Darren J. Kelly

The (Pro)Renin Receptor Site-Specific and Functional Linkage to the Vacuolar H+-ATPase in the Kidney

The (pro)renin receptor ([P]RR) is a transmembrane protein that binds both renin and prorenin with high affinity, increasing the catalytic cleavage of angiotensinogen and signaling intracellularly through mitogen-activated protein kinase activation. Although initially reported as having no homology with any known membrane protein, other studies have suggested that the (P)RR is an accessory protein, named ATP6ap2, that associates with the vacuolar H+-ATPase, a key mediator of final urinary acidification. Using in situ hybridization, immunohistochemistry, and electron microscopy, together with serial sections stained with nephron segment–specific markers, we found that (P)RR mRNA and protein were predominantly expressed in collecting ducts and in the distal nephron. Within collecting ducts, the (P)RR was most abundant in microvilli at the apical surface of A-type intercalated cells. Dual-staining immunofluorescence demonstrated colocalization of the (P)RR with the B1/2 subunit of the vacuolar H+-ATPase, the ion exchanger that secretes H+ ions into the urinary space and that associates with an accessory subunit homologous to the (P)RR. In collecting duct/distal tubule lineage Madin-Darby canine kidney cells, extracellular signal–regulated kinase 1/2 phosphorylation, induced by either renin or prorenin, was attenuated by the selective vacuolar H+-ATPase inhibitor bafilomycin. The predominant expression of the (P)RR at the apex of acid-secreting cells in the collecting duct, along with its colocalization and homology with an accessory protein of the vacuolar H+-ATPase, suggests that the (P)RR may function primarily in distal nephron H+ transport, recently noted to be, at least in part, an angiotensin II–dependent phenomenon.

Retinal Neovascularization Is Prevented by Blockade of the Renin-Angiotensin System

Both angiotensin II and vascular endothelial growth factor are angiogenic agents that have recently been implicated in the pathogenesis of proliferative diabetic retinopathy. In this study, retinal neovascularization was examined in a model of retinopathy of prematurity with the use of neonatal transgenic (mRen-2)27 rats, which overexpress renin in tissues, and Sprague-Dawley rats. Blockers of the renin-angiotensin system were administered during the neovascularization period. The ACE inhibitor lisinopril and the angiotensin type 1 receptor antagonist losartan both increased retinal renin levels and prevented inner retinal blood vessel growth. Quantitative in situ hybridization revealed that the expression of vascular endothelial growth factor and its type 2 receptor in the inner retina and proliferating blood vessels were increased in rats with retinopathy of prematurity. Lisinopril reduced both retinal vascular endothelial growth factor and its type 2 receptor mRNA in retinopathy of prematurity rats, whereas losartan had no effect. It is predicted that agents that interrupt the renin-angiotensin system may play an important role as retinoprotective agents in various forms of proliferative retinopathy.

Direct Actions of Urotensin II on the Heart. Implications for Cardiac Fibrosis and Hypertrophy

Abstract— Urotensin II (UII) is a somatostatin-like peptide recently identified as a potent vasoconstrictor. In this study, we examined whether UII promotes cardiac remodeling through nonhemodynamic effects on the myocardium. In a rat model of heart failure after myocardial infarction (MI), increased UII peptide and UII receptor protein expression was observed in both infarct and noninfarct regions of the left ventricle compared with sham. Moreover, post-MI remodeling was associated with a significant 75% increase in UII receptor gene expression in the heart (P <0.05 versus sham controls), with this increase noted in both regions of the left ventricle. In vitro, UII (10−7 mol/L) stimulation of neonatal cardiac fibroblasts increased the level of mRNA transcripts for procollagens &agr;1(I), &agr;1(III), and fibronectin by 139±15% (P <0.01), 59±5% (P <0.05), and 141±14% (P <0.01), respectively, with a concomitant 23±2% increase in collagen peptide synthesis as determined by 3H-proline incorporation (P <0.01). UII had no effect on cellular hypertrophy, as determined by changes in total protein content in isolated neonatal cardiomyocytes. However, expression of recombinant rat UII receptor in neonatal cardiomyocytes resulted in significant UII-dependent activation of hypertrophic signaling as demonstrated by increased total protein content (unstimulated, 122.4±4.0 &mgr;g/well; rat UII, 147.6±7.0 &mgr;g/well; P <0.01) and activation of the hypertrophic phenotype through G&agr;q- and Ras-dependent pathways. These results indicate that, in addition to potent hemodynamic effects, UII may be implicated in myocardial fibrogenesis through increased collagen synthesis by cardiac fibroblasts and may also be an important determinant of pathological cardiac hypertrophy in conditions characterized by UII receptor upregulation.

Angiotensin converting enzyme inhibition reduces retinal overexpression of vascular endothelial growth factor and hyperpermeability in experimental diabetes.

Aims/hypothesis. Angiotensin converting enzyme (ACE) inhibition has been recently suggested to have retinoprotective actions in diabetic patients but the mechanism of this effect is not known. In vitro, angiotensin II stimulates expression of vascular endothelial growth factor (VEGF), a permeability-inducing and endothelial cell specific angiogenic factor which has been implicated in the pathogenesis of diabetic retinopathy in humans and in experimental animals. We sought to determine the effects of ACE inhibition on retinal VEGF expression and permeability in experimental diabetic retinopathy.¶Methods. Streptozotocin-induced diabetic rats and control animals were assigned at random to receive ACE inhibitor treatment or vehicle. At 24 weeks the retinal VEGF protein gene expression was assessed by northern blot analysis and in situ hybridisation. Retinal permeability to albumin was measured using a double isotope technique.¶Results. Experimental diabetes was associated with cell specific two to fourfold increase in retinal VEGF protein gene expression (p < 0.01) and a 2-fold increase in retinal vascular permeability to albumin (p < 0.01). The localization of VEGF expression in the retina was not altered in animals with experimental diabetes. Angiotensin converting enzyme inhibitor treatment of diabetic rats reduced diabetes-associated changes in VEGF gene expression and vascular permeability.¶Conclusion/interpretation. These findings implicate the renin-angiotensin system in the VEGF overexpression and hyperpermeability which accompany diabetic retinopathy and provide a potential mechanism for the beneficial effects of ACE inhibition in diabetic retinal disease. [Diabetologia (2000) 43: 1360–1367]

Does indoxyl sulfate, a uraemic toxin, have direct effects on cardiac fibroblasts and myocytes?

AIMS Indoxyl sulfate (IS) is a uraemic toxin found at high concentration in patients with chronic kidney disease (CKD) co-morbid with chronic heart failure (CHF). The aim of this study was to determine direct effects of IS on cardiac cells as well as the pro-inflammatory effect of IS. METHODS AND RESULTS Indoxyl sulfate significantly increased neonatal rat cardiac fibroblast collagen synthesis (by 145.7% vs. control, P < 0.05) and myocyte hypertrophy (by 134.5% vs. control, P < 0.001) as determined by (3)H-proline or (3)H-leucine incorporation, respectively. Indoxyl sulfate stimulated tumour necrosis factor-alpha, interleukin-6 (IL-6), and IL-1beta mRNA expression in THP-1 cells as quantified by RT-PCR. Both p38 (RWJ-67657) and MEK1/2 (U0126) inhibitors suppressed all these effects by IS. Furthermore, western blot analysis showed that IS activated mitogen-activated protein kinase (MAPK) (p38, p42/44) and nuclear factor-kappa B (NFkappaB) pathways. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that IS exerted its effects without affecting cell viability. CONCLUSION This study has, for the first time, demonstrated that IS has pro-fibrotic, pro-hypertrophic, and pro-inflammatory effects, indicating that IS might play an important role in adverse cardiac remodelling mediated via activation of the p38 MAPK, p42/44 MAPK, and NFkappaB pathways. Targeting reduction of IS and/or the pathways it activates may represent a novel therapeutic approach to the management of CHF with concomitant CKD.

Obesity results in progressive atrial structural and electrical remodeling: implications for atrial fibrillation

BACKGROUND Obesity is associated with atrial fibrillation (AF); however, the mechanisms by which it induces AF are unknown. OBJECTIVE To examine the effect of progressive weight gain on the substrate for AF. METHODS Thirty sheep were studied at baseline, 4 months, and 8 months, following a high-calorie diet. Ten sheep were sampled at each time point for cardiac magnetic resonance imaging and hemodynamic studies. High-density multisite biatrial epicardial mapping was used to quantify effective refractory period, conduction velocity, and conduction heterogeneity index at 4 pacing cycle lengths and AF inducibility. Histology was performed for atrial fibrosis, inflammation, and intramyocardial lipidosis, and molecular analysis was performed for endothelin-A and -B receptors, endothelin-1 peptide, platelet-derived growth factor, transforming growth factor β1, and connective tissue growth factor. RESULTS Increasing weight was associated with increasing left atrial volume (P = .01), fibrosis (P = .02), inflammatory infiltrates (P = .01), and lipidosis (P = .02). While there was no change in the effective refractory period (P = .2), there was a decrease in conduction velocity (P<.001), increase in conduction heterogeneity index (P<.001), and increase in inducible (P = .001) and spontaneous (P = .001) AF. There was an increase in atrial cardiomyocyte endothelin-A and -B receptors (P = .001) and endothelin-1 (P = .03) with an increase in adiposity. In association, there was a significant increase in atrial interstitial and cytoplasmic transforming growth factor β1 (P = .02) and platelet-derived growth factor (P = .02) levels. CONCLUSIONS Obesity is associated with atrial electrostructural remodeling. With progressive obesity, there were changes in atrial size, conduction, histology, and expression of profibrotic mediators. These changes were associated with spontaneous and more persistent AF.

Pathological Expression of Renin and Angiotensin II in the Renal Tubule after Subtotal Nephrectomy: Implications for the Pathogenesis of Tubulointerstitial Fibrosis

The finding that the systemic renin-angiotensin system (RAS) is not activated in most types of chronic renal disease has led to the suggestion that a local, intrarenal RAS may be an important determinant in the relentless progression of renal disease. Therefore, cell specific changes in various components of the RAS in response to renal mass reduction and angiotensin converting enzyme (ACE) inhibition were examined. Thirty Sprague-Dawley rats were randomly assigned to sham surgery, subtotal nephrectomy (STNx) alone or STNx treated with the ACE inhibitor, perindopril, and sacrificed after 12 weeks. In sham rats, renin mRNA and protein were only present in the juxtaglomerular apparatus. In contrast, in STNx kidneys, renin and angiotensin II expression were noted predominantly in renal tubular epithelial cells in association with overexpression of the prosclerotic cytokine, transforming growth factor-beta1 (TGF-beta1). In perindopril-treated STNx rats expression of renin and TGF-beta1 were similar to control animals. These finding indicate that following renal mass reduction there is pathological tubular expression of various components of the RAS. Furthermore, in contrast to the juxtaglomerular apparatus, tubular renin expression was reduced with ACE inhibition. These changes within the intrarenal RAS may be pathogenetically linked to the development of tubulointerstitial injury.

Podocyte foot process broadening in experimental diabetic nephropathy: amelioration with renin-angiotensin blockade

Aims/hypothesis. Changes in podocyte number and morphology have been implicated in the pathogenesis of proteinuria and the progression of human and experimental kidney disease. This study sought to examine podocyte foot process and slit pore architecture in experimental diabetic nephropathy and to determine whether such changes were modified with renoprotective intervention by blockade of the renin-angiotensin system. Methods. The number of filtration slits per 100 μm of glomerular basement membrane was assessed by transmission electron microscopy and quantitated histomorphometrically in control animals and in rats with 24 weeks of streptozotocin-induced diabetes. Diabetic rats were either untreated or received the angiotensin converting enzyme inhibitor ramipril, or the angiotensin II type 1 receptor antagonist, valsartan. Results. When compared with control animals, diabetes was associated with a decrease in the number of slit pores per unit length of glomerular basement membrane, indicative of podocyte foot process broadening. Both ramipril and valsartan attenuated these ultrastructural changes to a similar degree. These differences remained after correcting for glomerular volume as a possible confounding variable. Conclusion/interpretation. Preservation of podocyte architecture could contribute to the renoprotective effects of renin-angiotensin system blockade in diabetic nephropathy. [Diabetologia (2001) 44: 878–882]

Effect of angiotensin II type 1 receptor blockade on experimental hepatic fibrogenesis.

BACKGROUND/AIMS The aim of this study was to investigate whether in the liver, as in other tissues, there is evidence that angiotensin II, acting via the angiotensin II type 1 receptor (AT1-R), plays a role in fibrogenesis. METHODS Sprague-Dawley rats were divided into three groups; sham, bile duct ligated (BDL) and BDL + AT1-R antagonist, irbesartan. Real time RT-PCR was utilised to assess gene expression of the AT1 receptor, TGF-beta1 and alpha1 (I) collagen in the liver. TGF-beta1 and alpha1 (I) collagen mRNA expression and localisation were also assessed by in situ hybridisation. TGF-beta1 activity was assessed by using the TGF-beta inducible gene product betaig-h3. Fibrosis was assessed by the Knodell scoring system, tissue hydroxyproline content and picro-sirius red staining. RESULTS Real time RT-PCR revealed that there was a 6-fold up-regulation in AT1 receptor expression in BDL animals compared with shams. This was associated with marked increases in TGF-beta1, betaig-h3 and alpha1 (I) collagen gene expression which were attenuated by AT1-RA treatment. However, AT1-RA therapy produced no significant change in liver histology or hydroxyproline content. CONCLUSIONS These results suggest that in the liver angiotensin II may play an important role in the fibrogenic response to injury. However, whether treatment with an AT1-RA will be of therapeutic benefit remains to be determined.

Role of VEGF in maintaining renal structure and function under normotensive and hypertensive conditions

Inhibiting the actions of VEGF is a new therapeutic paradigm in cancer management with antiangiogenic therapy also under intensive investigation in a range of nonmalignant diseases characterized by pathological angiogenesis. However, the effects of VEGF inhibition on organs that constitutively express it in adulthood, such as the kidney, are mostly unknown. Accordingly, we examined the effect of VEGF inhibition on renal structure and function under physiological conditions and in the setting of the common renal stressors: hypertension and activation of the renin–angiotensin system. When compared with normotensive Sprague–Dawley (SD) rats, glomerular VEGF mRNA was increased 2-fold in transgenic (mRen-2)27 rats that overexpress renin with spontaneously hypertensive rat (SHR) kidneys showing VEGF expression levels that were intermediate between them. Administration of either an orally active inhibitor of the type 2 VEGF receptor (VEGFR-2) tyrosine kinase or a VEGF neutralizing antibody to TGR(mRen-2)27 rats resulted in loss of glomerular endothelial cells and transformation to a malignant hypertensive phenotype with severe glomerulosclerosis. VEGFR-2 kinase inhibition treatment was well tolerated in SDs and SHRs; although even in these animals there was detectable endothelial cell loss and rise in albuminuria. Mild mesangial expansion was also noted in hypertensive SHR, but not in SD rats. These studies illustrate: (i) VEGF has a role in the maintenance of glomerular endothelial integrity under physiological circumstances, (ii) glomerular VEGF is increased in response to hypertension and activation of the renin–angiotensin system, and (iii) VEGF signaling plays a protective role in the setting of these renal stressors.