Expression of components of the renin-angiotensin system in autosomal recessive polycystic kidney disease.

Hypertension is a common complication in children with autosomal recessive polycystic kidney disease (ARPKD) who have survived the neonatal period. No information is available regarding the mechanism of hypertension in this condition. The renin-angiotensin system (RAS) is thought to play a role in hypertension associated with the more common autosomal dominant polycystic kidney disease (ADPKD). Occasional reports have documented increased activity of the intrarenal RAS in ADPKD, with ectopic renin expression within cysts and dilated tubules. Because of similarities between ARPKD and ADPKD, we hypothesized that increased intrarenal RAS activity might also be found in ARPKD. We performed immunohistochemical studies on kidney tissues from two infants with ARPKD and two control kidneys. The cystic dilated tubules showed staining with the peanut lectin arachis hypogaea, a marker of distal tubules and collecting ducts, but not with lotus tetragonolobus, a marker of proximal tubules. Strong renin staining was seen in many cysts and tubules of ARPKD kidneys, but only in the afferent arterioles of the normal control kidneys. Angiotensinogen staining was also observed in some cysts and in proximal tubules. Staining for angiotensin-converting enzyme, angiotensin II type 1 receptor, and angiotensin II peptide was present in many cystic dilated tubules. These immunohistochemical studies document for the first time ectopic expression of components of the RAS in cystic-dilated tubules of ARPKD and suggest that overactivity of RAS could result in increased intrarenal angiotensin II production, which may contribute to the development of hypertension in ARPKD.     

Renal functional responses to selective intrarenal renin inhibition in Cyp1a1-Ren2 transgenic rats with ANG II-dependent malignant hypertension

Angiotensin (ANG) II-dependent hypertension is characterized by increases in intrarenal ANG II levels, derangement in renal hemodynamics, and augmented tubular sodium reabsorptive capability. Increased nephron expression of renin-angiotensin system components, such as angiotensinogen by proximal tubule cells and renin by collecting duct principal cells, has been associated with an augmented ability of the kidney to form ANG II in hypertensive states. However, the contribution of de novo intrarenal ANG II production to the development and maintenance of ANG II-dependent hypertension remains unclear. The present study was performed to determine the effects of selective intrarenal renin inhibition on whole kidney hemodynamics and renal excretory function in Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension in the absence of the confounding influence of associated reductions in mean arterial pressure (MAP). Male Cyp1a1-Ren2 transgenic rats were induced to develop malignant hypertension, anesthetized, and surgically prepared for intrarenal administration of the direct renin inhibitor aliskiren (0.01 mg/kg). Following acute aliskiren treatment, urine flow and sodium excretion increased (10.5 ± 1.1 to 15.9 ± 1.9 μl/min, P < 0.001; 550 ± 160 to 1,370 ± 320 neq/min, P < 0.001, respectively) and ANG II excretion decreased (120 ± 30 to 63 ± 17 fmol/h, P < 0.05). There were no significant changes in MAP, glomerular filtration rate, estimated renal plasma flow, plasma ANG II levels, or protein excretion. The present findings demonstrate that selective renal renin inhibition elicits diuretic and natriuretic responses in Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension. Elevated intraluminal ANG II levels likely act to augment tubular reabsorptive function and, thereby, contribute to the elevated blood pressure in Cyp1a1-Ren2 rats with ANG II-dependent malignant hypertension.     

Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H⁺-ATPases and alternative functions in H⁺-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H⁺-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H⁺-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H⁺-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H⁺-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H⁺-ATPases. In mice treated with NH₄Cl, NaHCO₃, KHCO₃, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H⁺-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH₄Cl or NaHCO₃ treated mice demonstrated always colocalization of PRR/Atp6ap2 with H⁺-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H⁺-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H⁺-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H⁺-ATPase activity in intercalated cells.     

Increased renin excretion is associated with augmented urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats

Renin expression in principal cells of collecting ducts (CD) is upregulated in angiotensin II (ANG II)-dependent hypertensive rats; however, it remains unclear whether increased CD-derived renin undergoes tubular secretion. Accordingly, urinary levels of renin (uRen), angiotensinogen (uAGT), and ANG II (uANG II) were measured in chronic ANG II-infused Sprague-Dawley rats (80 ng/min for 14 days, n = 10) and sham-operated rats (n = 10). Systolic blood pressure increased in the ANG II rats by day 5 and continued to increase throughout the study (day 13; ANG II: 175 ± 10 vs. sham: 116 ± 2 mmHg; P < 0.05). ANG II infusion increased renal cortical and medullary ANG II levels (cortical ANG II: 606 ± 72 vs. 247 ± 43 fmol/g; P < 0.05; medullary ANG II: 2,066 ± 116 vs. 646 ± 36 fmol/g; P < 0.05). Although plasma renin activity (PRA) was suppressed in the ANG II-infused rats (0.3 ± 0.2 vs. 5.5 ± 1.8 ng ANG I·ml(-1)·h(-1); P < 0.05), renin content in renal medulla was increased (12,605 ± 1,343 vs. 7,956 ± 765 ng ANG I·h(-1)·mg(-1); P < 0.05). Excretion of uAGT and uANG II increased in the ANG II rats [uAGT: 1,107 ± 106 vs. 60 ± 26 ng/day; P < 0.0001; uANG II: 3,813 ± 431 vs. 2,080 ± 361 fmol/day; P < 0.05]. By day 13, despite suppression of PRA, urinary prorenin content increased in ANG II rats [15.7 ± 3 vs. 2.6 ± 1 × 10(-3) enzyme units excreted (EUE)/day, P < 0.01] as was the excretion rate of renin (8.6 ± 2 × 10(-6) EUE/day) compared with sham (2.8 ± 1 × 10(-6) EUE/day; P < 0.05). Urinary renin and prorenin protein levels examined by Western blot were augmented ～10-fold in the ANG II-infused rats. Concomitant AT(1) receptor blockade with candesartan prevented the increase. Thus, in ANG II-dependent hypertensive rats with marked PRA suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid. The greater availability of renin and AGT in the urine reflects the capability for intratubular ANG II formation which stimulates sodium reabsorption in distal nephron segments.     

Indoxyl Sulfate-Induced Activation of (Pro)renin Receptor Promotes Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells

Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs.

Conclusion

IS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.     

Novel expression and regulation of the renin-angiotensin system in metanephric organ culture

To evaluate the presence and regulation of the renin-angiotensin system (RAS) in metanephric organ culture, embryonic day 14 (E14) rat metanephroi were cultured for 6 days. mRNAs for renin and both ANG II receptors (AT(1) and AT(2)) are expressed at E14, and all three genes continue to be expressed in culture. Renin mRNA is localized to developing tubules and ureteral branches in the cultured explants. At E14, renin immunostaining is found in isolated cells scattered within the mesenchyme. As differentiation progresses, renin localizes to the ureteric epithelium, developing tubules and glomeruli. E14 metanephroi contain ANG II, and peptide production persists in culture. Renin activity is present at E14 (6.13 +/- 0.61 pg ANG I. kidney(-1). h(-1)) and in cultured explants (28.84 +/- 1. 13 pg ANG I. kidney(-1). h(-1)). Renin activity in explants is increased by ANG II treatment (70.1 +/- 6.36 vs. 40.97 +/- 1.94 pg ANG I. kidney(-1). h(-1) in control). This increase is prevented by AT(1) blockade, whereas AT(2) antagonism has no effect. These studies document an operational local RAS and a previously undescribed positive-feedback mechanism for renin generation in avascular, cultured developing metanephroi. This novel expression pattern and regulatory mechanism highlight the unique ability of developing renal cells to express an active RAS.     

New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT₁/AT₂) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT₄/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT₄/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT₁ receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.     

Neuronal nitric oxide synthase and cyclooxygenase-2 in diabetic nephropathy of type 2 diabetic OLETF rats.

Neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) regulate the tubuloglomerular feedback (TGF) and renin-angiotensin system (RAS) in the kidney. In type 1 diabetic rats, renal overproduction of these enzymes and their relationship to the pathogenesis of diabetic nephropathy has been demonstrated. In the present study, we histologically and immunohistochemically investigated the kidneys of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as a model of type 2 diabetes, at 62 weeks of age (chronic phase of diabetes). The kidneys of OLETF rats showed typical diabetic nephropathy. Quantitative scores for glomerulosclerosis and interstitial fibrosis in OLETF rats were significantly higher than those of age-matched control Long-Evans Tokushima Otsuka (LETO) rats. nNOS- and COX-2-positive immunoreactions were observed in the distal tubules and collecting ducts. These reactions appeared to be more widely distributed in OLETF, and the number of nNOS-and COX-2-positive sites in the OLETF were significantly more than those in LETO rats. Expression of renin, angiotensin II, and inducible nitric oxide synthase (iNOS) were also examined immunohistochemically, and no differences between OLETF and LETO rats were observed in the distributions and the number of immunoreactive-sites. In conclusion, the overproduction of nNOS and COX-2 in the kidney of OLETF rats was confirmed, suggesting that the overproduction of nNOS and/or COX-2 does not affect the intrarenal RAS or iNOS production but does affect TGF.     

Reciprocal changes in renal ACE/ANG II and ACE2/ANG 1-7 are associated with enhanced collecting duct renin in Goldblatt hypertensive rats

Alterations in the balance between ANG II/ACE and ANG 1-7/ACE2 in ANG II-dependent hypertension could reduce the generation of ANG 1-7 and contribute further to increased intrarenal ANG II. Upregulation of collecting duct (CD) renin may lead to increased ANG II formation during ANG II-dependent hypertension, thus contributing to this imbalance. We measured ANG I, ANG II, and ANG 1-7 contents, angiotensin-converting enzyme (ACE) and ACE2 gene expression, and renin activity in the renal cortex and medulla in the clipped kidneys (CK) and nonclipped kidneys (NCK) of 2K1C rats. After 3 wk of unilateral renal clipping, systolic blood pressure and plasma renin activity increased in 2K1C rats (n = 11) compared with sham rats (n = 9). Renal medullary angiotensin peptide levels were increased in 2K1C rats [ANG I: (CK = 171 ± 4; NCK = 251 ± 8 vs. sham = 55 ± 3 pg/g protein; P < 0.05); ANG II: (CK = 558 ± 79; NCK = 328 ± 18 vs. sham = 94 ± 7 pg/g protein; P < 0.001)]; and ANG 1-7 levels decreased (CK = 18 ± 2; NCK = 19 ± 2 pg/g vs. sham = 63 ± 10 pg/g; P < 0.001). In renal medullas of both kidneys of 2K1C rats, ACE mRNA levels and activity increased but ACE2 decreased. In further studies, we compared renal ACE and ACE2 mRNA levels and their activities from chronic ANG II-infused (n = 6) and sham-operated rats (n = 5). Although the ACE mRNA levels did not differ between ANG II rats and sham rats, the ANG II rats exhibited greater ACE activity and reduced ACE2 mRNA levels and activity. Renal medullary renin activity was similar in the CK and NCK of 2K1C rats but higher compared with sham. Thus, the differential regulation of ACE and ACE2 along with the upregulation of CD renin in both the CK and NCK in 2K1C hypertensive rats indicates that they are independent of perfusion pressure and contribute to the altered content of intrarenal ANG II and ANG 1-7.     

Immunoreactive renin and angiotensin II in the afferent glomerular arterioles of rats with hypertension due to unilateral renal artery constriction

Summary The immunoreactivity for renin and angiotensin II (ANG II) in the ischaemic and non-ischaemic kidney of rats with renovascular hypertension was compared with that of the kidneys of sham operated controls. In addition, the renin concentration of these kidneys and the plasma level of ANG II were determined in hypertensive and control animals. In parallel with the renin concentration of kidney cortex, the immunoreactivity, i.e. the JG-index for renin of the afferent arterioles from the ischaemic kidney was slighly increased, that from the nonischaemic kidney drastically decreased as compared to control kidneys. Similarly, the JG-index for ANG II was increased in the ischaemic and decreased in the non-ischaemic kidney although the plasma level of ANG II was elevated in the animals with renovascular hypertension. This difference in the immunocytochemically detectable ANG II and especially the decrease of ANG II in the non-ischaemic kidney in spite of elevated plasma ANG II levels is interpreted to result from similar differences in the local (extravascular) formation of ANG II by the intrarenal renin-angiotensin system.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

(Pro)renin promotes fibrosis gene expression in HEK cells through a Nox4-dependent mechanism

The (pro)renin receptor (PRR) has recently been demonstrated to bind equally well renin and its precursor, prorenin, leading to a similar intracellular signaling independent of angiotensin II. In this study, we report that human embryonic kidney cells (HEK) exposed to renin or prorenin for 24 h in the presence of a blocking concentration of the angtiotensin-converting enzyme inhibitor perindoprilate increased superoxide anion production as measured by luminescence (lucigenin) and electron spin resonance spectroscopy (hydroxylamine radical transition). Also, both renin and prorenin increased Nox4 expression while Nox2, p47(phox), and p67(phox) remained unchanged. In an investigation of the effects of renin and prorenin on fibrosis genes, it appeared that both proteins stimulated transforming growth factor-β (TGF-β), fibronectin, and plasminogen activator inhibitor type 1 (PAI-1) expression and therefore participated to an overall switch toward a profibrotic state of the kidney cells. When the cells were transfected with a siRNA targeting the PRR, Nox4 expression was efficiently prevented as well as the increase in superoxide production, TGF-β, fibronectin, and PAI-1. Finally, we demonstrated that transfection of the cells with a Nox4-specific small interfering (si) RNA also prevented fibrosis gene expression following treatment with renin or prorenin. The results demonstrate that renin and prorenin, through their specific membrane receptor and independently of angiotensin II, promote fibrosis gene expression via a Nox4-dependent mechanism.     

Fetal uninephrectomy in male sheep alters the systemic and renal responses to angiotensin II infusion and AT1R blockade

Fetal uninephrectomy (uni-x) at 100 days of gestation results in compensatory nephrogenesis in the remaining kidney, resulting in a 30% reduction in total nephron number in male sheep. Recently, we showed that uni-x males at 6 mo of age have elevated arterial pressure, reduced renal blood flow (RBF), glomerular filtration rate (GFR), and low plasma renin levels (Singh R, Denton K, Bertram J, Jefferies A, Head G, Lombardo P, Schneider-Kolsky M, Moritz K. J Hypertens 27: 386-396, 2009; Singh R, Denton K, Jefferies A, Bertram J, Moritz K. Clin Sci (Lond) 118: 669-680, 2010). We hypothesized this was due to upregulation of the intrarenal renin-angiotensin system (RAS). In this study, renal responses to ANG II infusion and ANG II type 1 receptor (AT1R) blockade were examined in the same 6-mo-old male sheep. Uni-x animals had reduced levels of renal tissue and plasma renin and ANG II. Renal gene expression of renin, and gene and protein levels of AT1R and AT2R, were significantly lower in uni-x animals. In response to graded ANG II infusion, sham animals had the expected decrease in conscious RBF and GFR. Interestingly, the response was biphasic in uni-x sheep, with GFR initially decreasing, but then increasing at higher ANG II doses (34 ± 7%; P(group × treatment) < 0.001), due to a paradoxical decrease in renal vascular resistance (P(group × treatment) < 0.001). In response to AT1R blockade, while GFR and RBF responded similarly between groups, there was a marked increase in sodium excretion in uni-x compared with sham sheep (209 ± 35 vs. 25 ± 12%; P < 0.001). In conclusion, in 6-mo-old male sheep born with a single kidney, these studies demonstrate that this is a low-renin form of hypertension, in which responses to ANG II are perturbed and the intrarenal RAS is downregulated.     

Intrarenal transfer of an intracellular fluorescent fusion of angiotensin II selectively in proximal tubules increases blood pressure in rats and mice

The present study tested the hypothesis that intrarenal adenoviral transfer of an intracellular cyan fluorescent fusion of angiotensin II (ECFP/ANG II) selectively in proximal tubules of the kidney increases blood pressure by activating AT(1) (AT(1a)) receptors. Intrarenal transfer of ECFP/ANG II was induced in the superficial cortex of rat and mouse kidneys, and the sodium and glucose cotransporter 2 (sglt2) promoter was used to drive ECFP/ANG II expression selectively in proximal tubules. Intrarenal transfer of ECFP/ANG II induced a time-dependent, proximal tubule-selective expression of ECFP/ANG II in the cortex, which peaked at 2 wk and was sustained for 4 wk. ECFP/ANG II expression was low in the glomeruli and the entire medulla and was absent in the contralateral kidney or extrarenal tissues. At its peak of expression in proximal tubules at day 14, ANG II was increased by twofold in the kidney (P < 0.01) and more than threefold in proximal tubules (P < 0.01), but remained unchanged in plasma or urine. Systolic blood pressure was increased in ECFP/ANG II-transferred rats by 28 ± 6 mmHg (P < 0.01), whereas fractional sodium excretion was decreased by 20% (P < 0.01) and fractional lithium excretion was reduced by 24% (P < 0.01). These effects were blocked by losartan and prevented in AT(1a) knockout mice. Transfer of a scrambled ECFP/ANG IIc had no effects on blood pressure, kidney, and proximal tubule ANG II, or sodium excretion. These results provide evidence that proximal tubule-selective transfer of an intracellular ANG II fusion protein increases blood pressure by activating AT(1a) receptors and increasing sodium reabsorption in proximal tubules.     

The effect of hypothalamo-pituitary disconnection on the renin-angiotensin system in the late-gestation fetal sheep

The activity of the renin-angiotensin system (RAS) increases significantly in the late-gestation fetal sheep. Fetal cortisol is also increased during this time, and it is thought that the increase in cortisol may modulate the RAS changes. Previous studies have examined the effects of cortisol infusion on RAS activity, but the effects of blocking the peripartum increase in cortisol concentrations on the developmental changes in the RAS are not known. Therefore, we utilized the technique of hypothalamic-pituitary disconnection (HPD), which prevents the cortisol surge from occurring, to investigate the importance of the late-gestation increase in cortisol on the ontogenic changes in RAS activity. HPD of fetal sheep was performed at 120 days of gestational age (dGA), and fetuses were delivered between 135 and 139 dGA. Control fetuses were sham operated. HPD blocked the late-gestation cortisol increase but did not alter renal renin mRNA, renal renin or prorenin protein content, nor plasma renin levels compared with sham operated. However, HPD fetuses had increased ANG II receptor subtype 1 (AT1) mRNA and protein expression in the kidney and lungs. ANG II receptor subtype 2 (AT2) expression was not altered in these tissues at either mRNA or protein level. HPD did not change AT1 or AT2 mRNA in the left ventricle but did result in decreased protein levels for both receptors. These studies demonstrate that blockade of the naturally occurring increase in fetal cortisol concentration in late gestation is associated with tissue-specific alterations in expression of AT1 and AT2 receptors. These changes may impact on fetal tissue maturation and hence have consequences in postnatal life.     

Vascular and tubular renin in the kidneys of mice

Summary Monospecific antisera and the immunocytochemical PAP-method have been used to localize renin in the kidneys of mice. With this procedure, reaction product was not only observed in the epitheloid cells of kidney vessels but also in kidney tubules: in the apical portion of proximal tubule cells and in some cells of the connecting and the cortical collecting tubule. To answer the question, whether the occurrence of renin in kidney tubule cells is the consequence of tubular synthesis or that of glomerular filtration of plasma renin followed by its uptake from the tubular lumen, tracer experiments with radioiodinated renin and with horseradish peroxidase were undertaken. The results of these studies as well as other arguments suggest reabsorptive pinocytosis of the filtered hormone as the source of tubular renin.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Receptor-dependent prorenin activation and induction of PAI-1 expression in vascular smooth muscle cells

Although elevated plasma prorenin levels are commonly found in diabetic patients and correlate with microvascular complications, the pathological role of these increases, if any, remains unclear. Prorenin/renin binding to the prorenin/renin receptor [(p)RR] enhances the efficiency of angiotensinogen cleavage by renin and unmasks prorenin catalytic activity. We asked whether plasma prorenin could be activated in local vascular tissue through receptor binding. Immunohistochemical staining showing localization of the (p)RR in the aorta to vascular smooth muscle cells (VSMCs). After cultured rat VSMCs were incubated with 10(-7) M inactive prorenin, cultured supernatant acquired the ability to generate ANG I from angiotensinogen, indicating that prorenin had been activated. Activated prorenin facilitated angiotensin generation in cultured VSMCs when exogenous angiotensinogen was added. Small interfering RNA (siRNA) against the (p)RR blocked this activation and subsequent angiotensin generation. Prorenin alone induced dose- and time-dependent increases in mRNA and protein for the profibrotic molecule plasminogen activator inhibitor (PAI)-1, effects that were blocked by siRNA, but not by the ANG II receptor antagonist saralasin. When inactive prorenin and angiotensinogen were incubated with cells, PAI-1 mRNA increased a striking 54-fold, 8-fold higher than the increase seen with prorenin alone. PAI-1 protein increased 2.75-fold. These effects were blocked by treatment with siRNA + saralasin. We conclude that prorenin at high concentration binds the (p)RR on VSMCs and is activated. This activation leads to increased expression of PAI-1 via ANG II-independent and -dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may contribute to the progression of fibrotic disease.     

Defective nitric oxide production impairs angiotensin II-induced Na-K-ATPase regulation in spontaneously hypertensive rats

Angiotensin (ANG) II via ANG II type 1 receptors (AT1R) activates renal sodium transporters including Na-K-ATPase and regulates sodium homeostasis and blood pressure. It is reported that at a high concentration, ANG II either inhibits or fails to stimulate Na-K-ATPase. However, the mechanisms for these phenomena are not clear. Here, we identified the signaling molecules involved in regulation of renal proximal tubular Na-K-ATPase at high ANG II concentrations. Proximal tubules from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were incubated with low concentrations of ANG II (pM), which activated Na-K-ATPase in both the groups; however, the stimulation was more robust in SHR. A high concentration of ANG II (μM) failed to stimulate Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) continued to stimulate Na-K-ATPase, which was sensitive to the AT1R antagonist candesartan. In the presence of N(G)-nitro-l-arginine methyl ester (l-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, ANG II (μM) caused stimulation of Na-K-ATPase in proximal tubules of WKY rats while having no further stimulatory effect in SHR. ANG II (μM), via AT1R, increased proximal tubular NO levels in WKY rats but not in SHR. In SHR, NOS was uncoupled as incubation of proximal tubules with ANG II and l-arginine, a NOS substrate, caused superoxide generation only in SHR and not in WKY rats. The superoxide production in SHR was sensitive to l-NAME. There was exaggerated proximal tubular AT1R-G protein coupling and NAD(P)H oxidase activation in response to ANG II (μM) in proximal tubules of SHR compared with WKY rats. In SHR, inhibition of NADPH oxidase restored NOS coupling and ANG II-induced NO accumulation. In conclusion, at a high concentration ANG II (μM) activates renal NO signaling, which prevents stimulation of Na-K-ATPase in WKY rats. However, in SHR ANG II (μM) overstimulates NADPH oxidase, which impairs the NO system and leads to continued Na-K-ATPase activation.     

Renin released from mast cells activated by circulating MCP-1 initiates the microvascular phase of the systemic inflammation of alveolar hypoxia

Reduced alveolar Po(2) in rats produces a rapid systemic inflammation characterized by reactive O(2) species generation, mast cell (MC) degranulation, leukocyte-endothelial interactions, and increased vascular permeability. The inflammation is not initiated by the low systemic Po(2) but rather by the release of monocyte chemoattractant protein-1 (MCP-1) from alveolar macrophages (AMO) activated by alveolar hypoxia. Circulating AMO-borne MCP-1 induces MC degranulation, which activates the local renin-angiotensin system (RAS) and mediates the microvascular inflammation. This study was directed to determine the mechanism of RAS activation by MCP-1-induced MC degranulation. Experiments in isolated rat peritoneal MCs showed the following: 1) Western blots and immunocytochemistry demonstrated the presence of renin and angiotensin-converting enzyme (ACE) in MCs and their release upon degranulation; 2) MCP-1-induced degranulation of MCs incubated in plasma produced an increase in angiotensin II (ANG II) concentration; and 3) this increase was inhibited completely by the following agents: the MCP-1 receptor antagonist RS-102895, the specific rat renin inhibitor WFML, or the ACE inhibitor captopril administered separately. Captopril also inhibited ANG II generation by MCs incubated in culture medium plus ANG I. The results show that peritoneal MCs contain active renin, which activates the RAS upon degranulation, and that peritoneal MCs are a source of ACE and suggest that conversion of ANG I to ANG II is mediated predominantly by ACE. This study provides novel evidence of the presence of active renin in rat peritoneal MCs and helps explain the mechanism of activation of the RAS during alveolar hypoxia.     

Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-β(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.