-3 inhibitor (Gal3i)] for 6 wk; untreated REN2 and Sprague-Dawley rats served as controls. We measured cardiac function with echocardiogram and invasive hemodynamics before termination. BP and proteinuria were measured at baseline and at 3 and 6 wk. Plasma creatinine was determined at 6 wk. Renal damage was assessed for focal glomerular sclerosis, glomerular desmin expression, glomerular and interstitial macrophages, kidney injury molecule-1 expression, and α-smooth muscle actin expression. Inflammatory cytokines and extracellular matrix proteinases were quantified by quantitative real-time PCR. Systolic BP was higher in control REN2 rats, with no effect of Gal3i treatment. Plasma creatinine and proteinuria were significantly increased in control REN2 rats; Gal3i treatment reduced both. Renal damage (focal glomerular sclerosis, desmin, interstitial macrophages, kidney injury molecule-1, α-smooth muscle actin, collagen type I, and collagen type III) was also improved by Gal3i. All inflammatory markers (CD68, IL-68, galectin-3, and monocyte chemoattractant protein-1) were elevated in control REN2 rats and attenuated by Gal3i. Markers of extracellular matrix turnover were marginally altered in untreated REN2 rats compared with Sprague-Dawley rats. In conclusion, galectin-3 inhibition attenuated hypertensive nephropathy, as indicated by reduced proteinuria, improved renal function, and decreased renal damage. Drugs binding to galectin-3 may be therapeutic candidates for the prevention of chronic kidney disease.

High glucose induces Smad activation via the transcriptional coregulator p300 and contributes to cardiac fibrosis and hypertrophy.

BackgroundDespite advances in the treatment of heart failure, mortality remains high, particularly in individuals with diabetes. Activated transforming growth factor beta (TGF-β) contributes to the pathogenesis of the fibrotic interstitium observed in diabetic cardiomyopathy. We hypothesized that high glucose enhances the activity of the transcriptional co-activator p300, leading to the activation of TGF-β via acetylation of Smad2; and that by inhibiting p300, TGF-β activity will be reduced and heart failure prevented in a clinically relevant animal model of diabetic cardiomyopathy.Methodsp300 activity was assessed in H9c2 cardiomyoblasts under normal glucose (5.6 mmol/L—NG) and high glucose (25 mmol/L—HG) conditions. 3H-proline incorporation in cardiac fibroblasts was also assessed as a marker of collagen synthesis. The role of p300 activity in modifying TGF-β activity was investigated with a known p300 inhibitor, curcumin or p300 siRNA in vitro, and the functional effects of p300 inhibition were assessed using curcumin in a hemodynamically validated model of diabetic cardiomyopathy – the diabetic TG m(Ren-2)27 rat.ResultsIn vitro, H9c2 cells exposed to HG demonstrated increased p300 activity, Smad2 acetylation and increased TGF-β activity as assessed by Smad7 induction (all p < 0.05 c/w NG). Furthermore, HG induced 3H-proline incorporation as a marker of collagen synthesis (p < 0.05 c/w NG). p300 inhibition, using either siRNA or curcumin reduced p300 activity, Smad acetylation and TGF-β activity (all p < 0.05 c/w vehicle or scrambled siRNA). Furthermore, curcumin therapy reduced 3H-proline incorporation in HG and TGF-β stimulated fibroblasts (p < 0.05 c/w NG). To determine the functional significance of p300 inhibition, diabetic Ren-2 rats were randomized to receive curcumin or vehicle for 6 weeks. Curcumin treatment reduced cardiac hypertrophy, improved diastolic function and reduced extracellular matrix production, without affecting glycemic control, along with a reduction in TGF-β activity as assessed by Smad7 activation (all p < 0.05 c/w vehicle treated diabetic animals).ConclusionsThese findings suggest that high glucose increases the activity of the transcriptional co-regulator p300, which increases TGF-β activity via Smad2 acetylation. Modulation of p300 may be a novel strategy to treat diabetes induced heart failure.

Dual protein kinaseC alpha and beta inhibitors and diabetic kidney disease: a revisited therapeutic target for future clinical trials.

Diabetic kidney disease is the primary cause of chronic kidney disease worldwide, which can progress to end-stage renal disease that requires chronic dialysis therapy or renal transplantation1. An improved therapeutic strategy to combat diabetic kidney disease might include blocking the mechanisms by which diabetes leads to renal injury; for example, activation of protein kinase C (PKC). The PKC family comprises a group of related serine/threonine kinases that are ubiquitously expressed and participate in a variety of intracellular signaling pathways. The PKC family is divided into classical (α, β I, β II, γ), novel (δ, ε, η, θ) and atypical (ζ, ι/λ) isoforms on the basis of the biochemical properties of the isoforms2. In diabetics, PKC activity is upregulated in vascular tissue including the retina and the renal glomeruli. Of the 10 PKC isoforms, the α, β I, β II, δ, and ζ isoforms have been reported to be activated in glomeruli and renal cells exposed to high concentrations of glucose3, 4. In previous preclinical studies, we showed the beneficial effects of oral treatment with the selective PKCβ inhibitor, ruboxistaurin, on diabetic kidney and eye diseases.Treatment with ruboxistaurin improved albuminuria, glomerular filtration rate and retinal circulation in diabetic rats when administered orally for 2–8 weeks. In a longer study in the db/db mouse, treatment with ruboxistaurin ameliorated albuminuria and mesangial expansion by reducing the expression of transforming growth factor (TGF)-β, fibronectin and type IV collagen5. Subsequently, in a study in diabetic transgenic Ren-2 rats, inhibition of PKCβ with ruboxistaurin resulted in amelioration of albuminuria, structural injury and TGF-β expression, despite continued hyperglycemia and hypertension. In short-term clinical trials, ruboxistaurin was shown to be effective in the treatment of diabetic kidney disease and advanced retinopathy, consistent with preclinical studies. However, the results of long-term clinical studies in patients with diabetic eye disease have been disappointing, despite some modest effect on albuminuria6, and further clinical trials of ruboxistaurin or other PKC β inhibitors are therefore warranted. Although a number of researchers have implicated PKCβ activation in the development and progression of diabetic kidney disease, other studies have implicated PKCα as a major underlying mechanism of diabetes-induced albuminuria. Specifically for streptozotocin (STZ)-induced diabetes, Kang et al.7 showed activation of PKCα and ε isoforms in the kidney without significant increase in PKCβ isoforms, in contrast to our findings. Using PKCα and β knockout mice, Haller et al.8 showed that PKCβ activation was involved in transforming growth factor (TGF)-β1-mediated renal hypertrophy and extracellular matrix expansion, whereas PKCα activation mediated the expression of perlecan, vascular endothelial growth factor (VEGF) and nephrin, resulting in albuminuria. Similarly, King et al.9 presented a longer study in diabetic PKCβ knockout mice carried out over 24 weeks that showed reduced glomerular and renal hypertrophy, although only a modest reduction in albuminuria was observed. A recent report in Diabetes clearly showed that deletion of both PKCα and β isoforms inhibits the development of diabetic kidney disease in STZ-induced diabetic mice, although albuminuria was not completely prevented as compared with exclusively PKCα knockout diabetic mice9. As further evidence for these findings, pharmacological inhibition of PKCα and β with CGP41252, an agent utilized as the classical PKC inhibitor in several cancer trials, ameliorated albuminuria, but failed to significantly reduce renal hypertrophy in the STZ-induced 129/SV and the db/db mice. Interpretation of these findings implicated CGP41252 as a broad-PKC inhibitor as opposed to a specific inhibitor of PKCα and β. Such an agent might inhibit novel PKC isoforms, such as PKCε. Deletion of the PKCε signaling pathway induces glomerulosclerosis and tubulointerstitial fibrosis in vivo, suggesting a protective role against diabetic kidney disease10. Diabetic kidney disease continues to be a major complication of type 1 and type 2 diabetes, and represents the major cause of end-stage renal disease globally. There is an urgent need for new therapeutic drugs, although intensified blood glucose and blood pressure control with inhibition of the renin–angiotensin system are critical for reducing albuminuria, and preserving or slowing decline of renal function in diabetics. However, this new study highlights the need for further development of isoform-specific PKC inhibitors specifically targeting both PKCα and β action without inhibition of other PKC isoforms (Figure 1). Discovery of such inhibitors could have potential use in the future treatment of diabetic kidney disease. There is no conflict of interest.

Role of the eNOS-NO System in Regulating the Anti-Proteinuric Effects of VEGF Receptor 2 Inhibition in Diabetes

Since the original description over a decade ago of the upregulation of the VEGF/VEGFR-2 system in the diabetic kidney, there has been intense interest in the pathogenetic role of this system in renal decline and its amenability to therapeutic interventions. Recently, it has become apparent that subtle perturbations in intraglomerular VEGF/VEGFR-2 signalling or in the influencing microenvironment can profoundly affect renal function, resulting in the apparently paradoxical observation that VEGF blockade attenuates proteinuria development in experimental diabetes despite exerting the opposite effect under other circumstances. In the present study, we sought to explore the role of eNOS-NO activity in regulating the differential response to VEGF blockade in the diabetic and non-diabetic settings. In a rodent model of accelerated renal injury, the transgenic (mRen-2)27 (Ren-2) rat, VEGFR-2 inhibition with the small molecule vandetanib resulted in an increase in urine protein excretion preceding a subsequent rise in systolic blood pressure. When compared to their normoglycemic counterparts, diabetic Ren-2 rats exhibited an increase in the renal expression of eNOS and in urinary excretion of nitric oxide (NO) metabolites. In contrast to the heavy proteinuria observed with vandetanib in non-diabetic TGR(mRen-2)27 rats, VEGFR-2 inhibition reduced urine protein excretion in diabetic animals, despite a comparable magnitude of histological injury. However, proteinuria was markedly increased by concomitant treatment of diabetic Ren-2 rats with vandetanib and the nitric oxide synthase inhibitor L-NAME. These observations highlight the pivotal role that the eNOS-NO system plays in regulating the biological response to VEGF within the glomerulus especially under diabetic conditions.

Salt loading exacerbates diastolic dysfunction and cardiac remodeling in young female Ren2 rats

ObjectiveRecent data would suggest pre-menopausal insulin resistant women are more prone to diastolic dysfunction than men, yet it is unclear why. We and others have reported that transgenic (mRen2)27 (Ren2) rats overexpressing the murine renin transgene are insulin resistant due to oxidative stress in insulin sensitive tissues. As increased salt intake promotes inflammation and oxidative stress, we hypothesized that excess dietary salt would promote diastolic dysfunction in transgenic females under conditions of excess tissue Ang II and circulating aldosterone levels. Materials/methodsFor this purpose we evaluated cardiac function in young female Ren2 rats or age-matched Sprague–Dawley (SD) littermates exposed to a high (4%) salt or normal rat chow intake for three weeks. ResultsCompared to SD littermates, at 10weeks of age, female Ren2 rats fed normal chow showed elevations in left ventricular (LV) systolic pressures, LV and cardiomyocyte hypertrophy, and displayed reductions in LV initial filling rate accompanied by increases in 3-nitrotyrosine content as a marker of oxidant stress. Following 3weeks of a salt diet, female Ren2 rats exhibited no further changes in LV systolic pressure, insulin resistance, or markers of hypertrophy but exaggerated increases in type 1 collagen, 3-nitrotryosine content, and diastolic dysfunction. These findings occurred in parallel with ultrastructural findings of pericapillary fibrosis, increased LV remodeling, and mitochondrial biogenesis. ConclusionThese data suggest that a diet high in salt in hypertensive female Ren2 rats promotes greater oxidative stress, maladaptive LV remodeling, fibrosis, and associated diastolic dysfunction without further changes in LV systolic pressure or hypertrophy.

Enhanced CB1 Cannabinoid Receptor Tone Contributes to Impaired Baroreflex Sensitivity in Hypertensive (mRen2)27 Transgenic Rats

We demonstrated that activation of CB1 cannabinoid receptors within the solitary tract nucleus (NTS) of the brainstem via bilateral microinjection of the CB1 agonist CP55,940 modulates baroreflex sensitivity (BRS) for control of heart rate, an index of vagus nerve function, in a dose‐related biphasic manner. The CB1‐selective antagonist SR141716 (36 pmol) prevented or reversed these changes, and there was no effect of the antagonist alone. We now determine the effects of CB1 receptor blockade in (mRen2)27 (Ren2) rats, a model of angiotensin (Ang) II‐dependent hypertension with impaired baseline BRS. Unlike in Sprague‐Dawley (SD) rats, SR141716 dose‐dependently improved BRS when microinjected into the NTS of Ren2 rats. Microinjection of 0.36 pmol SR141716 (n = 5) increased BRS by 59% (p &lt; 0.001), while 36 pmol SR141716 (n = 5) increased BRS by 124% (p &lt; 0.001), to a level similar to baseline BRS of SD rats. There was no change in BRS by 1.2 pmol CP55,940 (n = 3) in Ren2 rats. Immunoblots revealed a trend for higher CB1 expression in brainstem of the Ren2 strain compared to SD rats. Thus, elevated endocannabinoid actions in the NTS contribute to impaired BRS in Ang II‐dependent hypertension. It is not yet known whether this results from elevated receptors or endogenous levels of the cannabinoids, but CB1 receptor blockade may positively influence BRS in the context of hypertension. Supported by P01‐HL051952 and R01‐DA03690.

Renin Inhibition and AT1R blockade improve metabolic signaling, oxidant stress and myocardial tissue remodeling

ObjectiveStrategies that block angiotensin II actions on its angiotensin type 1 receptor or inhibit actions of aldosterone have been shown to reduce myocardial hypertrophy and interstitial fibrosis in states of insulin resistance. Thereby, we sought to determine if combination of direct renin inhibition with angiotensin type 1 receptor blockade in vivo, through greater reductions in systolic blood pressure (SBP) and aldosterone would attenuate left ventricular hypertrophy and interstitial fibrosis to a greater extent than either intervention alone. Materials/MethodsWe utilized the transgenic Ren2 rat which manifests increased tissue expression of murine renin which, in turn, results in increased renin–angiotensin system activity, aldosterone secretion and insulin resistance. Ren2 rats were treated with aliskiren, valsartan, the combination (aliskiren+valsartan), or vehicle for 21days. ResultsCompared to Sprague–Dawley controls, Ren2 rats displayed increased systolic blood pressure, elevated serum aldosterone levels, cardiac tissue hypertrophy, interstitial fibrosis and ultrastructural remodeling. These biochemical and functional alterations were accompanied by increases in the NADPH oxidase subunit Nox2 and 3-nitrotyrosine content along with increases in mammalian target of rapamycin and reductions in protein kinase B phosphorylation. Combination therapy contributed to greater reductions in systolic blood pressure and serum aldosterone but did not result in greater improvement in metabolic signaling or markers of oxidative stress, fibrosis or hypertrophy beyond either intervention alone. ConclusionsThereby, our data suggest that the greater impact of combination therapy on reductions in aldosterone does not translate into greater reductions in myocardial fibrosis or hypertrophy in this transgenic model of tissue renin overexpression.

Suicidal erythrocyte death, eryptosis, as a novel mechanism in heart failure-associated anaemia

Suicidal death of erythrocytes (eryptosis) is characterized by cell shrinkage and exposure of phosphatidylserine (PS) residues at the cell surface. Excessive eryptosis may lead to anaemia. We aimed to study the role of eryptosis in heart failure (HF)-associated anaemia. We measured eryptosis in rodent models of HF. Typical measures of eryptosis including PS-exposure, increased intracellular Ca(2+) levels, and decreased cell volume were determined by flow cytometry. Transgenic REN2 rats displayed mild anaemia which was associated with a two-fold increase in erythrocyte PS-exposure when compared with Sprague Dawley (SD) control rats (P < 0.01). Upon stimulation with eryptotic triggers such as oxidative stress, hyperosmotic shock and energy depletion, eryptosis was more prominent in REN2 as shown by increased PS-exposure, cytosolic Ca(2+) influx, and cell shrinkage (P < 0.05 vs. SD). Increasing cytosolic Ca(2+) levels resulted in a stronger increase in PS-exposure in REN2 erythrocytes (P < 0.01 vs. SD). Accordingly, inhibition of Ca(2+) entry blunted the increased PS-exposure upon oxidative stress. The REN2 rats had significantly higher reticulocytes (REN2: 10.6 ± 2.3%; SD: 5.4 ± 0.1%; P < 0.05) and erythrocyte turnover was increased, indicated by increased clearance of eryptotic erythrocytes. Eryptosis was also increased in a rat model of hypertensive cardiac remodelling (uninephrectomized rats implanted with deoxycorticosterone acetate pellets), in mice after transverse aortic constriction, as well as in a small proof-of-concept study in human HF patients. Eryptosis is increased during HF development and could contribute to HF-associated anaemia. Eryptosis may therefore become a novel target for therapy in HF-associated anaemia.