Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ramipril Improves Endothelial Function and Increases the Number of Endothelial Progenitor Cells in Patients With Systemic Lupus Erythematosus.

BACKGROUND/OBJECTIVES: Endothelial dysfunction and reduced number of endothelial progenitor cells (EPCs) in peripheral blood are contributing factors to cardiovascular disease in systemic lupus erythematosus (SLE) patients. Endothelial progenitor cell proliferation is regulated by vascular endothelial growth factor (VEGF). Angiotensin-converting enzyme inhibitors reduce cardiovascular mortality in patients with coronary heart disease. METHODS: This was a randomized trial including 37 female SLE patients without cardiovascular risk factors allocated into 2 groups: 19 patients received ramipril 10 mg/d for 12 weeks (IG) and 18 patients maintained without ramipril (CG). Endothelial function was assessed by brachial artery ultrasound measuring flow-mediated dilation, and EPCs were quantified by flow cytometry and cell culture, at baseline and after 12 weeks. Serum VEGF levels were measured by enzyme-linked immunosorbent assay. Statistical analysis was intention to treat. p < 0.05 was considered significant. RESULTS: After 12 weeks, higher flow-mediated dilation (6.17% vs. 11.14%, p < 0.001) was observed in IG, without change in CG (5.37% vs. 5.02%, p = 0.630). Higher number of EPC colony-forming units was also observed in IG (21.3 ± 10.4 vs. 31.6 ± 8.5, p < 0.001), without difference in CG ( p = 0.714). No difference was found in EPCs evaluated by flow cytometry. Vascular endothelial growth factor level increased after 12 weeks in IG ( p = 0.048), with no difference in CG ( p = 0.661). CONCLUSION: Ramipril improved endothelial function and increased the numbers of EPCs evaluated by cell culture and VEGF levels in SLE patients without cardiovascular risk factors. These data suggest that angiotensin-converting enzyme inhibitor bring an extra benefit beyond the hypotensive action and should be considered as a preferred antihypertensive drug in SLE patients.

Analysis of Binding Interactions of Ramipril and Quercetin on Human Serum Albumin: A Novel Method in Affinity Evaluation.

The aim of this study was to analyze the binding interactions between a common antihypertensive drug (ramipril, R) and the widely distributed plant flavonoid quercetin (Q), in the presence of human serum albumin (HSA). From the observed fluorescence spectra of the (HSA + R) system we can assume that ramipril is also one of the Site 3 ligands-similar to fusidic acid-the binding of which has been proven by RTG crystallography. Our claim is supported by near-UV CD spectroscopy, microscale themophoresis and molecular modeling. The presence of R slightly inhibited the subsequent binding of Q to HSA and, on the contrary, the pre-incubation of HSA with Q caused a stronger binding of R, most likely due to allosteric interactions. At high concentrations, R is also able to displace Q from its binding site. The dissociation constant KD for the binding of R is more than hundredfold larger than for Q, which means that R is a very weak binder to HSA. The knowledge of qualitative and quantitative parameters of R, as well as the methods used in this study, are important for future research into HSA binding. This study shows the importance of implementing other methods for KD determination. Microscale thermophoresis has proved to be a novel, practical and accurate method for KD determination on HSA, especially in cases when fluorescence spectroscopy is unable to produce usable results.

Binding interaction of ramipril with bovine serum albumin (BSA): Insights from multi-spectroscopy and molecular docking methods.

The binding interaction between a typical angiotensin-converting enzyme inhibitor (ACEI), ramipril, and a transport protein, bovine serum albumin (BSA), was studied in vitro using UV-vis absorption spectroscopy, steady-state fluorescence spectroscopic titration, synchronous fluorescence spectroscopy, three dimensional fluorescence spectroscopy, circular dichroism and molecular docking under the imitated physiological conditions (pH=7.4). The experimental results suggested that the intrinsic fluorescence of BSA was quenched by ramipril thought a static quenching mechanism, indicating that the stable ramipril-BSA complex was formed by the intermolecular interaction. The number of binding sites (n) and binding constant of ramipril-BSA complex were about 1 and 3.50×104M-1 at 298K, respectively, suggesting that there was stronger binding interaction of ramipril with BSA. The thermodynamic parameters together with molecular docking study revealed that both van der Waal's forces and hydrogen bonding interaction dominated the formation of the ramipril-BSA complex and the binding interaction of BSA with ramipril is enthalpy-driven processes due to |ΔH°|>|TΔS°| and ΔG°<0. The spatial distance between ramipril and BSA was calculated to be 3.56nm based on Förster's non-radiative energy transfer theory. The results of the competitive displacement experiments and molecular docking confirmed that ramipril inserted into the subdomain IIA (site I) of BSA, resulting in a slight change in the conformation of BSA but BSA still retained its secondary structure α-helicity.

In vitro drug metabolism by human carboxylesterase 1: focus on angiotensin-converting enzyme inhibitors.

Carboxylesterase 1 (CES1) is the major hydrolase in human liver. The enzyme is involved in the metabolism of several important therapeutic agents, drugs of abuse, and endogenous compounds. However, no studies have described the role of human CES1 in the activation of two commonly prescribed angiotensin-converting enzyme inhibitors: enalapril and ramipril. Here, we studied recombinant human CES1- and CES2-mediated hydrolytic activation of the prodrug esters enalapril and ramipril, compared with the activation of the known substrate trandolapril. Enalapril, ramipril, and trandolapril were readily hydrolyzed by CES1, but not by CES2. Ramipril and trandolapril exhibited Michaelis-Menten kinetics, while enalapril demonstrated substrate inhibition kinetics. Intrinsic clearances were 1.061, 0.360, and 0.02 ml/min/mg protein for ramipril, trandolapril, and enalapril, respectively. Additionally, we screened a panel of therapeutic drugs and drugs of abuse to assess their inhibition of the hydrolysis of p-nitrophenyl acetate by recombinant CES1 and human liver microsomes. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors: the dihydropyridine calcium antagonist, isradipine, and the immunosuppressive agent, tacrolimus. CES1 plays a role in the metabolism of several drugs used in the treatment of common conditions, including hypertension, congestive heart failure, and diabetes mellitus; thus, there is a potential for clinically relevant drug-drug interactions. The findings in the present study may contribute to the prediction of such interactions in humans, thus opening up possibilities for safer drug treatments.

Perindopril and ramipril phosphonate analogues as a new class of angiotensin converting enzyme inhibitors.

A series of phosphonate analogues related to perindopril and ramipril were prepared and tested to estimate their ability to inhibit angiotensin converting enzyme. These new synthesized compounds were active ACE inhibitors with a promising activity.

Loss of captopril-bound Fe by end-stage renal failure patients during hemodialysis.

BACKGROUND: Patients on chronic hemodialysis often suffer from severe anemia, the outcome of iron deficiency and inadequate response to erythropoietin. Antihypertensive treatment with captopril worsens anemia, erythropoietin production and iron balance in hemodialysis patients. We investigated the possibility that iron chelation by captopril in the blood may result in elimination of iron-captopril complexes during hemodialysis, thus minimizing the effect of both medications. METHODS: Twelve hypertensive hemodialysis patients (group 1) were treated with 12.5 mg/day captopril, while their 12 counterparts received 1.25 mg/day ramipril. Following two weeks of treatment and two weeks of "washout", captopril in group 1 was substituted with ramipril and ramipril in group 2 was replaced by captopril for an additional two week period. Blood and dialysate samples were procured at the beginning and the end of the dialysis, for iron, aluminum, transferin, ferritin, hemoglobin (Hb) and hematocrit (Htc) determination. RESULTS: Iron, ferritin, transferin, Hb and Htc were decreased in the captopril-treated group 1. They similarly decreased in group 2 following replacement of ramipril by captopril for an additional period of two weeks. Significant amounts of iron were detected in dialysates of captopril, but not ramipril-treated patients. At the end of the dialysis, iron content was further increased in dialysates of the captopril-treated groups. CONCLUSIONS: 1) Captopril-chelated iron is eliminated in dialysis fluid during the dialysis session, apparently contributing to captopril-related anemia in patients on chronic hemodialysis. 2) Antihypertensive treatment with angiotensin converting enzyme (ACE) inhibitors other than captopril might prove advantageous for this patient category.

Pharmacokinetics, metabolism and biliary and urinary excretion of oral ramipril in man.

In order to evaluate the pharmacokinetics and excretion of ramipril in man, 8 cholecystectomy patients aged between 53 and 68 years received 5 mg ramipril orally as a single dose. All patients had a T-drain inserted to permit bile collection; all gave their informed consent to participate in the trial. Serum samples were collected half-hourly until 2 hours, then hourly until 6 hours, then at 8, 10, 24 and 25 hours after intake. Urine was collected in 2-hour fractions until 8 hours, followed by a 4- and a 12-hour fraction. Bile was collected hourly until 6 hours, followed by a 6- and a 12-hour collecting fraction. Concentrations of ramipril and ramiprilat in serum, and determinations in urine and bile of ramipril, ramiprilat, ramipril glucuronide, ramiprilat glucuronide, diketopiperazine and diketopiperazine acid were made; total amounts excreted were calculated. Peak concentrations of ramiprilat in plasma (8.7 +/- 1.6 ng/ml) were reached after about 8 hours. AUC0-8 and AUC0-24-values were 36.5 and 111.9 ng.h/ml, respectively. Ramiprilat Cmax-concentrations were about 300-fold higher in bile than in plasma, the corresponding difference for ramipril between bile and plasma was about 4-fold. The main fractions excreted in the urine were diketopiperazine acid and ramiprilat amounting to 13.2 +/- 5.6 and 4.4 +/- 2.4%, respectively, of the dose administered. Only a very small fraction of the dose was excreted with urine as unchanged ramipril, on average 0.9 +/- 1.0%. The main fractions excreted in the bile were diketopiperazine acid, ramiprilat glucuronide and diketopiperazine, 9.0 +/- 5.3, 3.4 +/- 4.2 and 2.0 +/- 1.2% in 24 hours, respectively, of the dose administered. Only a negligible fraction of the dose (average 0.1 +/- 0.1%) was excreted with bile as unchanged ramipril. In conclusion, there is strong evidence that circulating ramipril and ramiprilat are eliminated by both the liver and the kidneys. For the patients studied it can be estimated from late collection periods that some 2/3 of circulating ramipril and ramiprilat are eliminated by the kidneys and 1/3 eliminated by the liver.

Effects of dietary protein and uninephrectomy on renal angiotensin converting enzyme activity in the rat.

This study examines the effects of dietary protein and of uninephrectomy on angiotensin converting enzyme (ACE) in the normotensive rat, with particular regard to the kidney. Male Wistar Kyoto rats were fed isocaloric diets containing 5, 16 or 50% protein for three weeks. Other groups of rats were subjected to either left unilateral nephrectomy or sham operations, and the rats were killed eight days after surgery. ACE activity was measured in the kidney medulla, cortex, proximal tubule brush border membrane and in the plasma, heart and lung. Renal cortex and brush border ACE activity increased in parallel with protein intake, whereas plasma and lung ACE activity decreased; heart and kidney medulla ACE activity did not vary significantly. Uninephrectomy also led to a high increase in brush border ACE activity in the contralateral kidney, with no effect in the renal medulla or in the other tissues. The increase in ACE activity in the brush border membrane corresponded to a similar increase in the maximum number of binding sites of 3H-ramiprilat. This suggested that the increase in ACE activity corresponded to an increase in ACE concentration. The increase in renal tubular ACE activity could result in higher angiotensin II levels, and could consequently play a role in the modification of sodium reabsorption and cellular growth which occurs in the proximal tubule in these experimental models.

Plasma renin activity and changes in tissue angiotensin converting enzyme.

OBJECTIVES: Recent evidence suggests that tissue generation of angiotensins I and II depends on the level of the plasma components of the renin-angiotensin system and on tissue-specific processes. The present study was undertaken to clarify the possible relationship between plasma renin activity (PRA) and tissue angiotensin converting enzyme (ACE) activity in the heart, lung, kidney cortex and kidney medulla of Wistar-Kyoto rats. In the kidney cortex particular attention was focused on renal brush-border ACE. METHODS: Different experimental models known to have opposite effects on PRA were used: changes in salt intake, deoxycorticosterone acetate (DOCA) with or without salt supplements, and the Goldblatt two-kidney, one clip (2-K,1C) model. Two weeks after the start of the experiments the rats were killed, and PRA, and plasma and tissue ACE activity, were measured. RESULTS: At the end of the study the blood pressure in the treated rats was not significantly different from control. As expected, the PRA were highest in the 2-K,1C and depleted-salt groups and lowest in the DOCA, DOCA-salt and high-salt groups. ACE responses were different in different types of tissue, with no relationship between PRA and plasma or tissue ACE activity. For example, DOCA treatment led to increased ACE activity in the heart and the kidney only if the rats were maintained on a high salt intake. DOCA or salt alone failed to have this effect. In the 2-K,1C model the unclipped kidneys did not show any significant variation in ACE activity, but the clipped kidneys exhibited increased ACE activity compared with sham-operated rats. This increase, coupled with increased renal renin secretion, could play a role in the acceleration of local angiotensin II formation, and could thus initiate and sustain the development of hypertension in this model. CONCLUSION: The present results show that variations in ACE activity were organ-specific and were not linked either to hypertension or to changes in PRA.

ACE inhibitor effect on bradykinin metabolism in the vascular wall.

In the isolated rabbit thoracic aorta the ACE inhibitor ramiprilat attenuated bradykinin degradation by enzymes localized on vascular endothelial cells as well as on vascular smooth muscle cells by 26% and 32%, respectively. We conclude that the ACE inhibitor can attenuate vascular bradykinin degradation not only by inhibition of endothelial ACE but also by inhibition of other bradykinin degrading enzymes in deeper layers of the vascular wall.