Prolylcarboxypeptidase Research Articles

Prolyl carboxypeptidase purified from human placenta: its characterization and identification as an apelin-cleaving enzyme

BackgroundThe proteolytic regulation of peptides involved in feeding behavior is poorly understood. Prolyl carboxypeptidase (PRCP) is particularly known for its role in body weight control by converting the anorexigenic peptide, α-melanocyte-stimulating hormone 1–13 into the inactive form 1–12. The purpose of this study was to characterize purified human PRCP, to investigate its substrate specificity and to discover novel substrates linked to obesity. Pyroglutamated apelin-13, ghrelin, enterostatin and obestatin were investigated since these are feeding-regulating peptides with potential cleaving sites for PRCP. MethodsPRCP was purified from human placenta and identified using western blotting and mass spectrometry. The kinetic parameters of purified and commercially available PRCP for known and potential peptide substrates were determined and compared using a RP-HPLC activity assay, isothermal titration calorimetry and mass spectrometry. ResultsPRCP was purified 575-fold from human placenta and succesfully identified as human lysosomal Pro-X carboxypeptidase. Purified and recombinant PRCP had similar substrate specificity with angiotensin III as the substrate of preference. Pyroglutamated apelin-13 was observed to be a novel substrate for human PRCP in vitro and PRCP-dependent cleavage was shown in a human umbilical vein endothelial cell culture experiment. Other potential substrates e.g. obestatin, ghrelin and enterostatin were not hydrolyzed by PRCP. ConclusionThese results show that placenta is a good source of human PRCP and that PRCP removes the C-terminal phenylalanine from pyroglutamated apelin-13. For the first time, PRCP is identified as an apelin-cleaving enzyme. This finding adds evidence to the hypothesis that PRCP plays a role in energy homeostasis.

Dysregulation of the renin-angiotensin system during lung ischemia-reperfusion injury

ABSTRACTAim/Purpose of the Study: Activation of the renin-angiotensin system leading to increased angiotensin-(1–7) (Ang-(1–7)) and decreased angiotensin 2 (Ang 2) levels may be a new therapeutic approach to reduce acute lung injury. Prolylcarboxypeptidase (PRCP) and prolyloligopeptidase (PREP) are capable of hydrolyzing Ang 2 into Ang-(1–7). However, their relation with circulating Ang 2 levels after lung ischemia–reperfusion injury (LIRI) has never been explored. This study determines whether the activity and expression of PRCP and PREP in plasma and lung tissue is related to circulating Ang 2 levels in a murine model of LIRI. Materials and Methods: LIRI in Swiss mice (6 animals per group) was induced by temporary left lung hilar clamping (1 h) followed by 0, 1 or 24 h of reperfusion. Animals in the sham group received thoracotomy only. PRCP activity was measured via RP-HPLC, PREP activity using a fluorogenic substrate and plasma Ang 2 levels via ELISA. Western blotting was used to determine the PRCP and PREP protein expression profiles in left lung tissue. Results: Plasma Ang 2 levels significantly rise after lung ischemia and remain increased after 1 h and 24 h of reperfusion compared to the sham group. While a significant decrease in plasma PREP activity was found after 24 h of reperfusion, a transient increase in plasma PRCP activity was observed after ischemia. However, no correlation with plasma Ang 2 levels could be demonstrated. The activity profiles of PRCP and PREP and the protein expression of PRCP in the lung tissues remained unchanged after LIRI. Conclusions: LIRI causes a dysregulation of circulating Ang 2 levels and plasma PREP activity, although no direct link between both phenomena could be shown. The activity profile of pulmonary PRCP and PREP was not significantly changed after LIRI, which implies a minor role for local PRCP and PREP in the ischemic lung itself.

A Cross-sectional Study of KLKB1 and PRCP Polymorphisms in Patient Samples with Cardiovascular Disease.

Plasma kallikrein formed from prekallikrein (PK) produces bradykinin from kininogens and activates factor XII. Plasma PK is activated by factors αXIIa, βXIIa, or prolylcarboxypeptidase (PRCP). A cross-sectional investigation determined if there is an association of PRCP and KLKB1 polymorphisms with cardiovascular disease (CVD). DNA was obtained from 2243 individuals from the Prevention of Events with Angiotensin Converting Enzyme trial. Two PRCP SNPs, rs7104980 and rs2298668, and two KLKB1 SNPs, rs3733402 and rs3087505, were genotyped. Logistic regression models were performed for history of diabetes, myocardial infarction, stroke, angina, angiographic coronary disease, CABG, intermittent claudication, percutaneous transluminal coronary angioplasty (PTCA), and transient ischemic attack. The PRCP SNP rs7104980 increased the odds of having a history of PTCA by 21% [odds ratio (OR) = 1.211; 95% confidence intervals (CI) = (1.008, 1.454)]; P = 0.041, but was non-significant after Bonferroni correction. Alternatively, having the G allele for rs3733402 (KLKB1 gene) decreased the odds of having a history of angiographic coronary disease by 24% [OR = 0.759; 95% CI = (0.622, 0.927)]; P = 0.007 that was statistically significant (P < 0.01) after Bonferroni correction for multiple hypothesis testing. When the best-fit model based on the Akaike information criterion controlled for age, weight, gender, hypertension, and history of angina, the G allele of KLKB1 rs3733402 that is associated with less plasma kallikrein activity correlated with reduced history of CVD.

Prolylcarboxypeptidase gene expression in the heart and kidney: Effects of obesity and diabetes.

Prolylcarboxypeptidase (PRCP) regulates plasma prekallikrein/high molecular weight kininogen/bradykinin axis. It also modulates angiotensin II (Ang II), angiotensin III (Ang III), and alpha-melanocyte stimulating hormone (α-MSH) physiological effects. Study suggests that increased plasma PRCP level is associated with cardiovascular risk factors, such as atherosclerosis, inflammation, and diabetes. Since expression pattern of PRCP in Zucker diabetic fatty (ZDF) rat vascular tissue remain unproved, we aimed to study its expression in the heart and kidney. The purpose of the present study was also to obtain systemic information of inflammation status with regard to PRCP expression and function in a high-fat diet (HFD)- fed ZDF rats. The ZDF rats were divided into 2 groups, which were fed a high-fat diet for 16 weeks or 32 weeks. Differential expression and pathological significance of PRCP expression during the consecutive stages of renal disease development were identified. After 16 weeks, ZDF rats exhibited early transiently altered PRCP expression in the heart and kidneys. After 32 weeks, ZDF rats showed continuously altered expression in PRCP and inflammatory markers, which was linked to severe hyperglycemia and nephropathy. Altered expression of PRCP associated with inflammatory mediators was illustrated to be functionally relevant. In further support of an important role of PRCP, we found PRCP protein to be highly elevated in rat plasma and in human plasma and the anti-diabetic agents reversed it. These findings indicate that impairment of tissues within the cardiovascular system influences PRCP expression and suggest that pathogenic mechanisms of deregulated PRCP expression warrant further investigation.

The Dipeptidyl Peptidase Family, Prolyl Oligopeptidase, and Prolyl Carboxypeptidase in the Immune System and Inflammatory Disease, Including Atherosclerosis.

Research from over the past 20 years has implicated dipeptidyl peptidase (DPP) IV and its family members in many processes and different pathologies of the immune system. Most research has been focused on either DPPIV or just a few of its family members. It is, however, essential to consider the entire DPP family when discussing any one of its members. There is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. In this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases DPPIV, FAP, DPP8, DPP9, dipeptidyl peptidase II, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. We highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease.

QSAR study of prolylcarboxypeptidase inhibitors by genetic algorithm: Multiple linear regressions

The predictive analysis based on quantitative structure activity relationships (QSAR) on benzimidazolepyrrolidinyl amides as prolylcarboxypeptidase (PrCP) inhibitors was performed. Molecules were represented by chemical descriptors that encode constitutional, topological, geometrical, and electronic structure features. The hierarchical clustering method was used to classify the dataset into training and test subsets. The important descriptors were selected with the aid of the genetic algorithm method. The QSAR model was constructed, using the multiple linear regressions (MLR), and its robustness and predictability were verified by internal and external cross-validation methods. Furthermore, the calculation of the domain of applicability defines the area of reliable predictions. The root mean square errors (RMSE) of the training set and the test set for GA-MLR model were calculated to be 0.176, 0.279 and the correlation coefficients (R 2) were obtained to be 0.839, 0.923, respectively. The proposed model has good stability, robustness and predictability when verified by internal and external validation. The predictive model using quantitative structure-activity relationships (QSAR) on benzimidazolepyrrolidinyl amides as prolylcarboxypeptidase (PrCP) inhibitors was derived based on internally and externally validated robust GA-MLR model.

Loss of prolyl carboxypeptidase in two-kidney, one-clip goldblatt hypertensive mice.

It is well documented that angiotensin (Ang) II contributes to kidney disease progression. The protease prolyl carboxypeptidase (PRCP) is highly expressed in the kidney and may be renoprotective by degrading Ang II to Ang-(1-7). The aim of the study was to investigate whether renal PRCP protein expression and activity are altered in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice. Left renal artery was constricted by using 0.12 mm silver clips. Blood pressure was measured using telemetry over the eleven weeks of study period and revealed an immediate increase in 2K1C animals during the first week of clip placement which was followed by a gradual decrease to baseline blood pressure. Similarly, urinary albumin excretion was significantly increased one week after 2K1C and returned to baseline levels during the following weeks. At 2 weeks and at the end of the study, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion and renal fibrosis. Renal PRCP expression and activity were significantly reduced in clipped kidneys. Immunofluorescence revealed the loss of renal tubular PRCP but not glomerular PRCP. In contrast, expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected, while angiotensin converting enzyme was elevated in unclipped kidneys and renin was increased in clipped kidneys. Results suggest that PRCP is suppressed in 2K1C and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PRCP.

Prolylcarboxypeptidase Is a Risk Factor for Cardiovascular Events

BACKGROUND: Prolylcarboxypeptidase (PRCP) is a membrane-associated serine protease that regulates biologic peptides bradykinin, angiotensin II, α-melanocyte stimulating hormone, and activates prekallikein. PRCP and its variants have been associated with metabolic syndrome in males, hypertension in preeclampsia, resistance to ACE inhibitors, and embryonic angiogenesis. Recent studies on PRCPgt/gt mice that have 25% normal PRCP levels show that these animals are hypertensive, have higher risk for arterial thrombosis, increased vascular inflammation, and reduced angiogenic repair after ischemia and injury (Blood 117:3929, 2011; Blood 122:1522, 2013). The hypothesis of this investigation is that polymorphisms in PRCP are associated with cardiovascular disease (CVD).METHODS: DNA from 2,243 subjects from the PEACE Trial (NEJM 351:2058, 2004) were genotyped at 2 PRCP SNPs (rs7104980, rs2298668) and 3 KLKB1 (prekallikrein) SNPs (rs4253252, rs3733402, rs3087505). These subjects were 82% female and had a history of (in decreasing frequency) angina (71%), angiographic coronary disease (66%), heart attack (58%), PTCA (44%), hypertension (42%), CABG (36%), diabetes (15%), stroke (4%), and TIA (3%). The association between single SNP alleles and different cardiovascular related phenotypes was assessed using logistic regression models. These models were adjusted for age, weight, gender, history of hypertension, and history of diabetes.RESULTS: When the logistic regression model was adjusted for age, weight, and gender, SNP rs710980 in PRCP had a 21% increased odds [odds ratio=1.211; 95% CI=(1.008, 1.454)] of having a history of PTCA if carrying the G allele (frequency of 66%) as compared to not having the G allele (frequency 34%). Additionally, adjusting the logistic regression model for history of hypertension and diabetes did not alter the odds ratio for PTCA history. However when the logistic regression model included history of hypertension and diabetes, the odds of having a history of a MI if carrying the same allele also is increased by 21% [odds ratio=1.21; 95% CI=(1.001, 1.455)]. In summary, the rs710980 intronic PRCP SNP conferred risk for CVD, while the PRCP exonic SNP rs2298668 showed no relationship in this population. Alternatively, the exonic KLKB1 SNP rs3733402 of Apple Domain 2 where high molecular weight kininogen binds conferred reduced odds of 24% [odds ratio=0.76; 95% CI=(0.622, 0.928)] of having a history of angiographic coronary disease if carrying the G allele (frequency 67%) as compared to not having the G allele (frequency of 33%). The other two KLKB1 SNPs showed no associations.DISCUSSION: These combined data on PRCP suggest that it is a risk factor for CVD. The present genetic data are consistent with biochemical, cell culture, and in vivo investigations showing that PRCP levels influence vascular biology, renal function, and metabolism. The extent of the genetic associations of PRCP polymorphisms with CVD may be resultant of the database interrogated. The PEACE trial consisted of individuals not severely ill with CVD. Its hypothesis to show that administration of ACE inhibitors leads to increased protection from mild CVD was not met. However, the present investigation showing several positive odds ratios with a certain PRCP SNP for CVD suggests that PRCP is a stronger risk factor for CVD than can be demonstrated in the PEACE subject population. PRCP should be interrogated in additional populations of subjects with CVD.Funding Support: NIH HL052779-17, HL112666-2 DisclosuresNo relevant conflicts of interest to declare.

Prolyl carboxypeptidase activity decline correlates with severity and short-term outcome in acute ischemic stroke.

Prolyl carboxypeptidase (PRCP) is an enzyme associated with cerebrovascular risk factors such as hypertension, diabetes mellitus, obesity and hyperlipidemia. We aim to evaluate the relation between serum PRCP activity and severity, evolution and outcome of acute ischemic stroke. We used a specific RP-HPLC activity assay to measure PRCP activity in serum of 50 stroke patients at admission, and at 24 h, 72 h and 7 days after stroke onset to assess correlations with stroke severity based on the National Institutes of Health Stroke scale score (NIHSS), infarct volume on brain MRI scan, stroke outcome based on the modified Rankin scale (mRS) and mortality at 3 months after stroke. The average PRCP activity in serum decreased significantly the first 24 h after stroke onset and returned to baseline values at day 7. High NIHSS scores and infarct volumes at admission were related with a more pronounced decrease of PRCP in the first 24 h after stroke (ΔPRCP24, r = 0.31, P < 0.05; r = 0.30, P < 0.05). In addition, patients who displayed a more pronounced decrease in PRCP levels during the first 24 h after stroke were more likely to be institutionalized upon discharge (n = 21) (ΔPRCP24 ± SD, 0.05 ± 0.10 U/L vs. 0.17 ± 0.14 U/L, P = 0.001). The decrease in PRCP levels in the first 24 h after stroke onset is associated with stroke severity and an unfavourable short-term stroke outcome.

Deficient prolylcarboxypeptidase gene and protein expression in left ventricles of spontaneously hypertensive rats (SHR)

Prolylcarboxypeptidase (PRCP), an endothelial cell membrane serine peptidase that inactivates angiotensin II and activates pre-kallikrein, is thought to have anti-hypertensive and anti-proliferative roles in cardiovascular homeostasis. We hypothesized that PRCP function may be altered in heart tissue under conditions that predispose to left ventricle hypertrophy (LVH) in rats. We therefore used real-time PCR and western-blotting to examine the mRNA and protein expression of PRCP in the hearts of spontaneously hypertensive rats (SHR) at pre-hypertensive (5-week-old) and hypertensive (16-week-old) stages compared with age-matched hypertensive (2 kidney-1 clip; 2K-1C) rats and normotensive Wistar rats. PRCP mRNA expression was significantly reduced in hearts of 5- and 16-week-old SHR compared with age-matched Wistar controls, 2K-1C hypertensive rats and sham-operated normotensive rats. There were no significant differences in the PRCP mRNA and protein expression levels in hearts from hypertensive renovascular and sham-operated normotensive rats. Prolonged treatment of SHR with the AT1 receptor antagonist losartan (40mg/kg, gavage for 8 weeks) reduced the left ventricular weight/body weight ratio (LVW/BW), as well as the mRNA expression of collagen type 1, collagen type 3 and MMP9 in left ventricular tissue, without affecting PRCP gene and protein expression. Our results suggest that diminished PRCP gene and protein expression might be constitutionally involved in the SHR phenotype. In addition, since neither the development of arterial hypertension in the 2K-1C model nor its successful treatment in SHR altered PRCP gene and protein expression in heart tissue, it appears unlikely that PRCP function is regulated by the renin–angiotensin system or by afterload conditions.

The Prolyl Peptidases PRCP/PREP Regulate IRS-1 Stability Critical for Rapamycin-induced Feedback Activation of PI3K and AKT

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells.

Angiotensin-(1–7)

In the last decade, a substantial change in our understanding of the renin–angiotensin system (RAS) occurred.1,2 It is now clear that the circulating and tissue RAS are far more complex than previously anticipated. The modern concept of the RAS includes, in addition to the classical components (renin, angiotensin [Ang]-converting enzyme [ACE], Ang II, Ang III, and Ang type 1 and type 2 receptors [AT1R and AT2R]), novel enzymes, peptides, and receptors (ACE2,3,4 Ang-(1–7), (pro)renin receptor,5 and Mas).6 More recently, novel putative components of the RAS (Ang A,7 alamandine,8 and the Mas-related G-protein–coupled receptor D)8 were identified. These new discoveries bring into perspective new possibilities of interaction among RAS components. Figure 1 shows a simplified updated view of the RAS. Figure 1. Simplified updated view of the renin–angiotensin system. ACE indicates angiotensin-converting enzyme; AMP, aminopeptidase; Ang, angiotensin; AT1, angiotensin type 1 receptor; AT2, angiotensin type 2 receptor; D-Amp, dipeptidyl aminopeptidase I–III; IRAP, insulin-regulated aminopeptidase; Mas, Mas receptor; MrgD, Mas-related G-protein–coupled receptor D; NEP, neutral endopeptidase; PCP, prolyl carboxypeptidase; and PEP, prolyl endopeptidase. Based on Lautner et al.8 The demonstration that Ang-(1–7) is a key component of the RAS represented one of the most significant conceptual changes of this important hormonal system. This review will focus on the cardiovascular actions of Ang-(1–7) and the potential of Ang-(1–7) and other Mas agonists as cardiometabolic therapeutic agents. Figure 2 shows most of known cardiovascular actions of Ang-(1–7). It should be pointed out that the actions of Ang-(1–7) are not restricted to the cardiovascular system. For example, Ang-(1–7) also impacts the renal system, which may play a role in cardiovascular diseases, such as hypertension. Because of space limitation, only a partial list of references was included in …

Pyrazoles as non-classical bioisosteres in prolylcarboxypeptidase (PrCP) inhibitors

Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.

Prolyl carboxypeptidase mRNA expression in the mouse brain

Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.

Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20h post oral dose.

Abstract 630: Blood Pressure and Kidney Function in PRCP deficient Mice

Changes in the enzymes involved in Ang II degradation may influence disease conditions such as hypertension and kidney disease. The enzyme prolylcarboxypeptidase (PRCP) catalyzes the conversion of Ang II to Ang(1-7) and its deficiency results in a phenotype characterized by a prothrombotic state, hypertension, and impaired wound healing. Here, we investigated kidney function and blood pressure in young mice, 10-16 weeks of age, with a subtotal PRCP deficiency (PRCP genetrap, PRCPgt). Enzyme activity based on a synthetic fluorogenic peptide was used to measure PRCP activity. The activity of recombinant PRCP was strongly pH-dependent with an optimum in the acidic range (pH 5). In keeping with this, recombinant PRCP degraded Ang II at pH 5, but not at pH 7.4 in-vitro. PRCP activity was detected in serum and in kidney of WT mice whereas PRCPgt/gt mice displayed highly diminished serum PRCP activity (about 5% of the WT). Kidney function was assessed by urinary protein/creatinine ratio and GFR. At 10 weeks of age, neither female nor male PRCPgt/gt animals showed elevations in urinary protein/creatinine-ratio (female PRCPgt/gt 19.9±1.4 vs. WT 23.5±1.9 and male PRCPgt/gt 32.4±3.5 vs. WT 34.8±1.8). At 16 weeks of age, the GFR (measured by clearance of FITC-Inulin) was not different (female PRCPgt/gt 4.10±0.32 vs. WT 4.33±0.29 and male PRCPgt/gt 4.41±0.50 vs. WT 4.28±0.31 ul/min/g). Systolic blood pressure was significantly elevated in PRCPgt/gt animals as compared to the respective WT controls (female PRCPgt/gt 147.8±3.4 vs. WT 117.3±7.1 mmHg, male 138.1±8.4 vs. 120.5±3.6 mmHg, p&lt;0.05). PRCP deficient mice have normal GFR but elevated basal blood pressure. Whether PRCP deficiency causes hypertension as a result of impaired Ang II degradation or other mechanisms needs to be determined.

Abstract 188: Loss of Renal Prolyl Carboxypeptidase in Mice With Chronic Kidney Disease

There are 26 million adults with chronic kidney disease (CKD) in the U.S. and the incidence continues to increase. It is well documented that the activation of the renin angiotensin system and the elevated formation of angiotensin (Ang) II both contribute to renal pathophysiology in CKD. Emerging evidence suggests that the Ang II degrading protease prolyl carboxypeptidase (PCP) is renoprotective. Thus, we investigated protein expression and activity of renal PCP using immunofluorescence, western blot and mass spectrometry in a mouse model of CKD. Renal injury in male C57Bl6 mice was caused by constriction of the left renal artery using silver clips (2K1C-method). Blood pressure measurements by radiotelemetry revealed a significant increase of 36.1 ± 3.9 mm Hg in 2K1C animals compared with control animals 1 week after clip placement (p&lt;0.0001). Using immunofluorescence and confocal microscopy, PCP was localized in the Bowman’s capsule of the glomerulus and in proximal and distal renal tubules. Western blot analysis showed PCP was significantly reduced in clipped 2K1C kidneys compared to unclipped kidneys of the 2K1C mice or compared to control mice (clipped 0.04 ± 0.02 vs unclipped 0.58 ± 0.16 vs control 0.65 ± 0.18, p &lt; 0.05). In addition, renal PCP enzyme activity was found to be markedly reduced in 2K1C kidneys as assessed by mass spectrometric based enzyme assays (clipped 37.1 ± 4.3 pmol Ang-(1-7)/h/μg vs unclipped 77.3 ± 12.3 pmol Ang-(1-7)/h/μg vs control 120.7 ± 14.7 pmol Ang-(1-7)/h/μg, p &lt; 0.01). In contrast, protein expression of prolyl endopeptidase, another enzyme capable of converting Ang II into Ang-(1-7), was not affected. Notably, renal pathologies were exacerbated in the 2K1C model as revealed by a significant increase in mesangial expansion (clipped 34.6 ± 3.1 vs unclipped 52.1 ± 4.0 vs control 1.2 ± 2.1, p &lt; 0.0001) and renal fibrosis (clipped 57.5 ± 0.9 vs unclipped 33.0 ± 0.7 vs control 3.3 ± 0.2, p &lt; 0.0001). Results suggest that PCP is suppressed in chronic kidney injury and that this downregulation may attenuate renoprotective effects via impaired Ang II degradation by PCP. Therefore, Ang II processing by PCP may have clinical implications in patients with renal pathologies.

Prolyl carboxypeptidase: a forgotten kidney angiotensinase. Focus on “Identification of prolyl carboxypeptidase as an alternative enzyme for processing of renal angiotensin II using mass spectrometry”

hypertension, which affects one-third of the population, is commonly treated with inhibitors of ANG II production or action, as this hormone triggers vasoconstriction and raises extracellular volume by increasing sodium reabsorption along the nephron. ANG II, an octapeptide [ANG-(1–8)], emanates

Discovery of new renal Ang II processing enzyme activity using mass spectrometry and gene deletion mouse models

Angiotensin (Ang) converting enzyme 2 (ACE2) is expressed in the kidney and exerts its renoprotection via conversion of the vasoconstrictor Ang II to the vasodilator Ang‐(1–7). To better characterize the tissue distribution and activity of enzymes involved in Ang II processing novel in situ and in vitro mass spectrometric methods were used. Mouse kidney sections (12 μm) or kidney homogenates (20 μg protein) were incubated with 0.1–10 nmol Ang II at pH 4–10 for 5–30 min at 37 ºC. Ang (1–7) was predominantly formed in renal cortex and dependent on amount of Ang II and pH. Further, the contribution of alternative renal peptidases to ACE2 independent formation of Ang‐(1–7) was explored using gene deletion mouse models deficient of ACE2, prolyl carboxypeptidase (PCP) or prolyl endopeptidase. Results suggest that ACE2 processes renal Ang II under neutral and basic conditions (pH&gt;;6), while PCP catalyzes this reaction at acidic pH (pH&lt;6). Using type 1 and type 2 diabetic animals, renal ACE2 was increased while renal PCP was decreased. This is the first report to demonstrate that renal Ang II processing to Ang‐(1–7) is independent of ACE2. Elucidation of renal Ang II processing pathways should be clinically important in patients with metabolic and renal disease.

Neuromodulatory role of angiotensin-(1–7) in the central nervous system

Ang-(1-7) [angiotensin-(1-7)] constitutes an important functional end-product of the RAS (renin-angiotensin system) endogenously formed from AngI (angiotensin I) or AngII (angiotensin II) through the catalytic activity of ACE2 (angiotensin-converting enzyme 2), prolyl carboxypeptidase, neutral endopeptidase or other endopeptidases. Ang-(1-7) lacks the pressor, dipsogenic or stimulatory effect on aldosterone release characteristic of AngII. In contrast, it produces vasodilation, natriuresis and diuresis, and inhibits angiogenesis and cell growth. At the central level, Ang-(1-7) acts at sites involved in the control of cardiovascular function, thus contributing to blood pressure regulation. This action may result from its inhibitory neuromodulatory action on NE [noradrenaline (norepinephrine)] levels at the synaptic cleft, i.e. Ang-(1-7) reduces NE release and synthesis, whereas it causes an increase in NE transporter expression, contributing in this way to central NE neuromodulation. Thus, by selective neurotransmitter release, Ang-(1-7) may contribute to the overall central cardiovascular effects. In the present review, we summarize the central effects of Ang-(1-7) and the mechanism by which the peptide modulates NE levels in the synaptic cleft. We also provide new evidences of its cerebroprotective role.