N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) has been shown to cause changes in cultured endothelial cells and smooth muscle function at concentrations from 5 to 25 mM. To determine whether HEPES also affects vascular permeability, the effects of two buffers, HEPES and phosphate, were compared in isolated perfused rabbit lungs. Hemodynamic parameters and vascular protein permeability-surface area products (PS) were measured after perfusion with the buffers. Endothelial permeability was measured for an anionic and a cationic albumin to assess the charge effects of the zwitterion buffer. With HEPES, there were no changes in vascular pressure or resistance but permeability was affected. Cationic albumin permeability increased with 12 mM HEPES (8.7(phosphate) → 30(12 mM HEPES) x ml · min-1 · g dry lung-1 x 10-2) as did the anionic albumin PS (2.7(phosphate) → 3.52(12 mM HEPES). The cationic PS returned to baseline (8.1(60 mM HEPES)) at 60 mM HEPES, but the anionic PS did not change from the 12 mM HEPES (4.01(60 mM HEPES)). In summary, we find that HEPES is not innocuous. Although hemodynamic parameters did not change, endothelial permeability was increased when HEPES was used at normal concentrations. Therefore, HEPES should be used with caution as a physiological buffer in perfused organ systems.

Vascular endothelial cells (VECs) play an essential role in regulating the passage of macromolecules and cells between the blood stream and underlying tissues. The second messenger 3’, 5’ cyclic adenosine monophosphate (cAMP) regulates numerous events in VECs, including permeability. Since human VECs express several distinct cAMP-hydrolyzing phosphodiesterases (PDEs), and these are the only enzymes that catalyze the inactivation of cAMP, we investigated if selective pharmacological inhibition of PDEs could impact VEC permeability. Interestingly, we found that PDE4 inhibitors decreased human aortic VEC (HAEC) permeability and PDE4 and PDE3 inhibitors decreased human microvascular VEC (HMVEC) permeability. Consistent with a role for both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in regulating VEC permeability, selective activators of these enzymes significantly decreased permeability. Since neither PDE4 nor PDE3 inhibitors significantly increased cAMP in these cells, our data are consistent with the idea that PDE inhibition causes small localized increases in “pools” of cAMP that regulate permeability. In order to test if PDE4 enzymes could act locally on pools of cAMP that regulated permeability, we selectively isolated the adherens junctional protein VE-cadherin from confluent monolayers of HAECs or HMVECs, and immunoblotted these isolates for cAMPeffectors and PDEs. Briefly, we found that each PKA-II, EPAC1, and a PDE4D variant, but not PDE3 enzymes, each could be isolated in VE-cadherin-based complexes from these cells. These novel findings identify PKA-II, EPAC1, and PDE4D as members of VE-cadherin-based signaling complexes in human VECs and are consistent with the idea that localized cAMP-signaling regulates permeability in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-09-14 15:52:20.216

The endothelial cells lining the inner surface of the tissue capillaries and post-capillary venules form a semi-permeable barrier between the blood circulation and interstitial compartments. The semi-permeable barrier in these vessels is the major site of blood-tissue exchange. A compromised endothelial barrier contributes to the pathological process such as edema, acute respiratory distress syndrome (ARDS) and tumor metastasis. Sphingosine-1-phosphate (S1P), an endogenous, bioactive lipid present in all cells, is a potential therapeutic agent that can restore compromised endothelial barrier function. On the other hand, S1P also has pleotropic effects and can either increase or decrease arterial tone and tissue perfusion under different conditions. The detailed mechanisms underlining S1P’s endothelial barrier protective effect are still largely unknown, but are suggested to depend on cell spreading termed “lamellipodia”. Therefore, to fully take advantage of the beneficial properties of S1P, it is important to first understand how S1P-induced lamellipodia protrusions correlate with its effect on endothelial barrier function. It is also important to know the underlining mechanisms that S1P enhances endothelial barrier function, including intracellular signaling and receptor signaling. To study local lamellipodia activities, we acquired time-lapse images of live endothelial cells expressing GFP-actin, and subsequently analyzed different lamellipodia parameters. Experiments were performed under baseline conditions, and during endothelial barrier disruption or enhancement. The compounds used in these experiments included thrombin and S1P. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function for in vitro studies. Changes of local lamellipodia dynamics and endothelial barrier function within the same time frame were studied. For mechanistic studies, we combined biochemical, immunological and pharmacological approaches. Rho family small GTPase activities were measured with an ELISA pull-down assay. Fluorescence Resonance Energy Transfer (FRET) was also used to study the localization of RhoA activation. Pharmacological compounds targeting intracellular signaling messengers were used to test the involvement of Rac1, RhoA, MLC-2 in endothelial local lamellipodia activity and S1P-mediated endothelial barrier enhancement. Receptor agonists and antagonists were used to study the involvement of S1P receptor signaling. Finally, for cell behavior and cytoskeleton studies, we utilized immunofluorescence labeling that enables direct visualization of changes in cytoskeleton, cell-cell junction and focal adhesions. We found that S1P increases both local lamellipodia protrusions and TER. The rapid increase in local lamellipodia protrusion frequencies also corresponded to the rapid increase in TER seen within the same time frame. Under the microscope, local lamellipodia protrusions from adjacent cells overlapped with each other and extended beyond junctional cell-cell contacts. Strikingly, S1P-induced lamellipodia protrusions carry VE-cadherin molecules to the cell-cell contact, established junctional adhesions. Combined with our previous published studies on thrombin induced lamellipodia activity changes, we think lamellipodia protrusions are a major component that regulates endothelial barrier function. Combined, our imaging studies revealed the mechanisms on how lamellipodia regulates endothelial barrier function: 1) lamellipodia overlap and increase the apical to basal diffusion distance, which in turn decreases permeability and upregulates endothelial barrier function. 2) Local lamellipodia protrusions contain VE-cadherin, which is delivered the to the cell-cell contact by the lamellipodia to increase junctional stability. S1P is effective for rescuing thrombin-induced endothelial barrier dysfunction. The known barrier disruptor thrombin, decreased local lamellipodia protrusions, disrupted VE-cadherin integrity, and caused a drop in TER. S1P increased local lamellipodia protrusions after thrombin challenge, and resulted in faster recovery towards baseline TER compared with vehicle controls. Interestingly, we also found that both thrombin and S1P increased MLC-2 phosphorylation at Thr18/Ser19. We subsequently accessed Rho family GTPase activity after thrombin and S1P. As expected, thrombin rapidly increased GTP-bound RhoA levels, and decreased GTP-bound Rac1 levels. Unexpectedly, S1P not only increased GTP-bound Rac1, but also increased GTP-bound RhoA to a more prominently levels (4-fold). Since Rac1 has been implicated in promoting lamellipodia protrusions, we tested the role of Rac1 on the local lamellipodia activities first. We found that Wild-Type (WT) Rac1 group had the highest local lamellipodia protrusion frequencies, protrusion distances, withdraw time and highest percentage of protrusions that lasted more than 5 min. WT Rac1 overexpression had greatest protrusion frequencies and lowest monolayer permeability to FITC-albumin compared to GFP and DN-Rac1 overexpression monolayers. These results suggest that Rac1 is important for baseline endothelial barrier function. This is also confirmed by the finding that pharmacological inhibition of Rac1 significantly decreased baseline TER. Although Rac1 is important for baseline endothelial barrier function, we noticed that it is dispensable in S1P-mediated endothelial barrier enhancement. Rac1 inhibitors, DN-Rac1 overexpression, and Rac1 siRNA knockdown all failed to abolish the S1P-mediated increase in TER. This is partially explained by the findings that S1P-induced Rac1 activation is short-lived and less pronounced in contrast to RhoA activation. We subsequently tested the role of RhoA in S1P-mediated endothelial barrier enhancement, based on our findings that both S1P and thrombin significantly activated RhoA and induced MLC-2 phosphorylation. Significant RhoA activation was found to be mainly at cell periphery and lamellipodia protrusions in HUVEC on FRET, after S1P was given. In addition, RhoA inhibitors significantly decreased the amplitude of S1P-induced MLC-2 phosphorylation, vinculin redistribution and barrier enhancement. The data suggest that the mechanisms involved in S1P-mediated endothelial barrier enhancement depend on RhoA activation and subsequent cytoskeletal rearrangement. We next investigated which receptor is responsible for the endothelial barrier enhancement of S1P. However, antagonism of S1P1, S1P2 or S1P3 alone with W146, JTE-013 or TY-52156 respectively all failed to attenuate S1P-mediated increase in TER. While agonism of S1P1 with CYM-5442 hydrochloride alone produced significant increase in TER, neither S1P2 nor S1P3 activation (CYM 5520 & CYM 5541) produced any change on TER. Interestingly, S1P1 antagonist failed to block the effect of S1P1 agonist on TER. This could be due to that the S1P1 agonist may not be very selective at concentrations tested. We also identified that S1P4 and S1P5 are present on endothelial cells. Further studies would be necessary to elucidate the roles of newly identified S1P4 or S1P5 alone on endothelial barrier function. It is also worth investigating in the future if multiple S1P receptors are involved in its endothelial barrier enhancing effect. In conclusion, we found that lamellipodia protrusions contribute to the endothelial barrier enhancement of S1P. While Rac1 is important for the maintenance of endothelial barrier function, it is dispensable in S1P-mediated endothelial barrier enhancement. On the other hand, RhoA activation appears to be, at least in part, responsible for the endothelial barrier enhancement of S1P. It is currently still unclear if S1P’s endothelial barrier enhancing effect is through one single receptor activation or activation of multiple receptors. Future studies are needed to elucidate the receptor signaling that contributes to S1P-mediated endothelial barrier enhancement.

L’endothélium préserve l'homéostasie vasculaire et tissulaire qui contrôle plusieurs processus physiologiques dans les corps humain. Lors de certaines pathologies, l'intégrité vasculaire peut être perturbée par une diversité de médiateurs de perméabilité qui interrompent la fonction barrière de cet épithélium, et provoquer des lésions tissulaires au cours de la progression de la maladie. Par conséquent, l'intégrité de la barrière endothéliale est essentielle pour l'homéostasie vasculaire. Dans le cas de la fièvre hémorragique due à la dengue, de nombreuses voies de signalisation induisent une perméabilité vasculaire qui résulte d'une rupture de la barrière hémato-encéphalique qui, dans certains cas, peuvent permettre la pénétration virale dans le système nerveux central (SNC). La plupart de ces voies de signalisation (y compris le TNF-alpha) sur-activent les enzymes appelées métalloprotéinases matricielles (MMP), qui induisent la dégradation de la matrice extracellulaire (MEC) et des protéines intercellulaires régissant la perméabilité vasculaire. Parmi ces enzymes, les MMP gélatinolytiques (MMP-9 et MMP-2) sont considérées comme la classe la plus importante de MMP puisqu'elles sont capables de dégrader le collagène de type IV, constituant principal de la MEC, et les jonctions cellulaires PECAM- 1 et VE-cadhérine. Ainsi, dans cette étude, nous avons développé deux approches pour inhiber l'activité de MMP-9 et protéger la perméabilité vasculaire in vitro. Ces approches incluent (i) des antagonistes de MMP-9 conçus in silico, tels que le composée HA048 (ii) des composés lipophénoliques synthétiques dérivés du resvératrol. Ce dernier existe naturellement dans les tiges de raisin, les arachides et plusieurs autres plantes et qui est capable d'inhiber l'expression et l'activité de la MMP-9. Bien que le resvératrol ait plusieurs activités biologiques testées in vitro, son utilisation dans des modèles animaux est limitée en raison de sa faible biodisponibilité, de son métabolisme rapide et de son élimination. Par conséquent, cette étude a trois objectifs principaux. Premièrement, de trouver un nouveau composé capable d'inhiber l'activité de MMP-9 in vitro. Deuxièmement, de vérifier s’il peut protéger l'intégrité de la couche endothéliale dans les HUVEC. Enfin, de passer à des études précliniques sur le modèle de souris dengue afin de vérifier son activité, sa toxicité et sa biodisponibilité. Outre le resvératrol et le SB-3CT (inhibiteur commercial de la MMP-9), nous avons constaté que le resvératrol-acide linoléique oméga 6 (RES-LA), le resvératrol-docosanoïque (RES-C22) et le HA048 ont montré la plus forte activité anti-MMP-9 parmi d'autres composés. Cependant, RES-LA a été préférentiellement choisi par rapport à RES-C22 en raison de sa meilleure solubilité. Par conséquent, nous avons utilisé un nouveau concept de formulation de ces composés en les solubilisant dans un solvant compatible et moins toxique pour des expériences in vivo, un solvant eutectique profond naturel (NADES), composé de 1,2-propanediol:ChCl: eau (1:1:1) (NADES/PCW). Les résultats de la perméabilité vasculaire in vitro ont révélé que les inhibiteurs de la MMP-9 ont diminué la perméabilité endothéliale exacerbée induite par le TNF-α dans les HUVEC, mesurée à la fois en temps réel et en fluorescence. En outre, l'examen microscopique des jonctions cellulaires adhérentes a montré qu’il y avait plus de CD31/PECAM-1 entre les HUVEC traitées à la fois par le TNF-α et les inhibiteurs de la gélatinase par rapport au témoin positif (TNF-alpha).Le mécanisme d'action des lipophénols dérivés du resvératrol a été évalué. Nos résultats ont révélé que les lipophénols dérivés du resvératrol ont inhibé l'expression de la MMP-9 en atténuant la phosphorylation des kinases MAPK ERK1/2 et JNK1/2. / The endothelial barrier preserves the vascular and tissue homeostasis that controls several physiological processes inside the body. In pathologies, vascular integrity could be disrupted by a diverse of permeability mediators that interrupt the barrier function and cause tissue damage during disease progression. Therefore, endothelial barrier integrity is critical for vascular homeostasis. Although the mechanisms leading to vascular leakage have been studied over several past decades, recent approaches have pointed new therapeutic targets in pre-clinical studies. In the pathogenesis of dengue hemorrhagic fever (DHF), many signaling pathways induce vascular permeability that result of disruption of blood brain barrier, that in some cases could allow viral penetration into the central nervous system (CNS). Most of these signaling pathways (including TNF-alpha) activate the overproduction active enzymes called matrix metalloproteinases (MMPs) that induce degradation of the extracellular matrix (ECM) and intercellular proteins governing vascular permeability. Among these enzymes, gelatinolytic MMPs, (MMP-9 and MMP-2), which are considered the most important class of MMPs since they are capable of degrading collagen type-IV, the main constituent of ECM, and cell junctions (PECAM-1 and VE-cadherin). Thus, in this study, we have developed two approaches to inhibit MMP-9 activity and protect the vascular permeability in vitro. Those approaches include, (i) in silico designed MMP-9 antagonists, where we had determined a lead MMP-9 blocker (HA048). The second approach is to synthesize derived lipophenolic compounds from resveratrol, that naturally exists in grape stalks, peanuts, and several other plants, which is capable of inhibiting the expression and activity of active MMP-9 inside the cells. Despite that resveratrol has several biological activities tested in vitro, its use in animal models is limited due to its poor bioavailability, rapid metabolism, and elimination. Therefore, this study has three main goals. First, is to find a novel compound capable of inhibiting MMP-9 activity in vitro. Second, is to verify whether it can preserve the endothelial monolayer integrity. Finally, is to move to the preclinical studies in dengue mouse model to verify its activity in vivo, toxicity, and bioavailability.Besides resveratrol and SB-3CT (commercial MMP-9 inhibitor), we found that resveratrol-linoleic acid, omega-6 (RES-LA), resveratrol-Docosanoic acid (RES-C22) and HA048 (in silico designed inhibitor) have demonstrated the highest MMP-9 inhibitory activity among dozens of synthesized compounds. However, RES-LA was preferably selected over RES-C22 due to for solubility reasons. Moreover, we formulated these compounds in novel solvents that could be compatible and less toxic for in vivo experiments, which is known as natural deep eutectic solvent (NADES) based on 1,2-propanediol:ChCl:water (1:1:1) (NADES/PCW).Results of in vitro vascular permeability revealed that MMP-9 inhibitors have decreased TNF-α induced-exacerbated endothelial permeability in HUVEC, measured in both real-time impedance sensing and fluorescence count. In addition, microscopic examination of cell junction proteins showed that CD31/PECAM-1 were more adherent and attached between HUVEC treated with both TNF-α and gelatinase inhibitors comparing to the positive control (TNF-alpha).The mechanisms of action of resveratrol derived lipophenols were assessed. Our findings revealed that resveratrol derived lipophenols have inhibited MMP-9 expression by attenuating the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinases (JNK) mitogen-activated protein kinases (MAPK).

Le TRALI représente un œdème pulmonaire lésionnel aigu survenant au cours d’une transfusion. Son mécanisme étiopathologique encore très imprécis conduit aujourd’hui à une sous-estimation de son incidence. Des études clinico-anatomiques ont souligné le rôle central des polynucléaires neutrophiles (PMN) en montrant que le TRALI résulte de l’accumulation de PMN au contact de l’endothélium lésé des capillaires pulmonaires. De nombreux investigateurs ont tenté de définir le facteur déclenchant présent dans le produit sanguin transfusé et évoquèrent l’existence d’un conflit immunologique par infusion d’anticorps anti-HLA. En appui avec les données de la littérature, ce travail a pour but d’apporter une meilleure connaissance du mécanisme du TRALI afin d’en améliorer son diagnostic, sa prévention et la prise en charge du patient. Tout d’abord, nous confirmons l’implication des anticorps anti-HLA dans la survenue du TRALI en validant pour la première fois l’hypothèse du modèle en deux étapes: une première étape préalable est requise chez le patient présentant une situation clinique ou thérapeutique prédisposante qui aboutit à une pré-stimulation des PMN, puis une seconde étape, dépendante de l’apport d’anticorps anti-HLA lors de la transfusion, entraîne l’activation de la NADPH oxydase phagocytaire. Cela conduit à l’activation des PMN et la libération de dérivés réactifs de l’oxygène qui sont directement responsables de la lésion endothéliale pulmonaire et provoque une augmentation de la perméabilité endothéliale. Nous démontrons en plus l’existence d’un seuil d’anticorps anti-HLA nécessaire pour déclencher une forte activation des PMN. Enfin, nous avons mis en évidence un mécanisme d’activation des PMN par les anticorps anti-HLA faisant intervenir la formation de complexes immuns antigène – anticorps à la surface des PMN. Ces complexes immuns sont reconnus avec une affinité plus grande que les anticorps seuls par les récepteurs Fc des PMN. Cette double interaction au sein d’un même PMN pourrait favoriser la formation de cluster de récepteurs Fc activés au niveau de radeaux lipidiques, ce qui induirait une activation optimisée de ces récepteurs, entraînant une cascade de signalisation aboutissant à l’activation de la NADPH oxydase des PMN. Nos résultats constituent un rationnel scientifique solide pour accéder à une meilleure connaissance du TRALI. / TRALI represents an acute non-cardiogenic pulmonary oedema following blood transfusion. The unknown etiopathological mechanism of TRALI leads to an underestimation of the incidence. Clinical and anatomical studies highlighted the major role of neutrophils (PMN) and showed that TRALI results from an increased number of neutrophils within the pulmonary capillary endothelium. Many evidence suggest that antibodies recognizing human leukocyte antigens (HLA) present in the blood transfusion are the predominant trigger leading to TRALI. Towards theses findings, we investigated the precise mechanism in TRALI in order to get a better knowledge of its diagnosis, its prevention and the patient care. We confirm the major role of anti-HLA antibodies and validate for the first time the two-hit model: the first-hit related to the patient clinical condition leads to their PMN stimulation, followed in the second-hit by the infusion of blood products containing anti-HLA antibodies that activate the phagocytic NADPH oxidase. This event induces PMN activation and the release of reactive oxygen species that are directly responsible for the pulmonary endothelial damage and cause the endothelial permeability increase. We also demonstrate the cut-off of anti-HLA antibodies that raises PMN activation. Finally, we showed that both the antigen-binding and the Fc-binding systems to antibodies are needed to induce a major PMN activation. We found that the binding of anti-HLA antibodies to HLA antigens promote the formation of cluster of Fc receptors within lipid rafts. The translocation of Fc receptors into lipid rafts improve Fc receptors activation, leading to intracellular signal transduction and activation of effector functions, such as NADPH oxidase activation and release of reactive oxygen species involved in tissue damage.

L’insuffisance rénale aiguë (IRA) est une complication clinique associée à une mortalité significative. Parmi les diverses causes d'IRA, l'ischémie-reperfusion (IRI) est une étiologie importante, en particulier dans le contexte de la transplantation rénale. Les types de mort cellulaire programmée (MCP) activées dans l'IRA induite par IRI ont été étudiées par des nombreux groupes. L’atteinte tubulaire épithéliale est classiquement considérée comme le principal contributeur à l'IRA.En effet, plusieurs morts programmées de cellules tubulaires ont été démontrées dans la littérature. Cependant, les lésions endothéliales microvasculaires rénales attirent davantage l'attention en tant qu'inducteurs cruciaux de dysfonctionnement microvasculaire et de fibrose rénale progressive. Ainsi, certaines équipes de recherche, dont la nôtre a rapporté le développement de l'apoptose endothéliale rénale en association avec l’IRI. Le but de mon travail était donc de caractériser les types de mort cellulaire microvasculaires secondaires à l’IRI et leur contribution à la dysfonction rénale. Pour évaluer l'importance de l'apoptose dans l'IRA induite par IRI, nous avons utilisé un modèle murin d’IRI chez des souris caspase-3 knock-out (KO) et sauvages, avec clampage de l'artère rénale pendant 30 minutes (modèle IRA légère) ou 60 minutes (modèle IRA sévère). Dans le modèle IRA légère, notre résultat montre que la carence en caspase-3 empêche la mort apoptotique des cellules endothéliales dans toutes les phases de l'IRA, atténuant la raréfaction microvasculaire, le dépôt de collagène et la fibrose rénale. L’absence de caspase-3 favorise aussi le maintien d’une perméabilité endothéliale microvasculaire normale à long terme. Toutefois, l’invalidation de la caspase-3 aggrave la mort cellulaire tubulaire à court terme en favorisant la nécroptose, mais améliore l’homéostasie tubulaire à long terme grâce à la préservation des capillaires péritubulaires (PTCs) permettant un maintien de la perfusion tubulaire. En outre, le déficit en caspase-3 est également associé à un effet protecteur contre la raréfaction microvasculaire rénale, la fibrose rénale progressive, ainsi qu'une perméabilité endothéliale améliorée et une préservation de la fonction rénale dans le modèle d’IRA sévère. En conclusion, nos résultats démontrent l'effet crucial de l’apoptose endothéliale microvasculaire en tant qu'inducteur de dysfonctionnement microvasculaire rénal, de raréfaction microvasculaire et de fibrose rénale progressive dans la physiopathologie de l'IRA légère et sévère induite par l'IRI. Ils établissent aussi l’importance prédominante de l’atteinte microvasculaire plutôt que tubulaire épithéliale dans la prédiction de la perte de fonction rénale à long terme suite à une IRI. / Acute kidney injury (AKI) is a crucial clinical event, with increasing incidence and mortality. Among various pathogenesis of AKI, ischemia-reperfusion injury (IRI) is an important etiology, especially in the renal post-transplant scenario. The complex of programmed cell deaths (PCD) developed in IRI-induced AKI has been proven in a number of investigations. Renal tubular epithelial injury has been considered as the major contributor in AKI and multiple programmed tubular epithelial cell (TECs) deaths have been demonstrated in the literature. However, renal microvascular endothelial injury is attracting more attention as an important inducer of microvascular dysfunction and renal progressive fibrosis. Some investigators, including our team, have reported the development of renal endothelial apoptosis in the condition of ischemia. Apoptosis, a commonly known programmed cell death, has been elucidated in both renal TECs and microvascular endothelial cells (ECs) post-IRI and the activation of caspase-3 functions as the key effector of caspase-dependent apoptosis. To verify the importance of apoptosis in IRI- induced AKI, we applied the in vivo murine renal IRI model in wild-type and caspase-3 KO mice, with clamping the renal artery for 30 minutes (mild AKI model) or 60 minutes (severe AKI model). In regard to the mild AKI model, our result demonstrates that caspase-3 deficiency prevents ECs apoptotic death in all phases of AKI, attenuating microvascular rarefaction, collagen deposition, and renal fibrosis, while maintaining physical endothelial permeability in the long-term. Meanwhile, caspase-3 deletion aggravates tubular injury in the short-term by promoting TECs necroptosis but ameliorates long-term tubular injury through preserved peritubular capillaries (PTCs) function. Furthermore, caspase-3 deficiency also demonstrated a protective effect against renal microvascular rarefaction, progressive renal fibrosis, as well as enhanced endothelial permeability in the severe AKI model. Conclusively, our findings determine the crucial effect of microvascular endothelial apoptosis as an inducer of renal microvascular dysfunction, microvascular rarefaction, and progressive renal fibrosis in the pathophysiology of mild and severe AKI induced by IRI. Additionally, our results demonstrate the predominant importance of microvascular endothelial injury over tubular epithelial injury in predicting renal function loss at long-term post-IRI.