Activation and measurement of plasma prorenin in the rat.

Prorenin determination in rat plasma has been problematic from the outset. Consequently, its existence is questioned by some and its quantity by others, making it difficult for knowledge to advance as to its function relative to the renin system. The present study examines major variables in the determination of rat plasma prorenin and renin, notably different prorenin activation protocols involving blood samples obtained under various conditions from animals under different anesthetics. We found that a trypsin activation step with 5 mg/mL plasma, 60 min at 23 degrees C, followed by a PRA step of 10 min at 37 degrees C, resulted in the highest prorenin estimates, up to approximately 400 ng.mL-1.h-1 in terms of angiotensin I, as compared with published values of 0-190, based on other protocols. These estimates were obtained despite considerable destruction of angiotensinogen (renin substrate) by trypsin. Cryoactivation of prorenin was much less effective than in human plasma but, when followed by trypsin, it facilitated greater activation than with trypsin alone. Comparable fresh and fresh-frozen plasmas had similar prorenin-renin values, but lower values were observed in plasmas that had been repeatedly frozen and thawed. Conscious rats and those anesthetized with Inactin or ether had higher renins and prorenins than those anesthetized with methoxyflurane or halothane. Rats with kidneys in place during blood collection had higher renins (but not prorenins) than those whose kidneys were clamped off, suggesting that last-minute renin release during blood collection had occurred. We conclude that (i) trypsin generates increased renin, or renin-like, activity in plasma, suggesting activation of a precursor; (ii) on this basis, high prorenin levels exist in normal rat plasma; (iii) renin and prorenin levels are variously influenced by different anesthetics and blood handling procedures; (iv) variation in prorenin levels suggests that it is a dynamic (functional?) component of the renin system; (v) prorenin measurements are heavily influenced by methodological variations during the trypsin step or the subsequent PRA step; (vi) using standardized methodology, the rat can serve as a model for investigating the function of prorenin in normotension and hypertension.     

Renin substrate (angiotensinogen) preparations in the determination of prorenin and renin: evidence for extrarenal plasma prorenin and its renal "convertase"

Standard methods for determining prorenin-renin concentrations in plasma (PRC) and other tissues require the addition of exogenous renin substrate (angiotensinogen) to improve the kinetics of the renin reaction. We studied the effects of substrate prepared from normal human plasma fraction Cohn IV-4, or from nephrectomized (2NX) sheep plasma, on PRC of normal and 2NX human plasmas before and after prorenin activation by acid, cold, and trypsin, and compared the results with plasma renin activities (PRA, no added substrate). Plasmas from 2NX men exhibited negligible basal PRA, indicating that very little, if any, renin had been formed from the extrarenal prorenin they contained, and suggesting the lack of an endogenous prorenin activating mechanism, or "convertase," of probable renal origin. Prorenin was demonstrable by tryptic activation, more than by acid or cold, at up to about 30% of normal. Addition of Cohn IV-4 substrate to 2NX plasma unexpectedly produced (i) a basal PRC value higher than in normal plasma, (ii) total renin values after activation by acid, cold, and trypsin that were much closer to normal values than reflected by PRA methodology, without a commensurate increase (if anything a decrease) in prorenin as a percentage of total renin estimated by all activation methods, and (iii) substantial equalization of activation effects such that trypsin was no longer more effective than acid and cold (and this was also noted with normal plasma). The skewing effect of adding Cohn IV-4 substrate on the PRC of 2NX plasma was much greater than in normal plasma, even though 2NX plasma already had an above normal level of endogenous substrate and should have been influenced less. Enhancement of PRC was very pronounced even when Cohn IV-4 was added to make up only 9% of total (endogenous + exogenous) substrate in the incubation system, suggesting that it was not the added substrate but a renin-generating contaminant that inflated the PRC. Such inflation could be blocked by adding protease inhibitors, suggesting that the responsible protease(s) acted as a prorenin "convertase" that generated new renin from renal and (or) extrarenal prorenin contributed by the added substrate, as well as by the plasma being assayed. One component of convertase could be kallikrein, which was identified by chromogenic assay, the importance of which relative to total convertase activity is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trypsin activation of human and cat prorenin: a comparative study.

Prorenin can be converted to renin by limited proteolysis with trypsin. In the current study we compared conditions for activation of human renal and ovarian prorenin and cat renal prorenin with either liquid-phase trypsin or trypsin bound to sepharose (solid phase). Higher concentrations of trypsin were required to activate cat prorenin than human prorenin. Human prorenin was destroyed by high concentrations of trypsin, while cat prorenin was not destroyed by up to 2 mg/mL solid-phase trypsin. For both human and cat prorenin, addition of the competitive serine protease inhibitor benzamidine--HCl increased the concentration of trypsin needed to activate prorenin, resulting in slightly higher levels of human prorenin but lower levels of cat prorenin. For human samples, activation with solid-phase trypsin resulted in slightly higher estimates of prorenin than liquid-phase trypsin. These results demonstrate species differences in the susceptibility of prorenin to trypsin cleavage. Cat prorenin requires more trypsin to be activated and is less susceptible to destruction than human prorenin.     

Evidence for beta-adrenergic regulation of renal and extrarenal plasma prorenin and renin in dogs

Beta blockade with propranolol for 7 days in healthy normotensive dogs produced a sustained 20-25% drop in heart rate, but only a transient suppression of blood pressure. Plasma renin activity and prorenin were also suppressed transiently, suggesting that both are under beta-receptor regulation. Bilateral nephrectomy (2NX) was followed by rapid clearance of renin from the circulation, at a rate that was minimally influenced by beta blockade. In contrast, the plasma prorenin level rose markedly to a peak within an hour after surgery, leveled off during the next 24 hr, dropped almost toward the pre-2NX baseline by 48 hr, but proceeded to rise again between 48 and 120 hr. Propranolol administration before and during the 2NX period reduced the detectable prorenin, suggesting that its extrarenal source is under beta-adrenergic regulation. The rapid increment of prorenin after 2NX suggests that extrarenal prorenin may have constituted part of the total plasma prorenin before 2NX, and/or had developed sufficiently quickly afterwards to replace and exceed the disappearing renal prorenin. Any fresh increment beyond 48 hr could presumably have been only extrarenal. These observations suggest the existence of a rich beta-regulated extrarenal source of prorenin capable of rapidly supplying the plasma. However, no renin-angiotesin was apparently produced from this prorenin in the nephrectomized state, implying the lack of renal "convertase," without which the prorenin convertase mechanism as a whole was rendered ineffective. The source of the extrarenal prorenin and the identity of the renal convertase remain to be established.     

N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells

Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.     

Prorenins activation by an enzyme from rat plasma (PreR-Co)

The aim of the present research was to explore the capacity of PreR-Co to process prorenin purified from kidney and corpora lutea (CL) and to study its action on extrarenal tissues. The PreR-Co was obtained from plasma as a single electrophoretic band by (NH4)2SO4 precipitation, gel filtration, anti-rat albumin immunoaffinity, and ion-exchange chromatography. Prorenin free of renin was obtained after (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography by a passage through an affinity gel of H-77 Sepharose. SDS-PAGE of supernatant and of acidic elution from gel, exhibited a single band of 43 kDa and 35 kDa, respectively; both recognized by the specific anti rat renin antibody. The isolated renin was not attacked by PreR-Co; on the contrary prorenin was completely activated. The product of PreR-Co-activated prorenin showed an analogous MW to that of renin and was recognized by the specific antibody. In addition to processing kidney prorenin, PreR-Co was able to cleave inactive renin from ovary, CL, uterus and adrenal gland homogenates. However, the amount of active renin generated from these tissues was lower than those produced by trypsin activation. PreR-Co is a good candidate for the role of the enzyme involved in tissues prorenin activation.     

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.     

The dominant role of the prosegment of prorenin in determining the rate of activation by acid or trypsin: studies with molecular chimeras

Human prorenin activation by acid or trypsin is faster than rat prorenin by two orders of magnitude. No plausible mechanism exists to explain the difference. Two chimeric mutant prorenins were produced in CHO cells. A chimera, hPro/rRen, composed of human prorenin prosegment and rat active renin segment, was activated as fast as wild-type human prorenin at pH 3.3 and 25 degrees C or by trypsin (1 microg/ml). The other chimera, rPro/hRen, composed of rat prorenin prosegment and human active renin segment, was activated as slowly as wild-type rat prorenin at pH 3.3 and 25 degrees C or by trypsin (50 microg/ml). These results indicate that the rate of activation of prorenin is predominantly determined by the N-terminal pro-sequence. Plausible mechanisms are discussed.     

Activation and function of prorenin: different viewpoints.

This Symposium includes 15 presentations and an editorial review dealing with prorenin activation and function. It comes 20 years after prorenin was first reported in various contexts and attracted attention because of its connection with renin--angiotensin, its high concentration relative to renin in the blood, and its presence in extrarenal, as well as renal, tissues. Intriguing changes in plasma prorenin have been reported after treatment with antihypertensive and other drugs, following various physiological stimuli, and in pathophysiological states such as Wilms' tumor, Bartter's syndrome, and diabetic nephropathy. Lately, very high prorenin concentrations have been found in human and animal ocular fluid, ovarian follicular fluid, and in association with angiogenesis and microangiopathy. High circulating prorenin concentrations and fulminant hypertension have been reported in rats harbouring the mouse Ren-2 gene. However, what prorenin does in all these extrarenal fluids, tissues, and conditions is not well understood. Among the reasons for this lack of understanding are the difficulties in measuring prorenin and in establishing good animal models. We have not answered the critical question as to whether prorenin itself is bioactive like a hormone, and if so, what its action(s) might be. Nor have we established the main alternative, i.e., whether the function of prorenin is indirect, through renin--angiotensin, be it in the circulation or in the extrarenal tissues. This Symposium provides only partial methodological advances and answers, but we hope it will stimulate the breakthrough work needed to supply more complete answers.     

Reversible cryoactivation of recombinant human prorenin.

Cleavage of prorenin's prosegment causes irreversible formation of renin. In contrast, renin activity is reversibly exposed when prorenin is acidified to pH 3.3. Nonetheless, acidification of plasma results in irreversible activation of prorenin, because endogenous proteases cleave the prosegment of acid-activated prorenin. Chilling of plasma results in irreversible cryoactivation of prorenin. In this study we investigated whether cryoactivation of purified prorenin is reversible. The intrinsic renin activity of recombinant human prorenin was measured by an enzyme kinetic assay using partially purified human angiotensinogen as substrate. Results are expressed as a percent (mean +/- S.E.) of the maximal activity exposed after limited proteolysis by trypsin. The intrinsic renin activity of two pools (0.3 and 0.06 Goldblatt units/ml) was 1.5% +/- 0.3 and 1.2% +/- 0.6 at 37 degrees C. Activity increased to 19% +/- 0.3 and 26% +/- 0.5 after incubation at 0 degrees C and to 5.4% +/- 0.5 and 2.1% +/- 1.2 at room temperature. Cryoactivation did not occur in buffers containing more than 1 M NaCl. It took 8 min at 37 degrees C or 180 min at room temperature for cryoactivated prorenin to lose half of its intrinsic renin activity. It took 48 and 26 h, respectively, at 0 degree C for the two pools of prorenin at 37 degrees C to regain half of their maximum intrinsic activity at 0 degrees C. A direct immunoradiometric assay that detects active renin but not prorenin was able to detect cryoactivated prorenin. These results show that human prorenin can be reversibly cryoactivated in buffers of low ionic strength and has greater intrinsic activity at room temperature than at 37 degrees C.     

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.     

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.     

Immunological evidence that inactive renin is prorenin

Antibody raised to a synthetic dodecapeptide, corresponding to the C-terminal portion of the human renin pro-segment, was tested for its ability to recognize highly purified human inactive or active (mature) renins; immune complexes were detected by precipitation with protein A-Sepharose. Serial antibody dilutions caused identical binding of renal or plasma inactive renins but failed to bind active renin. In contrast, antibody to active renin recognized both active and inactive forms. Reversible acid activation of inactive renin enhanced its binding to the anti-prorenin antibody, whereas irreversible trypsin activation significantly reduced binding. Binding was abolished following prolonged exposure to trypsin or if inactive renin was acidified prior to trypsin treatment. These results indicate that inactive renin shares immunochemical determinants with prorenin; they suggest that acidification alters the conformation of the pro-segment and that trypsin can convert the molecule both to fully mature renin and to intermediate form(s).     

Biochemical and immunological differences between plasma inactive renin from normal and nephrectomized rats.

Using immunological techniques, we have demonstrated that about half the trypsin-activatable renin in normal rat plasma is prorenin, while the other is not, and that inactive renin in nephrectomized rat plasma is not prorenin. In the present study, the trypsin-induced angiotensin I generating activity not related to prorenin from normal rat plasma disappeared after HPLC on G3000SW. HPLC analysis of trypsin-treated plasma showed the generation of active renin by trypsin for normal rat plasma, while it did not for nephrectomized rat plasma. These results indicate that trypsin treatment of crude plasma results in the generation of angiotensin I generating activity not due to prorenin, as well as activation of prorenin. HPLC on G3000SW is a useful tool for the determination of plasma prorenin.     

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.     

Site-directed mutagenesis of human prorenin. Substitution of three arginine residues in the propeptide with glutamine residues yields active prorenin

Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.     

A new form of renin in normal human plasma: “big renin” is a mixture of inactive prorenin and the new active high molecular weight renin

A new form of active renin was separated from inactive prorenin in normal human plasma by a new affinity chromatographic method on a column of Cibacron Blue F3GA-agarose. This active renin has a molecular weight of 54,000, considerably higher than the hitherto recognized active renin of 40,000 dalton in human plasma. The molecular weight of inactive prorenin was 56,000±2,000. Active renin produced from the inactive prorenin by trypsin or pepsin digestion or by acid treatment in $\text{in vitro}$ experiments showed a molecular weight of 54,000±2,000. Active renin with a molecular weight of 40,000 was not found in 6 samples of untreated plasma of normal human subjects nor was it formed by treatment with trypsin, pepsin, or acid pH. It is concluded that a large form of active renin (54,000 dalton) exists in normal human plasma which is distinct from a smaller form and that the activatable “big renin” is a mixture of this active renin and totally inactive prorenin. This explains the absence of molecular weight change during the activation of “big renin”.     

The purification and characterization of recombinant human renin expressed in the human kidney cell line 293

The cDNA encoding human preprorenin has been introduced into the adenovirus-transformed human kidney cell line 293. The recombinant 293 cells expressed and secreted prorenin; trypsin was used to activate the secreted prorenin to renin in vitro. The recombinant protein was purified to homogeneity by a single affinity chromatographic step. Using synthetic tetradecapeptide, the Km was 57.1 +/- 9.3 microM and the kcat was (7.48 +/- 1.57) x 10(3)/hr. Activation with trypsin resulted in a secondary cleavage between Arg53 and Leu54 generating a two chain form held together via a disulfide between Cys51 and Cys58. This secondary cleavage did not affect enzyme activity as determined by the ability of renin to degrade a synthetic tetradecapeptide substrate. Our paper demonstrates the potential for producing large quantities of renin from human kidney cells and also suggests that the use of trypsin, which has been widely used to convert prorenin to renin in vitro, causes a secondary cleavage in the renin peptide chain.     

Prorenin in plasma and kidney

Circulating prorenin is an enzymatically inactive form of renin, also present in kidney, which can be activated in vitro. Its biochemical properties and physiological behavior suggest that it may be a biosynthetic precursor of active renin. However, in contrast to typical prohormones, the normal plasma concentrations of prorenin are much higher than the active hormone. The purposes and functions of prorenin are unclear. It may have no further role after its secretion into the circulation. On the other hand, it may be a transport form of renin that can enter or exit cells more easily than the active form. It is also possible that the activity of the renin-angiotensin system may be regulated by the conversion of prorenin to renin in the kidney (which may be under beta-adrenergic control) or at other possible sites. Irreversible activation of prorenin appears to be a proteolytic process. In addition, acidification causes reversible activation, perhaps through a change in molecular conformation. Such reversible activation might occur in vivo by unknown mechanisms. Future studies are needed to define the biochemical processes by which increased physiological demand for renin is translated into the production of more active enzyme.     

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.