Renal Endothelium Research Articles

Antihypertensive Potential of Pistacia lentiscus var. Chia: Molecular Insights and Therapeutic Implications.

Background: Hypertension poses a significant global health burden and is associated with cardiovascular morbidity. Chios mastic gum (CMG), derived from Pistacia lentiscus var. Chia, shows potential as a phytotherapeutic agent, due to its multifaceted beneficial effects. However, its anti-hypertensive effects and vascular, circulatory, and renal-related dysfunction, have not been thoroughly investigated. Herein, we aimed to explore the antihypertensive potential of CMG, focusing on vascular and renal endothelium, in vivo. Methods: Two models of hypertension in male rats, induced by Angiotensin II and Deoxycorticosterone acetate (DOCA)-high-salt administration, were utilized. CMG was administered at 220 mg/kg daily for four weeks after hypertension onset and blood pressure was measured non-invasively. Whole blood RNA sequencing, metabolomics, real-time PCR, and Western blot analyses of kidney and aorta tissues were additionally performed. Results: CMG significantly lowered systolic, diastolic, and mean blood pressure in both models. RNA sequencing revealed that CMG modulated immunity in the Angiotensin II model and metabolism in the DOCA-HS model. CMG downregulated genes related to oxidative stress and endothelial dysfunction and upregulated endothelial markers such as Vegfa. Metabolomic analysis indicated improved endothelial homeostasis via lysophosphatidylinositol upregulation. Conclusions: CMG emerges as a potent natural antihypertensive therapy, demonstrating beneficial effects on blood pressure and renal endothelial function.

O237: Conversion of human kidney tissue to Bombay type using FucOB enzyme

Abstract Introduction The Bombay phenotype (Oh) is characterised by an absence of H antigen expression on red blood cells and vascular endothelium. The Oh phenotype is rare but most prevalent in the Indian subcontinent where it poses significant challenges in performing blood transfusions and kidney transplants, as Oh recipients can only receive from Oh blood type donors. The enzyme α-1,2-fucosidase (FucOB) derived from Akkermansia muciniphila bacteria has been shown to hydrolyse blood group H antigens. This study examined the efficiency of the FucOB enzyme on O type human kidney tissue to obtain Bombay phenotype. Methods H antigen removal was investigated in formalin-fixed paraffin embedded (FFPE) human kidney cortical biopsies from blood group O-type donors (n=8). Biopsies were incubated at 37OC for 1h with FucOB enzyme at concentrations 0µg/ml, 0.05µg/ml, 0.1µg/ml, 0.5µg/ml and 5µg/ml and inactive enzyme (iFucOB; 5µg/ml). Quantitative immunofluorescence was used to determine co-localisation of H antigen and vascular endothelium (Ulex). Results Increasing concentrations of FucOB progressively removed H antigen from FFPE sections of renal cortex. When compared to untreated control and iFucOB, a FucOB concentration of 5µg/ml caused 95% loss of H antigen expression on renal vascular endothelium (p<0.0001; Friedman test with Dunn’s multiple comparisons test). Conclusion This is the first description of enzymatic cleavage of H antigen from human O-type kidney tissue. The next step is to deliver FucOB enzyme to human kidneys during normothermic machine perfusion. This strategy has the potential to dramatically increase the availability of ABO compatible kidneys from individuals with Bombay phenotype.

The Bioluminescent Modified Ham Test Identifies the Classical Pathway As the Major Driver of Complement Activation in Atypical Hemolytic Uremic Syndrome and Primary Antiphospholipid Syndrome

Introduction: The role of complement is increasingly recognized in disease pathogenesis, however diagnostic assays are limited by low predictive value, technical requirements, and inability to assess relative contributions of each pathway of complement (classical, alternative, or lectin). The traditional mHam was developed as a functional assay of complement-mediated eukaryotic cell death in atypical hemolytic uremic syndrome (aHUS). However, broader use of the assay has been limited by inter-operator dependence and technical requirements. We have developed a bioluminescent mHam that is robust and easily performed (no cell washes or exogenous reagent addition) and utilizes an autonomously bioluminescent HEK293 cell line to allow for real-time assessment of changes in metabolic health due to complement activation. Given both HEK293 and endothelial cells lack complement receptor 1 (CR1), a crucial regulator of the classical pathway (CP), this assay measures complement activity of greater relevance to disease pathogenesis. Methods: The bioluminescent mHam uses LiveLight (LL) HEK293 cells (490 BioTech) engineered to stably express the bacterial luciferase ( lux) and flavin reductase genes ( luxF) to create autonomous bioluminescence. A PIGA null clone (LL PIGA- HEK293) was created through proaerolysin selection (removing GPI-anchored complement regulators CD55 and CD59). To perform the assay, 40,000 cells were harvested with trypsin/EDTA and plated as triplicates in gelatin veronal buffer (GVB ++,80 µL). Serum (20 µL) was added after specified treatments (heat inactivation or inhibitor addition). Activity was monitored by serial luminescence measurements every 5 min at 37 °C. A relative luminescence of <25% at one hour is designated as positive based upon lowest replicate of any healthy control. Results: The bioluminescent mHam, unlike the original mHam, provides continuous measurement of complement activity (example tracing Figure 1). The complement specificity of the luminescent changes were demonstrated with heat inactivation, terminal complement blockade with eculizumab, and calcium/magnesium dependence. The relative contribution of CP to changes in luminescence was confirmed with the specific CP inhibitor sutimlimab as well as use of pathway specific buffers. Healthy control (HC) samples show a relative luminescence of 53.7% (95%CI 44.0-63.4) with no difference observed in acute thrombotic thrombocytopenic purpura (TTP) (mean 56.2%, 95%CI 41.2-71.2, p=>0.9999). We next utilized samples of aHUS patients known to be in remission, off complement inhibitor therapy. A large proportion of these patients have ongoing assay positivity even in the traditional mHam (9 of 13 in one unpublished cohort). The bioluminescent mHam shows that 5 of 6 aHUS patients in remission have ongoing positivity (mean 17.56%, 95%CI -10.5-45.6, p=0.0131). An acute aHUS sample showed similar results (17.8% after plasmapheresis). Unexpectedly, the relative luminescence is rescued by addition of either C1s or C5 inhibitors, but use of alternative pathway (AP) inhibitors has no significant rescuing effect ( Figure 1-2). Of aHUS patients tested while on complement inhibition, 91% (10/11) show greater than 88% blockade ( Figure 2 aHUS on therapy) . Finally, in one cohort, the traditional mHam was positive in 52% (14/27) of primary antiphospholipid syndrome (PAPS) cases and 15% (10/66) of secondary APS (SAPS) (unpublished data). Thus far, the bioluminescent mHam shows abnormal complement activity in 92% (12/13) of PAPS and 17% (2/12) of patients in SAPS ( Figure 2). Again, complement inhibition with C1s or C5 inhibitors abrogated the effect in a dose dependent manner. Conclusions: The bioluminescent mHam test is an exquisitely sensitive real-time measure of abnormal complement activity. It is easy to perform, more closely approximates the complement regulators on renal endothelium, can be utilized for therapeutic monitoring, and has already offered novel disease insights through the use of specific pathway inhibitors. Although aHUS is traditionally categorized as a disorder of the AP, these results suggest the “trigger” mediating disease pathology is the classical pathway. Also, we show that a large proportion of primary APS patients have abnormal CP activation responsive to C1s and C5 inhibition, suggesting the disease may be responsive to complement-based therapeutics.

Targeting Pharmacotherapies for Inflammatory and Cardiorenal Endpoints in Kidney Disease.

Inflammation is an important contributor to excess cardiovascular risk and progressive renal injury in people with chronic kidney disease (CKD). Dysregulation of the innate and adaptive immune system is accelerated by CKD and results in increased systemic inflammation, a heightened local vascular inflammatory response leading to accelerated atherosclerosis, and dysfunction of the cardiac and renal endothelium and microcirculation. Understanding and addressing the dysregulated immune system is a promising approach to modifying cardiorenal outcomes in people with CKD. However, targeted pharmacotherapies adopted from trials of non-CKD and cardiorheumatology populations are only beginning to be developed and tested in human clinical trials. Pharmacotherapies that inhibit the activation of the NOD-like receptor protein 3 inflammasome and the downstream cytokines interleukin-1 and interleukin-6 are the most well-studied. However, most of the available evidence for efficacy is from small clinical trials with inflammatory and cardiorenal biomarker endpoints, rather than cardiovascular event endpoints, or from small CKD subgroups in larger clinical trials. Other pharmacotherapies that have proven beneficial for cardiorenal endpoints in people with CKD have been found to have pleiotropic anti-inflammatory benefits including statins, mineralocorticoid receptor antagonists, sodium-glucose cotransporter 2 inhibitors, and glucagon-like peptide-1 agonists. Finally, emerging therapies in CKD such as interleukin-6 inhibition, small-interfering RNA against lipoproteins, aryl hydrocarbon receptor inhibitors, and therapies adopted from the renal transplant population including mammalian target of rapamycin inhibitors and T regulatory cell promoters may have benefits for cardiorenal and inflammatory endpoints but require further investigation in clinical trials.

#4941 A METABOLOMIC FINGERPRINT PERSPECTIVE OF GUT-DERIVED METABOLITES ON EARLY DIABETIC KIDNEY DISEASE IN TYPE 2 DIABETES MELLITUS PATIENTS

Abstract Background and Aims Type 2 diabetes mellitus (T2DM) is the first cause of end stage renal disease worldwide. Metabolite profiling is an emerging field of great significance, as T2DM prevalence is continuously rising. The subtle dynamics of gut microbiota derived metabolites may have important implications in diabetic kidney disease (DKD) development. Nitrogen metabolic pathway and retinoic acid signaling pathway seem to be involved in early pathogenic mechanisms of DKD. The aim of study was to discover new putative gut-derived biomarkers that interfere with early physio-pathological mechanisms in DKD, regarding the podocyte, tubule, and the renal endothelium. Method The serum and urine of 90 T2DM patients (P group) and 20 healthy subjects (C group), were assessed in a cross-sectional study, by ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UHPLC-QTOF-ESI+-MS) techniques. P group was divided in 3 subgroups (normoalbuminuria-P1, microalbuminuria-P2, macroalbuminuria-P3). Statistical analysis was performed by untargeted multivariate (PLSDA, VIP scores, Random Forest, Biomarker analysis) and univariate (One Way ANOVA, Biomarker Analysis) methods. Results Multivariate analysis, by which the P group vs. C group were compared, displayed: (1) in serum samples: increased levels (P>C) of all-trans retinoic acid, cysteine-S sulfate, oleoylglicine, threonylglycine and decreased levels (P<C) of phenylalanine, tyrosine, kynurenic acid; (2) in urine samples, there were increased levels of: all-trans retinoic acid, indoxyl suflate, serotonin sulfate and glycylprolylarginine. Subsequently, by applying univariate analysis, the biomarkers derived from gut microbiota were selected: (1) in serum: all-trans retinoic acid (C<P1<P2<3), phenylalanine, tyrosine, and kynurenic acid with levels decreasing progressively from C to P1-P2-P3 (2) in urine: all trans retinoic acid, indoxyl sulfate, and serotonin sulfate, showing good differentiation between C and P1, and slight differences between P1∼P2∼P3 subgroups. The study suggests that ATRAs dynamic is disturbed in the normoalbuminuric stage of DKD. While its urinary levels correlated significantly with albuminuria, the changes observed in P1 subgroup, in serum and urine, may be correlated to the loss of renal tubular tight junction as a result of claudin downregulation. Phenylalanine and tyrosine, a part of nitrogen metabolic pathway, are indicators of the new-onset DKD. Their levels were upregulated in serum vs. urine, indicating their possible involvement in early DKD when comparing C vs. P1-P2-P3. Kynurenic acid and indoxyl-sulfate result from tryptophan metabolism. Low levels in serum of kynurenic acid may suggest its possible implication in incipient endothelial dysfunction in DKD. Indoxyl-sulfate (IS) urinary excretion is dependent on albuminuria. The study shows an elevated excretion of IS, in the normoalbuminuric group compared to controls and may suggest a tubular damage that precedes the glomerular modifications in DKD. Conclusion UHPLC-QTOF-ESI+-MS untargeted analysis reveals a particular metabolite fingerprint in early DKD. The study describes all-trans retinoic acid, phenylalanine, tyrosine, kynurenic acid, and indoxyl sulfate, as potential key biomarkers involved in early DKD pathogenesis, their levels being expressed in P1 subgroup compared with C group, and P2, P3 subgroups.

Translocation of outer membrane vesicles from enterohemorrhagic Escherichia coli O157 across the intestinal epithelial barrier.

Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeny During Injury and Regeneration in a Murine Model of Renal Thrombotic Microangiopathy

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeny During Injury and Regeneration in a Murine Model of Renal Thrombotic Microangiopathy

Cyclocarya paliurus triterpenoids attenuate glomerular endothelial injury in the diabetic rats via ROCK pathway

Ethnopharmacological relevanceCyclocarya paliurus (Batal.) Iljinskaja. (C. paliurus) is a distinctive traditional Chinese herb, with remarkable hypoglycemic capacity. Emerging evidence suggested that glomerular endothelial injury is a crucial pathological process of diabetic kidney disease (DKD). Our previous research found that C. paliurus triterpenoids fraction (CPT) has ameliorative effects on DKD. However, whether C. paliurus could counteract the glomerular endothelial injury of DKD is still undefined. Aim of the studyWe aimed to investigate the effects of CPT on glomerular endothelial function and explore its underlying mechanisms with in vivo and in vitro experiments. Materials and methodsThe effects and possible mechanisms of CPT on glomerular endothelial injury in streptozotocin (STZ)-induced diabetic rats and H2O2-challenged primary rat glomerular endothelial cells were successively investigated. ResultsIn vivo, we found that CPT treatment obviously decreased the levels of blood glucose, microalbumin, BUN and mesangial expansion. Additionally, CPT could ameliorate renal endothelium function by reducing the content of VCAM-1 and ICAM-1, and blocking the loss of glycocalyx. In vitro, CPT could also alleviate H2O2-induced endothelial injury. Mechanistically, CPT remarkably increased the phosphorylation levels of Akt and eNOS, decreased the expression of ROCK and Arg2in vivo and in vitro. Noticeably, the favorable effects mediated by CPT were abolished following ROCK overexpression with plasmid transfection. ConclusionThese findings suggested that CPT could be sufficient to protect against glomerular endothelial injury in DKD through regulating ROCK pathway.

Urinary SARS-CoV-2 RNA Is an Indicator for the Progression and Prognosis of COVID-19.

Background: We aimed to analyze clinical characteristics and find potential factors to predict poor prognosis in patients with coronavirus disease 2019 (COVID-19). Methods: We analyzed the clinical characteristics and laboratory tests of COVID-19 patients and detected SARS-CoV-2 RNA in urine sediments collected from 53 COVID-19 patients enrolled in Renmin Hospital of Wuhan University from 31 January 2020 to 18 February 2020 with qRT-PCR analysis. Then, we classified those patients based on clinical conditions (severe or non-severe syndrome) and urinary SARS-CoV-2 RNA (URNA− or URNA+). Results: We found that COVID-19 patients with severe syndrome (severe patients) showed significantly higher positive rate (11 of 23, 47.8%) of urinary SARS-CoV-2 RNA than non-severe patients (4 of 30, 13.3%, p = 0.006). URNA+ patients or severe URNA+ subgroup exhibited higher prevalence of inflammation and immune discord, cardiovascular diseases, liver damage and renal dysfunction, and higher risk of death than URNA− patients. To understand the potential mechanisms underlying the viral urine shedding, we performed renal histopathological analysis on postmortems of patients with COVID-19 and found severe renal vascular endothelium lesion characterized by an increase of the expression of thrombomodulin and von Willebrand factor, markers to assess the endothelium dysfunction. We proposed a theoretical and mathematic model to depict the potential factors that determine the urine shedding of SARS-CoV-2. Conclusions: This study indicated that urinary SARS-CoV-2 RNA detected in urine specimens can be used to predict the progression and prognosis of COVID-19 severity.

Heme Induced Progressive Loss of Endothelial Protein C Receptor Promotes Development of Chronic Kidney Disease in Sickle Cell Disorders

Chronic kidney disease (CKD), widespread among the individuals with sickle cell disease (SCD), is a major contributor to early death in this patient population. The progressive deterioration of renal health in SCD is associated with chronic persistent intravascular hemolysis leading to anemia. We have previously reported that extracellular heme, released during acute intravascular hemolysis triggers clinically relevant acute kidney injury (AKI) in SCD mice (SS) (Blood (2020) 135 (13): 1044-1048). Although AKI is reversible, it is considered as a risk factor for CKD. The mechanistic approach elucidating the hemolysis driven pathogenesis of AKI-to-CKD transition in SCD is unknown. We found that CKD develops in the SS mice by progressive (1-10 months of age) increase in albuminuria (urinary albumin and creatinine ratio, uACR) and decrease in glomerular filtration rate (GFR) (n=5; p<0.001). Histopathology of the kidney showed age-dependent deterioration in renal peritubular vascular congestion in SS mice compared to that of the AA mice. Alongside, Evan's blue extravasation experiments showed that the SS mice are susceptible to vascular leakage that is correlated positively with age (Pearson r=0.98, p<0.001) and negatively with anemia (Pearson r= -0.46, p<0.05). We hypothesized that multiple acute hemolytic events may instigate persistent endothelial damage that ensues CKD development in SCD. To test this hypothesis, we intravenously infused vehicle or heme (14 μmoles/kg body weight; 5 times on alternate days) to 1-month old SS mice and monitored for 3 weeks following first heme injection. These mice developed severe albuminuria (n=5; p<0.01) with substantial loss of GFR (n=5; p<0.001), indicating heme induced CKD development. Next, we used ultrasound super-resolution (USR) imaging technology to determine renal microvascular changes in older SS mice (6-months) without heme challenge and in young SS mice (1-month) challenged with heme. Analysis of the USR data showed reduced renal blood volume (rBV) and substantial loss of vessel density in renal cortex as well as in corticomedullary areas of the older SS mice compared to the age-matched AA mice. Accordingly, multiple heme challenge reduced rBV and vessel density extensively in young SS mice comparable to the older SS mice without heme challenge. Since endothelial protein C receptor (EPCR) maintains vascular barrier integrity by activating protease activated receptor-1 (PAR1) signaling in the endothelium, we tested whether alterations in EPCR expression contribute to progressive endothelial damage in SS mice during CKD development. Using immunofluorescence microscopy, we determined that renal endothelium lacks expression of EPCR in older SS mice while younger SS mice retains EPCR cellular expression. In corroboration, infusion of heme in younger SS mice results in loss of renal endothelial EPCR. Shedding of EPCR from endothelium often results in a soluble form of EPCR (sEPCR). We found that SS mice had higher plasma levels of soluble EPCR (sEPCR) compared to their AA counterparts. While age dependent increase in plasma and urinary sEPCR were evident in SS mice (n=6; p<0.01), infusion of heme in younger SS mice results in significant increase in plasma sEPCR (n=6; p<0.01). In consistent with the mouse data, we discovered that the plasma sEPCR was significantly elevated in SCD patients compared to normal individuals (n=8-16; p<0.05). Moreover, the plasma sEPCR was significantly associated with the baseline albuminuria in a cohort of SCD patients (n=16; Pearson r=0.64; p<0.01). This study supports the conclusion that multiple hemolytic events may trigger CKD development in SCD by gradual loss of renal microvascular EPCR expression. Clinically, the sEPCR can be developed as risk factor for sickle CKD. Finally, our data suggest that restoration of EPCR function may protect SCD patients from CKD. DisclosuresNo relevant conflicts of interest to declare.

Зміни ендотелію судин нирок і показники ендотеліальної васкулярної дисфункції при системних автоімунних ревматичних хворобах

Метою роботи стала оцінка характеру морфологічних змін ендотелію капілярів й артеріол нирок при системному червоному вовчаку, ревматоїдному артриті, геморагічному васкуліті та мікроскопічному поліангіїті, зв’язок з клініко-лабораторними чинниками перебігу захворювань і вплив системної ендотеліальної дисфункції судин. Остання в цих групах хворих відповідно має місце в 39, 35, 52 та 100 % випадків, беручи участь в патогенетичних побудовах нефропатії, щільно пов’язана з системними клініко-інструментальними проявами ангіопатії, а проліферацію ендотелію капілярів й артеріол нирок, відкладання в ньому імуноглобулінів і компонентів комплементу залежно від нозологічних форм системних автоімунних ревматичних захворювань визначають рівні в крові судинного ендотеліального фактора зростання, ендотеліну-1, гомоцистеїну, Е- і Р-селектинів.

Slit2-Robo Signaling Promotes Glomerular Vascularization and Nephron Development.

Kidney function requires continuous blood filtration by glomerular capillaries. Disruption of glomerular vascular development or maintenance contributes to the pathogenesis of kidney diseases, but the signaling events regulating renal endothelium development remain incompletely understood. Here, we discovered a novel role of Slit2-Robo signaling in glomerular vascularization. Slit2 is a secreted polypeptide that binds to transmembrane Robo receptors and regulates axon guidance as well as ureteric bud branching and angiogenesis. We performed Slit2-alkaline phosphatase binding to kidney cryosections from mice with or without tamoxifen-inducible Slit2 or Robo1 and -2 deletions, and we characterized the phenotypes using immunohistochemistry, electron microscopy, and functional intravenous dye perfusion analysis. Only the glomerular endothelium, but no other renal endothelial compartment, responded to Slit2 in the developing kidney vasculature. Induced Slit2 gene deletion or Slit2 ligand trap at birth affected nephrogenesis and inhibited vascularization of developing glomeruli by reducing endothelial proliferation and migration, leading to defective cortical glomerular perfusion and abnormal podocyte differentiation. Global and endothelial-specific Robo deletion showed that both endothelial and epithelial Robo receptors contributed to glomerular vascularization. Our study provides new insights into the signaling pathways involved in glomerular vascular development and identifies Slit2 as a potential tool to enhance glomerular angiogenesis.

MO038SCARF EXPRESSION IN KIDNEY DISEASE

Background and AimsChronic kidney disease (CKD) is the most common risk factor for lethal COVID19 and the risk factor that most increases the risk of death of COVID19 patients. Additionally, acute kidney injury (AKI) is frequent in COVID19 and AKI increases the risk of death. However, the underlying cellular and molecular mechanisms of such increased risk are unclear. SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) are required for and/or regulate (in a positive or negative manner) coronary cell entry and/or viral replication. We have now studied changes in the expression of genes encoding for SCARF in the context of acute and chronic kidney disease.MethodData mining of in-house (experimental models of AKI -folic acid nephropathy- and CKD -Unilateral ureteral obstruction- in mice) and publicly available databases (Nephroseq, published single cell transcriptomics studies) of kidney tissue transcriptomics as well as the Protein Atlas database.ResultsOut of 28 SCARF genes identified by Singh et al (Cell Reports 2020), 26 were represented in the experimental AKI database. Of them 7 (27%) were differentially expressed during AKI (FDR <0.05), 4 of them upregulated and 3 downregulated (Figure 1.A). Additionally, 27 were represented in the experimental CKD database. Of them 17 (63%) were differentially expressed during experimental CKD, 6 of them upregulated and 11 downregulated (Figure 1.B). Two genes were consistently upregulated (Ctsl and Ifitm3) and two consistently downregulated (Tmprss2 and Top3b) in both experimental AKI and CKD (Figure 1.A and B). They encode cathepsin L, interferon induced transmembrane protein 3, transmembrane serine protease 2, DNA topoisomerase III beta, respectively. Single cell transcriptomics databases localized Ctsl expression mainly to podocytes and tubular cells while protein atlas showed clear tubular staining. The main site of Ifitm3 was endothelium in both datasets and it was also localized to leukocytes by single cell transcriptomics. Tmprss2 was mainly localized to tubular cells in both datasets while Top3b was widely expressed in parenchymal renal cells, endothelium and leucocytes in single cell transcriptomics. Increased kidney expression of Ifitm3 and decreased expression of Tmprss2 and Top3b were confirmed in diverse CKD datasets in Nephroseq.ConclusionBoth AKI and CKD are associated with differential expression of SCARF genes in kidney tissue, the impact of CKD appearing to be larger. Characterization of these changes and their functional impact in kidney tissue and beyond the kidneys may provide clues to the increased risk of severe or lethal COVID19 in kidney disease patients.Kidney SCARF gene expression

Phenotypic diversity and metabolic specialization of renal endothelial cells.

Complex multicellular life in mammals relies on functional cooperation of different organs for the survival of the whole organism. The kidneys play a critical part in this process through the maintenance of fluid volume and composition homeostasis, which enables other organs to fulfil their tasks. The renal endothelium exhibits phenotypic and molecular traits that distinguish it from endothelia of other organs. Moreover, the adult kidney vasculature comprises diverse populations of mostly quiescent, but not metabolically inactive, endothelial cells (ECs) that reside within the kidney glomeruli, cortex and medulla. Each of these populations supports specific functions, for example, in the filtration of blood plasma, the reabsorption and secretion of water and solutes, and the concentration of urine. Transcriptional profiling of these diverse EC populations suggests they have adapted to local microenvironmental conditions (hypoxia, shear stress, hyperosmolarity), enabling them to support kidney functions. Exposure of ECs to microenvironment-derived angiogenic factors affects their metabolism, and sustains kidney development and homeostasis, whereas EC-derived angiocrine factors preserve distinct microenvironment niches. In the context of kidney disease, renal ECs show alteration in their metabolism and phenotype in response to pathological changes in the local microenvironment, further promoting kidney dysfunction. Understanding the diversity and specialization of kidney ECs could provide new avenues for the treatment of kidney diseases and kidney regeneration.

Endothelial-Derived miR-17∼92 Promotes Angiogenesis to Protect against Renal Ischemia-Reperfusion Injury.

Damage to the renal microvasculature is a hallmark of renal ischemia-reperfusion injury (IRI)-mediated AKI. The miR-17∼92 miRNA cluster (encoding miR-17, -18a, -19a, -20a, -19b-1, and -92a-1) regulates angiogenesis in multiple settings, but no definitive role in renal endothelium during AKI pathogenesis has been established. Antibodies bound to magnetic beads were utilized to selectively enrich for renal endothelial cells from mice. Endothelial-specific miR-17∼92 knockout (miR-17∼92endo-/- ) mice were generated and given renal IRI. Mice were monitored for the development of AKI using serum chemistries and histology and for renal blood flow using magnetic resonance imaging (MRI) and laser Doppler imaging. Mice were treated with miRNA mimics during renal IRI, and therapeutic efficacies were evaluated. miR-17, -18a, -20a, -19b, and pri-miR-17∼92 are dynamically regulated in renal endothelial cells after renal IRI. miR-17∼92endo-/- exacerbates renal IRI in male and female mice. Specifically, miR-17∼92endo-/- promotes renal tubular injury, reduces renal blood flow, promotes microvascular rarefaction, increases renal oxidative stress, and promotes macrophage infiltration to injured kidneys. The potent antiangiogenic factor thrombospondin 1 (TSP1) is highly expressed in renal endothelium in miR-17∼92endo-/- after renal IRI and is a target of miR-18a and miR-19a/b. miR-17∼92 is critical in the angiogenic response after renal IRI, which treatment with miR-18a and miR-19b mimics can mitigate. These data suggest that endothelial-derived miR-17∼92 stimulates a reparative response in damaged renal vasculature during renal IRI by regulating angiogenic pathways.

USO DE ESTATINAS NO CONTROLE DA HIPERLIPIDEMIA PÓS-TRANSPLANTE RENAL: FATORES ASSOCIADOS E POSSÍVEIS BENEFÍCIOS EM UMA REVISÃO DO TIPO NARRATIVA

Preservation of graft function is the major concern in the follow-up of kidney transplant recipients. In contrast, those patients are subjected to cardiovascular disease, hypertension, dyslipidemia, diabetes mellitus, adverse effects of immunosuppressant drugs with altered lipoprotein levels and other metabolic disorders, which can contribute to graft loss and increased morbidity and mortality. Lipid lowering therapy with statins can offer protection to the renal graft endothelium, with consequently decreased rejection rates and improved quality of life. The present study aimed to approach the use of statins as lipid-lowering therapy in kidney transplant recipients, through a narrative review of the literature. A search was conducted on the following platforms: Pubmed, Scielo, LILACS, Cochrane Library and MedRxiv, which was followed by explanatory reading. Eleven scientific articles with the descriptors: ‘Kidney Transplantation’, ‘Hydroxymethylglutaryl-CoA Reductases Inhibitors’ and ‘Hypercholesterolemia’ were included. Of those studies, 45.6% (5) discuss the benefits of using statins to control lipid rates, 36.4% (4) describe the risks and benefits of using statins for damage progression, and 18% (2) report the effectiveness of statins associated with other drugs. The use of Hydroxymethylglutaryl-CoA Reductase Inhibitors (statins) has clinical relevance in kidney transplant recipients, with the main purpose to lowering the lipid levels, thus increasing the safety of treatment and promoting benefits for kidney transplant recipients.

Endothelial KCa 1.1 and KCa 3.1 channels mediate rat intrarenal artery endothelium-derived hyperpolarization response.

Endothelium-derived hyperpolarization (EDH)-mediated response plays an essential role in the control of kidney preglomerular circulation, but the identity of the K+ channels involved in this response is still controversial. We hypothesized that large- (KCa 1.1), intermediate- (KCa 3.1) and small (KCa 2.3) -conductance Ca2+ -activated K+ (KCa ) channels are expressed in the endothelium of the preglomerular circulation and participate in the EDH-mediated response. We study the functional expression of different K+ channels in non-cultured, freshly isolated native endothelial cells (ECs) of rat intrarenal arteries using immunofluorescence and the patch-clamp technique. We correlate this with vasorelaxant responses ex vivo using wire myography. Immunofluorescence revealed the expression of KCa 1.1, KCa 3.1 and KCa 2.3 channels in ECs. Under voltage-clamp conditions, acetylcholine induced a marked increase in the outward currents in these cells, sensitive to the blockade of KCa 1.1, KCa 3.1 and KCa 2.3 channels respectively. Isometric myography experiments, under conditions of endothelial nitric oxide synthase and cyclooxygenase inhibition, showed that blockade either of KCa 1.1 or KCa 3.1 channels was able to reduce the endothelium-derived vasorelaxation of isolated interlobar arteries, while their combined blockade completely abolished it. In contrast, blockade of KCa 2.3 channels did not reduce this vasorelaxant response, despite being functionally expressed in the endothelial cells. This study shows that KCa 1.1 and KCa 3.1 channels are functionally expressed at the renal vascular endothelium and play a central role in the EDH-mediated relaxation of kidney preglomerular arteries, which is important in the control of renal blood flow and glomerular filtration rate.

Carfilzomib-associated renal toxicity is common and unpredictable: a comprehensive analysis of 114 multiple myeloma patients

Carfilzomib (CFZ) is a non-reversible proteasome inhibitor approved for the treatment of patients with relapsed and refractory myeloma (RRMM). Its use has been associated with cardiovascular toxicity but although recently a signal of clinically significant renal complications has also been identified, it is less extensively investigated. We analyzed data of 114 consecutive patients with RRMM who received CFZ-based regimens. Renal complications not related to MM progression were observed in 19 (17%) patients; thrombotic microangiopathy (TMA) was seen in 6 (5%) patients, albuminuria >1 gr/day in 7 patients (6%) and at least grade 3 acute kidney injury (AKI) which could not be otherwise explained in 6 patients (5%). A total of 15 patients discontinued CFZ and dosing was reinitiated at a lower level in one patient with AKI. Albuminuria was associated with focal segmental glomerulosclerosis in the renal biopsy (performed in a total of 6 patients). Renal complications during CFZ therapy are common, occur mostly early and are unpredictable. A potential effect of CFZ on the renal endothelium could be implicated in the pathogenesis of these complications and may also share common pathophysiology with cardiovascular effects of CFZ.

UNCOMMON AMONGST COMMON: ANGIOTENSIN II-INDUCED PERIPHERAL ISCHEMIA IN REFRACTORY SEPTIC SHOCK

SESSION TITLE: Medical Student/Resident Critical Care Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Angiotensin (AT) II, a naturally occurring hormone, modulates blood pressure through direct vasoconstriction of the circulatory system and through stimulation of various catecholamines and vasopressin. Derived from the hydrolysis of AT I via the AT-converting enzyme (ACE) in the pulmonary & renal endothelium, AT II has been studied for cases of vasodilatory shock since the 1930’. Most recently, the ATHOS-3 trial demonstrated AT II’s utility as a rescue vasopressor agent, highlighting its catecholamine-sparing effects. CASE PRESENTATION: 34-year-old African American male presented with 3-day history of dyspnea, productive cough and an episode of hemoptysis. Upon presentation, he was febrile, tachycardic, hypotensive and hypoxic prompting endotracheal intubation and vasopressor support. Laboratory studies were significant for leukocytosis with bandemia, lactic acidosis, acute kidney injury, transaminitis & positive for influenza A. Despite aggressive critical care management with maximum ventilator requirements, broad-spectrum antibiotics, renal replacement therapy & proning, his overall condition continued to worsen. AT II was initiated on Day 2 of ICU admission for refractory hypotension secondary to septic shock. Approximately 48-72 hours after the initiation of AT II, he developed skin discoloration and blistering in all extremities secondary to both arterial and venous thromboemboli despite prophylaxis. The lesions ultimately progressed leading to gangrenous necrosis extending proximally to the torso. Unfortunately, on day 7 of ICU admission, patient suffered a cardiac arrest and expired despite ACLS efforts. DISCUSSION: In our patient, we exhibited similar results to those described in the ATHOS-3 trials. Administration of the AT II resulted in an overall improvement in the patient’s mean arterial pressure, systemic vascular resistance and reduced norepinephrine requirements. We also observed a concomitant improvement in serum lactate, indicating improvement in overall organ perfusion. There are no absolute contraindications to AT II administration, but ATHOS-3 did report an increased incidence of both venous and arterial thromboembolic events in patients who received AT II. Although AT II has been FDA approved for the management of refractory septic shock, more studies are necessary to define those populations most at risk of the adverse effects. CONCLUSIONS: AT II is an important vasopressor agent used in refractory shock; however, more studies are needed to understand the adverse effects in populations most at risk. Reference #1: National Center for Biotechnology Information. PubChem Database. Angiotensin II, CID=172198, https://pubchem.ncbi.nlm.nih.gov/compound/Angiotensin-II (accessed on Mar. 26, 2020) DISCLOSURES: No relevant relationships by Krupa Gandhi, source=Web Response No relevant relationships by Mohit Mody, source=Web Response

Abstract P060: High Salt Induces An Endothelial HDAC1-stimulating Circulating Factor Leading To Disrupted Renal Microvascular Nitric Oxide Signaling

High salt diet (HS) decreases endothelial nitric oxide (NO) signaling as described in human and rodent studies. We have previously shown that inhibition of HDAC1 restores NO signaling in the afferent arteriole of HS fed rats. It remains unknown, however, how HS initiates endothelial dysfunction and activates HDAC1. We first investigated if HS induced endothelial dysfunction is acutely regulated by a circulating factor. To test this, male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or 2 weeks of HS (4.0% NaCl). Afferent arteriole vasoconstrictor responses to the NOS inhibitor, L-NAME, were monitored using the in vitro, blood perfused juxtamedullary nephron preparation. Perfusing arterioles from NS fed rats with HS blood blunted constriction to L-NAME (88 ± 1% of Con) compared to perfusing with NS blood (76 ± 2% of Con; P=0.0003), but not completely as seen in arterioles from HS fed rats perfused with HS blood (96 ± 2% of Con; P=0.0095). HS arterioles perfused with NS blood had similar L-NAME-induced constriction (75 ± 2% of Con) compared to arterioles from NS fed rats perfused with NS blood (P=0.9107). These results suggest HS induces endothelial function through an acute-acting circulating factor. This was not due to increased plasma arginase activity (NS: 7.96 ± 3.73 U/L vs HS: 5.42 ± 1.83 U/L P=0.5813) which competes with NO synthase 3 for arginine, nor was it due to decreased superoxide scavenging capacity of the plasma (NS: 7.96 ± 3.73 U/L vs HS: 5.42 ± 1.83 U/L P=0.5813) as measured by a cytochrome c reduction based assay. We then investigated if HS increased endothelial HDAC1 activity. Renal endothelial cells were isolated via magnetic activated cell sorting from NS and HS fed rats and incubated in plasma from the same rat. HDAC1 activity was monitored as the MS-275 (HDAC1 specific inhibitor) inhibitable portion of total HDAC activity. We found HS significantly increased renal endothelium HDAC1 activity (NS: 0.38 ± 0.03 pmol/min vs HS:0.94 ± 0.19 pmol/min, P=0.01). We conclude that HS disruption of renal microvascular NO signaling is initiated by a circulating factor(s) that is dependent upon increased endothelial HDAC1 activity.