Glomerular Endothelial Injury Research Articles

Histopathologic and ultrastructural findings in oak (Quercus spp.) toxicity on 2 beef cattle farms in Colorado.

Natural oak toxicity, a phenomenon sporadically reported in the United States, is due to consumption of any part of most oak trees (Quercus spp.). Ruminants, mainly cattle, are disproportionately susceptible to oak toxicity. Toxicity is attributed to degradation of the oak plant hydrolysable tannins by rumen microbes and enzymes into absorbable low-molecular-weight metabolites, which are postulated to bind and damage endothelial cells by unknown mechanisms. The clinical manifestations of acute toxicosis are nonspecific or broadly suggestive of renal disease due to acute tubular injury. Here we document the clinical, gross, histopathologic, and novel ultrastructural features of natural acute oak nephrotoxicity in 3 beef calves on 2 farms in Colorado, USA. Gross postmortem findings included perirenal edema with renomegaly and hemorrhagic gastroenteritis. Histologically, renal tubular epithelial necrosis was severe, with hemorrhage and intratubular hyaline casts. Transmission electron microscopy revealed extensive involvement of proximal and distal convoluted tubules, with predominantly intact basement membranes, and glomerular and interstitial endothelial injury and necrosis. The ultrastructural details of toxic nephropathy and vasculopathy induced by oak metabolites in natural cases of bovine oak toxicosis have not been described previously, to our knowledge.

#1071 The effect and mechanism of SGLT2 inhibitor on improving inflammatory injury of glomerular endothelial cells in diabetes

Abstract Background and Aims ATP-binding cassette transporter A1 (ABCA1), which reduces intracellular cholesterol accumulation, is highly expressed in the kidney. Studies have shown that sodium-glucose co-transporter 2 inhibitors (SGLT2) inhibitors promote ABCA1 expression in multiple tissues and organs. However, the effect of SGLT2 inhibitors on ABCA1 expression in diabetic kidney disease (DKD) is unclear. In this study, the effect of empagliflozin, an SGLT2 inhibitor, on ABCA1 in glomerular endothelial cells(GECs) of DKD and the possible mechanisms linking ABCA1 to GECs inflammatory injury were investigated. Method Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. db/db mice were used as animal models of type 2 diabetes and the effects of SGLT2 inhibitors on ABCA1 were analyzed by treatment with empagliflozin. The effects of ABCA1 overexpression on glomerular lipid deposition and kidney injury were examined in a type 2 diabetic mouse model with ABCA1 overexpression (db/db + ABCA1 mice), and human renal glomerular endothelial cells (HRGECs) cultured in high glucose and high cholesterol conditions, which simulated type 2 diabetes in vitro. Results The expression of ABCA1 in the glomeruli of DKD patients and db/db mice was significantly decreased. Enhancing ABCA1 expression in the kidney tissue of diabetic mice by adenovirus transduction improved renal cholesterol deposition, inhibited the overactivation of C3a/C5a, and improved renal inflammatory injury. In addition, in cultured HRGECs under high glucose and high cholesterol conditions, ABCA1 deficiency increased the deposit of cellular cholesterol, contributed to overactivation of C3a/C5a, and induced inflammatory injury. Overexpression of ABCA1 enhancing cholesterol efflux or inhibition of C3a/C5a activation in vitro significantly protected against glomerular endothelial injury stimulated by high glucose and high cholesterol. Moreover, SGLT2 inhibitor empagliflozin up-regulated the expression of ABCA1 and down-regulated the levels of cholesterol, C3a/C5a and inflammatory cytokines in diabetic mice and HRGECs. Conclusion SGLT2 inhibitor (empagliflozin) might play a renal protective role by up-regulating ABCA1-mediated cholesterol outflow, inhibiting the activation of C3a/C5a, and then ameliorating the inflammatory injury of GECs.

Xanthine oxidoreductase inhibition ameliorates high glucose-induced glomerular endothelial injury by activating AMPK through the purine salvage pathway

Xanthine oxidoreductase (XOR) contributes to reactive oxygen species production. We investigated the cytoprotective mechanisms of XOR inhibition against high glucose (HG)-induced glomerular endothelial injury, which involves activation of the AMP-activated protein kinase (AMPK). Human glomerular endothelial cells (GECs) exposed to HG were subjected to febuxostat treatment for 48 h and the expressions of AMPK and its associated signaling pathways were evaluated. HG-treated GECs were increased xanthine oxidase/xanthine dehydrogenase levels and decreased intracellular AMP/ATP ratio, and these effects were reversed by febuxostat treatment. Febuxostat enhanced the phosphorylation of AMPK, the activation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1α and PPAR-α and suppressed the phosphorylation of forkhead box O (FoxO)3a in HG-treated GECs. Febuxostat also decreased nicotinamide adenine dinucleotide phosphate oxidase (Nox)1, Nox2, and Nox4 expressions; enhanced superoxide dismutase activity; and decreased malondialdehyde levels in HG-treated GECs. The knockdown of AMPK inhibited PGC-1α–FoxO3a signaling and negated the antioxidant effects of febuxostat in HG-treated GECs. Despite febuxostat administration, the knockdown of hypoxanthine phosphoribosyl transferase 1 (HPRT1) also inhibited AMPK–PGC-1α–FoxO3a in HG-treated GECs. XOR inhibition alleviates oxidative stress by activating AMPK–PGC-1α–FoxO3a signaling through the HPRT1-dependent purine salvage pathway in GECs exposed to HG conditions.

Thrombomodulin Protects Against Cell-Free Hemoglobin Mediated Acute Kidney Injury in Sickle Cell Anemia

Thrombomodulin (TM) is a transmembrane protein that plays a pivotal role in maintaining microvascular health. Functions of TM include 1) binding thrombin and inhibiting its interaction with fibrinogen, 2) augmenting protein C function, and 3) down-regulating complement activation. Reduced TM function has been implicated in several thrombotic microangiopathies, including atypical hemolytic uremic syndrome and disseminated intravascular coagulation (DIC) (PMID 29866818). Previous in vitro studies have demonstrated that cell-free hemoglobin (Hb) mediates loss of TM from the endothelial surface which, in turn, is associated with a higher risk of kidney injury in patients with sickle cell disease (PMID 34929051). Infusion of intact, soluble TM may restore TM's function in the microvasculature, as observed in critically ill patients with DIC (PMID 17059423). In transgenic sickle mice (Townes model, Jackson Laboratory; Bar Harbor, Maine), we investigated whether 1) cell-free Hb reduces TM function leading to glomerular endothelial and kidney injury and 2) if infusion of intact, soluble TM (Asahi Kasei Pharma ©; Tokyo, Japan) can restore TM function and reduce the toxic effects of cell-free Hb in the kidney. Sickle mice (4 male, 4 females per condition) were challenged with cell-free Hb (0.24g/kg intravenous [iv]) or normal saline followed 2 hours later by either infusion of TM (5mg/kg subcutaneous, 1mg/kg iv) or normal saline. Blood and urine samples were collected 24 hours after the challenge and the mice were sacrificed for histopathologic evaluation. In a separate set of similar experiments, sickle mice (3 males, 3 females per condition) received iv microbubbles (Vevo MicroMarker TM Contrast) 24 hours after the challenge to assess renal cortical blood flow by contrast-enhanced ultrasound (Vevo2100 system). Mean ± standard error of mean values are provided. Comparisons between Hb-only and Hb+TM conditions were made using the Mann-Whitney test. Vasculopathy: Infusion of cell-free Hb resulted in decreased TM antithrombotic function within the glomerulus; infusion of TM 2 hours after the challenge restored these functions (fibrinogen relative fluorescent intensity [RFI]: control, 24.2 ± 0.9; Hb-only, 40.6 ± 1.7; Hb+TM: 26.8 ± 1.6) and anti-complement function (C3 RFI: control, 12.9 ± 0.4; Hb-only, 19.3 ± 1.4; Hb+TM, 14.1 ± 0.5) (P≤0.007). Circulating vascular injury biomarkers rose in the cell-free Hb challenged sickle mice and this was abated with TM rescue for p-selectin (control, 271 ± 25 ng/mL; Hb-only, 533 ± 47 ng/mL; Hb+TM, 356 ± 34 ng/mL), VEGF (control, 59 ± 4 pg/mL; Hb-only, 93 ± 7 pg/mL; Hb+TM 69 ± 3 pg/mL), and vWF (control: 91 ± 9 ng/mL, Hb-only: 299 ± 31 ng/mL, Hb+TM: 183 ± 22 ng/mL) (P≤0.01). Kidney Damage: Urine biomarkers of tubular injury increased after the cell-free Hb challenge and improved with TM (KIM-1: control, 317 ± 66 pg/day; Hb-only, 1201 ± 142 pg/day; Hb+TM, 638 ± 78 pg/day; P=0.003) (NGAL: control, 234 ± 34 ng/day; Hb-only, 1624 ± 145 ng/day; Hb+TM, 767 ± 118 ng/day; P=0.003). An elevation in serum creatinine concentration is used to define acute kidney injury. The cell-free Hb challenge led to an acute rise in serum creatinine while the TM rescued mice had serum creatinine concentrations that were significantly lower than the Hb-only challenged mice (P=0.03) and similar to the control conditions (Figure 1A). Preliminary testing of TM administered 24 hours after the cell-free Hb challenge has also led to kidney protective effects as assessed by serum creatinine, p-selectin, and vWF (P < 0.05). Cortical Blood Flow: Peak enhancement, a measure of relative blood volume, was reduced in the cell-free Hb challenged mice compared to control conditions by contrast-enhanced ultrasound. The TM-rescued mice had improved peak enhancement compared to the Hb-only treated mice (P=0.03) and similar to control conditions (Figure 1B). In conclusion, cell-free Hb reduces TM function resulting in increased vascular and kidney injury and decreased cortical blood flow. Infusion of soluble TM 2 hours after the cell-free Hb challenge restores TM function, protects against vascular and kidney injury, and preserves cortical blood flow. Our data highlights TM as a potential candidate therapy to preserve vascular function and abate kidney injury in patients with sickle cell disease during severe vaso-occlusive or hyperhemolytic episodes, when concentrations of cell-free Hb increase several fold.

GPR56 Promotes Diabetic Kidney Disease Through eNOS Regulation in Glomerular Endothelial Cells.

Although glomerular endothelial dysfunction is well-recognized to contribute to the pathogenesis of diabetic kidney disease (DKD), the molecular pathways contributing to DKD pathogenesis in glomerular endothelial cells (GECs) are only partially understood. To uncover pathways that are differentially regulated in early DKD that may contribute to disease pathogenesis, we recently conducted a transcriptomic analysis of isolated GECs from diabetic NOS3-null mice. The analysis identified several potential mediators of early DKD pathogenesis, one of which encoded an adhesion G-protein coupled receptor-56 (GPR56), also known as ADGRG1. Enhanced glomerular expression of GPR56 was observed in human diabetic kidneys, which was negatively associated with kidney function. Utilizing cultured mouse GECs, we observed that GPR56 expression was induced with exposure to advanced glycation endproducts, as well as in high glucose conditions, and its overexpression resulted in decreased phosphorylation and expression of endothelial nitric oxide synthase (eNOS). This effect on eNOS by GPR56 was mediated by coupling of Gα12/13-RhoA pathway activation and Gαimediated cAMP/PKA pathway inhibition. The loss of GPR56 in mice led to a significant reduction in diabetes-induced albuminuria and glomerular injury, which was associated with reduced oxidative stress and restoration of eNOS expression in GECs. These findings suggest that GPR56 promotes DKD progression mediated in part through enhancing glomerular endothelial injury and dysfunction.

ELABELA/APJ Axis Prevents Diabetic Glomerular Endothelial Injury by Regulating AMPK/NLRP3 Pathway

ELABELA (ELA), a recently discovered peptide, is highly expressed in adult kidneys and the endothelium system. It has been identified as a novel endogenous ligand for the apelin receptor (APJ). This study aims to investigate the role of ELA in diabetic glomerular endothelial pyroptosis and its underlying mechanism. Initially, a significant decrease in ELA mRNA levels was observed in the renal cortex of db/db mice and high glucose–treated glomerular endothelial cells (GECs). It was also found that ELA deficiency in ELA+/− mice significantly accelerated diabetic glomerular injury, as shown by exacerbated glomerular morphological damage, increased serum creatine and blood urea nitrogen, and elevated 24-h urinary albumin excretion. In addition, in vivo overexpression of ELA prevented diabetic glomerular injury, reduced von Willebrand factor expression, restored endothelial marker CD31 expression, and attenuated the production of adhesive molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Furthermore, in vitro studies confirmed that treatment with ELA inhibited GEC injury by regulating the NOD-like receptor protein 3 (NLRP3) inflammasome, as indicated by blocking NLRP3 inflammasome formation, decreasing cleaved Caspase-1 production, and inhibiting interleukin-1β and interleukin-18 production. Moreover, in vitro experiments demonstrated that the protective effects of ELA in GECs during hyperglycemia were diminished by inhibiting adenosine monophosphate–activated protein kinase (AMPK) using Compound C or by APJ deficiency. Taken together, this study provides the first evidence that ELA treatment could prevent diabetic glomerular endothelial injury, which is partly mediated by the regulation of the AMPK/NLRP3 signaling pathway. Therefore, pharmacologically targeting ELA may serve as a novel therapeutic strategy for diabetic kidney disease.

#6294 UTILITY OF KIDNEY BIOPSY AND SPECTRUM OF BIOPSY PROVEN KIDNEY DISEASES IN PATIENTS WITH MALIGNANCY: A RETROSPECTIVE SINGLE-CENTER EXPERIENCE

Abstract Background and Aims Kidney biopsy may improve diagnostic accuracy and aid in the management of cancer patients with kidney diseases. However, literature on the utility of kidney biopsy in cancer patients and survivors is lacking. We aimed to evaluate the clinical characteristics of cancer patients who underwent kidney biopsy, as well as the histological spectrum of biopsy proven kidney diseases in this cohort. Method This was a single-center retrospective study of all patients who had native kidney biopsies at the Singapore General Hospital between 1st October 2015 to 31st December 2022. Demographic, clinical, laboratory and histological data were retrieved from electronic medical records to identify patients with solid-organ or hematological malignancies who underwent kidney biopsy. Results Out of 1208 patients who underwent kidney biopsy between 1st October 2015 to 31st December 2022, 116 (9.6%) patients had diagnoses of solid-organ or hematological malignancies. Approximately two-thirds (79/116; 68.1%) had solid-organ malignancies, of which 21.5% had metastatic disease at time of biopsy. Approximately one-third (36/116; 31.0%) had hematological malignancies, of which 10 (8.6%) had a hematological stem-cell transplant (HSCT). One (0.9%) patient had both solid-organ and hematological malignancies. Sixty-eight (58.6%) patients were in remission at time of biopsy, comprising of 52 (44.8%) and 16 (13.8%) patients with solid -organ and hematological malignancies, respectively. Median age at biopsy was 65.7 years (IQR 56.5, 71.4) and majority was female (61/116; 52.6%). Comorbidities of hypertension, type 2 diabetes mellitus, autoimmunity, and liver transplant were found in 36.2% (42/116), 23.3% (27/116), 8.6% (10/116) and 1.7% (2/116) patients respectively. Median serum creatinine at time of biopsy was 152.0μmol/L (IQR 101.8, 220.5). Micro-hematuria was present in approximately two-thirds (68.1%) of patients. Nephrotic range proteinuria and nephrotic syndrome were observed in one-quarter (25.9%) and one-third (31.0%) of the patients, respectively. Approximately half (49.1%) of the patients had acute kidney injury at time of biopsy, while one-fifth (19.8%) had progressive chronic kidney disease. A wide spectrum of histological diagnoses was observed. Glomerulonephritides, tubulointerstitial inflammation and/or glomerular endothelial injuries were diagnosed in 60.8% (48/79) of solid-organ and 80.6% (29/37) of hematological malignancies. Amongst patients with active solid-organ malignancies (n = 20), acute tubulointerstitial nephritis (ATIN) (5/20, 25.0%), IgA nephropathy (3/20; 15.0%) and lupus nephritis (3/20; 15.0%) were the most common primary diagnoses. Amongst patients with active hematological malignancies (n = 17), dysproteinemic kidney disease and/or leukemic infiltration was most frequently diagnosed (6/17; 35.9%). Compared to remission status, active malignancy was not associated with an increased likelihood of an overall diagnosis of glomerulonephritides, tubulointerstitial inflammation and/or glomerular endothelial injuries (p = 0.16). Specifically, the incidence of membranous nephropathy was similar in patients with active malignancy and those in remission (p = 0.42). Amongst patients with HSCT, thrombotic microangiopathy (40.0%) was observed most frequently. Overall, onco-therapeutics-related kidney injuries were present in 13 (11.2%) patients, of which ATIN (38.4%) was most commonly encountered, followed by glomerular endothelial injury (30.7%). Conclusion Kidney biopsy provides valuable diagnostic information amongst cancer patients. While a broad spectrum of histological diagnoses can be observed, glomerular diseases and onco-therapeutics-related kidney injuries remain the most common.

Kidney injury molecule-1 and podocalyxin dysregulation in an arginine vasopressin induced rodent model of preeclampsia

ObjectiveTo assess renal injury in an arginine vasopressin (AVP) rodent model of preeclampsia. Study designUrinary expression of kidney injury molecule-1 (KIM-1), urinary protein and creatinine was determined in rodents (n = 24; pregnant AVP, pregnant saline, non-pregnant AVP and non-pregnant saline), which received a continuous dose of either AVP or saline via subcutaneous mini osmotic pumps for 18 days, using a Multiplex kidney toxicity immunoassay. Renal morphology was assessed using haematoxylin and eosin staining and transmission electron microscopy. The immunolocalization of KIM-1 and podocalyxin was qualitatively evaluated using immunohistochemistry. ResultsUrinary KIM-1 and urinary protein levels were significantly increased in treated vs. untreated rats on gestational days 8 (p < 0.05), 14 (p < 0.001) and 18 (p < 0.001). The pregnant rats displayed a lower trend of creatinine compared to the non-pregnant groups, albeit non-significantly. KIM-1 was immunolocalized in the proximal convoluted tubules in AVP treated vs. untreated groups. In contrast, podocalyxin was weakly immunostained within glomeruli of pregnant AVP treated vs. pregnant untreated rats. Histological evaluation revealed reduced Bowman’s space, with some tubular and blood vessel necrosis in the pregnant treated group. Ultrastructural observations included effacement and fusion of podocyte foot processes, glomerular basement membrane abnormalities, podocyte nuclear crenations, mitochondrial oedema and cristae degeneration with cytoplasmic lysis within treated tissue. ConclusionOur findings demonstrate region-specific kidney injury particularly glomerular impairment and endothelial injury in AVP-treated rats. The findings highlight the utility of this model in studying the mechanisms driving renal damage in a rodent model of preeclampsia.

PLVAP as an Early Marker of Glomerular Endothelial Damage in Mice with Diabetic Kidney Disease.

Plasmalemma vesicle-associated protein (PLVAP) is the main component of endothelial diaphragms in fenestrae, caveolae, and transendothelial channels. PLVAP is expressed in the adult kidney glomerulus upon injury. Glomerular endothelial injury is associated with progressive loss of kidney function in diabetic kidney disease (DKD). This study aimed to investigate whether PLVAP could serve as a marker for glomerular endothelial damage in DKD. Glomerular PLVAP expression was analyzed in different mouse models of DKD and their respective healthy control animals using automatic digital quantification of histological whole kidney sections. Transgenic mice expressing a dominant-negative GIP receptor (GIPRdn) in pancreatic beta-cells as a model for diabetes mellitus (DM) type 1 and black and tan brachyuric (BTBR) ob/ob mice, as a model for DM type 2, were used. Distinct PLVAP induction was observed in all diabetic models studied. Traces of glomerular PLVAP expression could be identified in the healthy control kidneys using automated quantification. Stainings for other endothelial injury markers such as CD31 or the erythroblast transformation-specific related gene (ERG) displayed no differences between diabetic and healthy groups at the time points when PLVAP was induced. The same was also true for the mesangial cells marker α8Integrin, while the podocyte marker nephrin appeared to be diminished only in BTBR ob/ob mice. Glomerular hypertrophy, which is one of the initial morphological signs of diabetic kidney damage, was observed in both diabetic models. These findings suggest that PLVAP is an early marker of glomerular endothelial injury in diabetes-induced kidney damage in mice.

PS-BPC02-3: EVALUATION OF VASCULAR FUNCTION FOR PREDICTION OF RENAL PROGNOSIS IN JAPANESE PATIENTS WITH LIFESTYLE-RELATED DISEASES.

Background: Since chronic kidney disease (CKD) is associated with the development of serious cardiovascular diseases such as stroke, coronary artery disease and heart failure, prevention and early diagnosis of CKD are important clinical issues. Many risk factors including diabetes, hypertension, dyslipidemia, obesity, and cigarette smoking are known to be involved in the development of CKD; however, there is no available biomarker that can predict the progression of CKD at an early stage. Although it has been reported that glomerular endothelial injury causes development of CKD, it remains unclear whether evaluation of vascular endothelial function can predict renal prognosis. Therefore, in the present study, we examined the association between vascular endothelial function and longitudinal eGFR change in patients with lifestyle-related diseases. Methods: We retrospectively evaluated 341 Japanese patients (176 males and 165 females; mean age, 63.87 ± 12.02 years) with lifestyle-related diseases in whom baseline vascular function was evaluated by flow-mediated dilation (FMD) and brachial-ankle pulse wave velocity (baPWV) and in whom 24-month longitudinal eGFR values were recorded after vascular testing at Tokushima University Hospital and Anan Medical Center, Tokushima Japan. The association between values of FMD and eGFR slope and the association between values of baPWV and eGFR slope were statistically analyzed. Statistical significance was set at p &lt; 0.05. Results: Single regression analysis showed that value of FMD was positively associated with eGFR slope in females (p &lt; 0.001) and non-smoking males (p = 0.033) but not in smoking males (p = 0.133). Multiple regression analysis including confounding factors demonstrated that the value of FMD remained a contributor against annual eGFR decline in females (p = 0.002) and non-smoking males (p = 0.046) but not in smoking males (p = 0.266). On the other hand, the value of baPWV had no significant association with eGFR slope regardless of sex and smoking. Conclusions: In patients with lifestyle-related diseases, FMD value can be used for prediction of renal prognosis represented by the eGFR slope in females and non-smoking males. Thus, early comprehensive intervention for improving vascular endothelial function might lead to a better renal prognosis in female and non-smoking male patients with lifestyle-related diseases.

ABCA1 deficiency-mediated glomerular cholesterol accumulation exacerbates glomerular endothelial injury and dysfunction in diabetic kidney disease

BackgroundHyperglycemia and dyslipidemia are two major characteristics of diabetes. In this study, the effects of glomerular cholesterol accumulation primarily due to ABCA1 deficiency on glomerular endothelial injury in diabetic kidney disease (DKD) and the possible mechanisms were investigated. MethodsThe effects of ABCA1 deficiency on glomerular lipid deposition and kidney injury were examined in a type 2 diabetic mouse model with ABCA1 deficiency in glomerular endothelial cells (DM-ABCA1−/− mice) and human renal glomerular endothelial cells (HRGECs) cultured in high glucose and high cholesterol conditions, which simulated type 2 diabetes in vitro. ResultsABCA1 deficiency in glomerular endothelial cells exacerbated renal lipid deposition and kidney injuries in type 2 diabetic mice and manifested as increased creatinine levels, more severe proteinuria, mesangial matrix expansion and fusion of foot processes, and more pronounced renal inflammatory injury and cell death. In HRGECs cultured under high glucose and high cholesterol conditions, ABCA1 deficiency increased the deposition of cellular cholesterol, contributed to inflammation and apoptosis, damaged the endothelial glycocalyx barrier, and induced endoplasmic reticulum stress (ERS). Conversely, ABCA1 overexpression enhancing cholesterol efflux or inhibition of ERS in vitro, significantly protected against glomerular endothelial injury stimulated by high glucose and high cholesterol. ConclusionsThese findings establish a pathogenic role of ABCA1 deficiency in glomerular endothelium injury and dysfunction and imply that ABCA1 may represent a potential effective therapeutic target for early diabetic kidney disease.

Overexpression of Plasmalemmal Vesicle-Associated Protein-1 Reflects Glomerular Endothelial Injury in Cases of Proliferative Glomerulonephritis with Monoclonal IgG Deposits

Proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) occasionally presents refractory nephrotic syndrome resulting in poor renal prognosis, but its etiology is not fully elucidated. Given that glomerular endothelial cell (GEC) stress or damage may lead to podocytopathy and subsequent proteinuria, as in thrombotic microangiopathy (TMA), diabetic kidney disease, and focal segmental glomerulosclerosis, we investigated the evidence of glomerular endothelial injury by evaluating the expression of plasmalemmal vesicle-associated protein-1 (PV-1), a component of caveolae in the cases of PGNMID. We measured the immunofluorescent PV-1 intensities of 23 PGNMID cases and compared with those of primary membranoproliferative glomerulonephritis (MPGN) (n= 5) and IgA nephropathy (IgAN) (n= 54) cases. PV-1 localization was evaluated with Caveolin-1, and CD31 staining, and the ultrastructural analysis was performed using a low-vacuum scanning electron microscope (LVSEM). To check the association of podocyte injury, we also conducted 8-oxoguanine and Wilms tumor 1 (WT1) double stain. We then evaluated PV-1 expression in other glomerulitis and glomerulopathy such as lupus nephritis and minimal change disease. The intensity of glomerular PV-1 expression in PGNMID is significantly higher than that in the other glomerular diseases, although the intensity is not associated with clinical outcomes such as urinary protein levels or renal prognosis. Immunostaining and LVSEM analysis revealed that glomerular PV-1 expression is localized in GECs in PGNMID. 8-oxoguanine accumulation was detected in WT1-positive podocytes but not in PV-1-expressing GECs, suggesting GEC-derived podocyte injury in PGNMID. PV-1 overexpression reflects glomerular endothelial injury, which could be associated with podocyte oxidative stress in PGNMID cases.

MO165: The Controversial Role of Proteinuria and Urinary Output After Radical Nephrectomy in the Development Of Acute Kidney Injury: Double Agents

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is increasingly common and associated with adverse short- and long-term outcomes in oncological patients who require radical nephrectomy (RN) for the presence of renal cancer. As defined by the KDIGO Criteria classification system, the diagnosis of AKI is nowadays based on changes in the serum creatinine levels (sCr) or urine output. However, these parameters often underestimate the severity of renal damage and therefore recent insights have resulted in the implementation of proteinuria as means of more accurately assessing for AKI. Proteinuria represents indeed an indicator of both glomerular and renal endothelial injury in chronic disease (CKD), whereas in AKI the presence of proteinuria could be derived from a tubular dysfunction. In the RN scenario, it is still debatable if the development of a mild to severe proteinuria after surgery could be considered a marker of renal damage resulting in AKI or, on the opposite, could be a physiological sign of adaptive compensatory hyperfiltration promoted by the remanent counterlateral kidney. The aim of our study was to investigate if the development of a postoperative proteinuria plays a positive or negative role in the prevention of AKI in a consecutive cohort of nephrectomized patients affected by renal cancer. METHOD We enrolled a consecutive cohort of 131 patients who underwent RN due to the presence of a kidney mass suspected of malignancy from 2018 to 2021 in a tertiary care urological Institution. The following data were considered: age, gender, body mass index (BMI), smoke, TNM staging, Fuhrman grading, hypertension, diabetes, cardiovascular events, dyslipidaemia, medical therapy (ACE-inhibitors (ACEi), angiotensin II receptor blockers (ARBs), calcium antagonists, beta blockers and diuretics), proteinuria in 24 h, urine volume in 24 h, preoperative haematocrit and haemoglobin. Serum creatinine (s-Cr) values [Kinetic Picrate standardized (COBAS C 800) for IDMS] were collected before surgery (t 0) and at 24, 48 and 72 h after surgery and at dismissal (respectively t 1, t 2 and tf) to detect renal function fluctuations and the subsequent risk of AKI. GFR was estimated at baseline using CKD-EPI 2012 and MDRD formula. The level of proteinuria was measured at 24 h postoperative and was standardized on the urine volume. Each operated patient underwent the same pre-peri- and postoperative medical protocol in terms of surgical and anesthesiologic management. Patients affected by pre-existing medical nephropathies with proteinuria were excluded. Comparisons between groups were performed using the Kruskal–Wallis rank sum test for numerical variables and Pearson's Chi-squared test for categorical variables. Logistic regression was used to identify variables odds ratio for AKI onset after surgery. RESULTS Descriptive analysis is shown in Table 1. Our analysis surprisingly underlined that 66% of overall patients experienced AKI after surgery. Moreover, the analysis showed a significant difference (P value &amp;lt; .05) between the AKI cohort and the non-AKI group concerning basal serum creatinine (sCr), eGFR using both CKD-EPI and MDRD and postoperative urine volume. In particular, the lower the basal sCr or eGFR, the higher was the risk of developing AKI. Concerning the urine output, the median volume of AKI population was significantly less in respect to their counterpart, but far from the critical definition of oliguria or anuria (Table 1 and Figure 1). On the contrary, no evidence related to a possible prognostic role of proteinuria was underlined. CONCLUSION Our work underlined that proteinuria does not represent a valid biomarker for AKI development after RN. On the contrary, basal eGFR, serum creatinine and postoperative urinary volume could be promising tools to predict and follow the acute renal damage after RN.

Endothelial-specific loss of Krüppel-Like Factor 4 triggers complement-mediated endothelial injury

Thrombotic microangiopathy (TMA) in the kidney represents the most severe manifestation of kidney microvascular endothelial injury. Despite the source of the inciting event, the diverse clinical forms of kidney TMA share dysregulation of endothelial cell transcripts and complement activation. Here, we show that endothelial-specific knockdown of Krüppel-Like Factor 4 (Klf4)ΔEC, an anti-inflammatory and antithrombotic zinc-finger transcription factor, increases the susceptibility to glomerular endothelial injury and microangiopathy in two genetic murine models that included endothelial nitric oxide synthase knockout mice and aged mice (52 weeks), as well as in a pharmacologic model of TMA using Shiga-toxin 2. In all models, Klf4ΔEC mice exhibit increased pro-thrombotic and pro-inflammatory transcripts, as well as increased complement factors C3 and C5b-9 deposition and histologic features consistent with subacute TMA. Interestingly, complement activation in Klf4ΔEC mice was accompanied by reduced expression of a key KLF4 transcriptional target and membrane bound complement regulatory gene, Cd55. To assess a potential mechanism by which KLF4 might regulate CD55 expression, we performed in silico chromatin immunoprecipitation enrichment analysis of the CD55 promotor and found KLF4 binding sites upstream from the CD55 transcription start site. Using patient-derived kidney biopsy specimens, we found glomerular expression of KLF4 and CD55 was reduced in patients with TMA as compared to control biopsies of the unaffected pole of patient kidneys removed due to kidney cancer. Thus, our data support that endothelial Klf4 is necessary for maintenance of a quiescent glomerular endothelial phenotype and its loss increases susceptibility to complement activation and induction of prothrombotic and pro-inflammatory pathways.

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.

Glomerular endothelial injury following vaccination for SARS-CoV-2.

Glomerular endothelial injury following vaccination for SARS-CoV-2.

Relationship between Elevated Maternal Serum Podocalyxin Concentrations with Blood Pressure Values and Routine Laboratory Parameters in Preeclampsia

Summary Podocalyxin (PCX) is a glycoprotein member of the CD34 transmembrane sialomucin family and covers the surface of podocytes. It is assumed to be a marker of glomerular endothelial injury. In conditions, podocalyxin excretion in the urine is increased as a result of podocyte injury. Recent studies show that PCX is expressed not only in kidneys but also in the endothelial cells of other organs. Preeclampsia (PE) is characterized by new-onset hypertension, generalized endothelial injury, and glomerular alteration. Having this in mind, in the present study, we aimed to: (1) determine circulating PCX levels in sera of women with preeclampsia and normal pregnancy; (2) explore for an association between PCX and blood pressure and (3) investigate a possible relationship between PCX and routine laboratory markers of cardiac and renal injury/dysfunction. Fifty-five women with preeclampsia were examined. The mean age of patients was 24.9±6 years, and the mean age of the control group of 35 women with normal pregnancies was 24.7±5.4 years. The enzyme-linked immunosorbent assay (ELISA) was used to determine concentrations of PCX. Levels of serum PCX in preeclamptic women were statistically significantly higher than those in women with normal pregnancy: 2.66±0.67 vs. 2.40±0.33 ng/ml (p=0.03). Podocalyxin correlated with SBP (r=0.30; p=0.004), DBP (r=0.35; p=0.0007), uric acid (r=0.32; p=0.002), CPK (r=0.22; 0.03) and its isoenzyme CK-MB (r=0.21; p=0.04). Our results showed significantly higher levels of serum PCX in women with preeclampsia than in healthy pregnancy. Elevated podocalyxin levels are associated with an increase in blood pressure in preeclampsia. We found a relationship between PCX and routine laboratory indicators of cardiac (CPK and CK-MB) and renal injury/dysfunction (uric acid). PCX has the potential as a future preeclampsia diagnostic biomarker.

New Endothelial Mechanisms in Glomerular (Patho)biology and Proteinuria Development Captured by Intravital Multiphoton Imaging.

In the past two decades, intravital imaging using multiphoton microscopy has provided numerous new visual and mechanistic insights into glomerular biology and disease processes including the function of glomerular endothelial cells (GEnC), podocytes, and the development of proteinuria. Although glomerular endothelial injury is known to precede podocyte damage in several renal diseases, the primary role of GEnCs in proteinuria development received much less attention compared to the vast field of podocyte pathobiology. Consequently, our knowledge of GEnC mechanisms in glomerular diseases is still emerging. This review highlights new visual clues on molecular and cellular mechanisms of GEnCs and their crosstalk with podocytes and immune cells that were acquired recently by the application of multiphoton imaging of the intact glomerular microenvironment in various proteinuric disease models. New mechanisms of glomerular tissue remodeling and regeneration are discussed based on results of tracking the fate and function of individual GEnCs using serial intravital multiphoton imaging over several days and weeks. The three main topics of this review include (i) the role of endothelial injury and microthrombi in podocyte detachment and albumin leakage via hemodynamic and mechanical forces, (ii) the alterations of the endothelial surface layer (glycocalyx) and its interactions with circulating immune cells in lupus nephritis, and (iii) the structural and functional remodeling and regeneration of GEnCs in hypertension, diabetes, and other experimental injury conditions. By the comprehensive visual portrayal of GEnCs and the many other contributing glomerular cell types, this review emphasizes the complexity of pathogenic mechanisms that result in proteinuria development.

Myeloperoxidase immunohistochemical staining can identify glomerular endothelial cell injury in dense deposit disease

Previous studies have demonstrated residual complement-mediated deposits in repeat kidney biopsies of C3 glomerulopathies (C3G) (dense deposit disease (DDD) and C3 glomerulonephritis) following eculizumab treatment, despite some clinical improvement. With residual complement deposition, it is difficult to determine whether there is a reduced complement-mediated endothelial cell injury. We validated that myeloperoxidase (MPO) immunohistochemical staining identified glomerular endothelial cell injury in crescentic glomerulonephritis and C3G. We report that MPO staining in the glomerular endothelium of the post-treatment kidney biopsy was significantly reduced after 3years of eculizumab treatment and clinical improvement in a 5-year-old boy with initial DDD and secondary crescent formation. We find that immunostaining for MPO is a useful method to compare glomerular endothelial injury in C3G following eculizumab treatment. This finding also supports the notion that eculizumab, a C5 blocker, may not mainly block C3 deposits in the glomeruli but significantly blocks final activation of the complement cascade, thus reducing glomerular endothelial cell injury.

Roles of glomerular endothelial hyaluronan in the development of proteinuria.

Vascular endothelial cells are covered with glycocalyx comprising heparan sulfate, hyaluronan, chondroitin sulfate, and associated proteins. Glomerular endothelial glycocalyx is involved in protecting against induction of proteinuria and structural damage, but the specific components in glycocalyx that represent therapeutic targets remain unclear. Anti‐vascular endothelial growth factor (VEGF) therapy is associated with an increased risk of glomerular endothelial injury. This study investigated whether hyaluronan could provide a therapeutic target to protect against proteinuria. We conducted ex vivo and in vivo experiments to explore the effects of degrading glomerular hyaluronan by administering hyaluronidase and of supplementation with hyaluronan. We investigated hyaluronan expression using biotin‐labeled hyaluronan‐binding protein (HABP) in human kidney specimens or serum hyaluronan in endothelial injuries under inhibition of VEGF signaling. We directly demonstrated hyaluronan in glomerular endothelial layers using HABP staining. Ex vivo and in vivo experiments showed the development of proteinuria after digestion of hyaluronan in glomerular capillaries. Supplementation with hyaluronan after hyaluronidase treatment suppressed proteinuria. Mice in the in vivo study developed albuminuria after intraperitoneal injection of hyaluronidase with decreased glomerular hyaluronan and increased serum hyaluronan. In human kidneys with endothelial cell dysfunction and proteinuria due to inhibition of VEGF, glomerular expression of hyaluronan was reduced even in normal‐appearing glomeruli. Serum hyaluronan levels were elevated in patients with pre‐eclampsia with VEGF signaling inhibition. Our data suggest that hyaluronan itself plays crucial roles in preventing proteinuria and preserving the integrity of endothelial cells. Hyaluronan could provide a therapeutic target for preventing glomerular endothelial glycocalyx damage, including VEGF signaling inhibition.