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Polycystic kidney disease (PKD) is the most common monogenic kidney disorder and an important cause of end stage renal disease. Vascular abnormalities are the most important non‐cystic complication and contribute to renal disease progression. Homocysteine (Hcy)‐mediated endothelial dysfunction (ED) and oxidative stress are evident early on, but the underlying mechanisms remain unknown. We hypothesized that abnormalities in Hcy metabolism are responsible for the increased levels, leading to Hcy‐induced ED from early stages of PKD. Methods We investigated the Hcy metabolic pathway (transulfuration/remethylation fig. 1) and downstream effects ‐main sources of renal tubular (RT) and endothelial cell (EC) reactive oxygen species (ROS), NADPH oxidase 4, (NOX4) and mitochondria‐ in 4‐week‐old PCK and Sprague‐Dawley (SD) control rats (n=12 each). Kidneys were harvested, frozen in liquid nitrogen, or preserved in formalin for metabolomics and ex‐vivo studies. Twenty‐four‐hour urine and terminal blood samples were collected for metabolite analysis and chemistries. Plasma folic acid, vitamin B12, and Hcy were determined by ELISA. Hcy pathway metabolites (betaine, dimethylglycine) were determined by 1 HNMR. Endothelial NO (eNOS) and oxidative stress were assessed by double immunofluorescence staining for CD31/eNOS and CD31/NOX4, respectively. RT and EC mitochondrial structure was assessed by electron microscopy. Results Serum creatinine and BUN remained unchanged, yet kidney weight/body weight ratio were increased in PCK. eNOS immunoreactivity was lower in PCK vs SD. Plasma Hcy and urine excretion of methyl donor betaine were elevated in PCK, and Hcy positively correlated with urine betaine. Plasma betaine was similar between the groups, but its tissue concentration was lower in PCK, which may lead to betaine insufficiency, impaired betaine dependent remethylation, and hyperhomocysteinemia. Contrarily, tissue glutathione concentration was higher in PCK vs SD, and plasma folate and vitamin B12 were similar, arguing against a defect in the transsulfuration or folate‐dependent remethylation pathways. RT NOX4 expression and immunoreactivity were increased in PCK compared to SD, in association with renal oxidative stress. PCK had increased number of CD31/NOX4 positive cells in peritubular capillaries, indicating increased EC ROS production. Collecting duct and EC mitochondria were swollen and showed cristae remodeling. Conclusion In PKD, early elevation in Hcy is likely related to betaine insufficiency and impaired betaine‐dependent remethylation may be responsible for the early elevation of Hcy. NOX4 and mitochondria abnormalities may be important contributors to oxidative stress and disease severity. Our data suggests these may not only be early vascular disease markers, but also play an important pathogenic role in vascular complications associated with PKD, and contribute to renal disease progression. Support or Funding Information R25‐DK101405, NIDDK P30 DK090728, NIDDK P30 DK 106912 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background Mesenchymal stem/stromal cells (MSCs) release extracellular vesicles (EVs), which shuttle proteins to recipient cells, promoting cellular repair. We hypothesized that cardiovascular risk factors may alter the pattern of proteins packed within MSC‐derived EVs, blunting their capacity to preserve the post‐stenotic kidney in pigs with metabolic syndrome (MetS) and renal artery stenosis (RAS). Methods We compared the protein cargo of EVs to their parent MSCs obtained from pigs with MetS and Lean controls. Porcine MSCs were harvested from abdominal fat after 16 weeks of Lean‐ or MetS‐diet (n=5 each), and their EVs isolated. Following LC‐MS/MS proteomics, clustering analysis was performed to identify primary functional categories of proteins enriched in Lean‐ and MetS‐EVs. In addition, MetS pigs were studied after 16 weeks of RAS untreated or treated 4 weeks earlier with a single intrarenal delivery of allogeneic labeled Lean‐ or MetS‐EVs (2.5x10^5/Kg). Lean and MetS Sham served as controls (n=6 each). Single‐kidney volume, renal blood flow (RBF), and glomerular filtration rate (GFR) were studied in‐vivo (multi‐detector CT), and tubular injury (Periodic Acid Schiff) and fibrosis (Trichrome) ex‐vivo. Results Proteomics analysis identified 6,690 and 6,790 distinct proteins in Lean‐ and MetS‐EVs, respectively. Differential expression analysis revealed that 91 proteins were upregulated in Lean‐EVs and 130 proteins in MetS‐EVs compared to their parent MSCs (FDR<0.05 and log2 fold‐change EVs/MSCs>2). Proteins enriched exclusively in Lean‐EVs modulate pathways related to the MSC reparative capacity, including cell proliferation, differentiation, and activation, as well as transforming growth factor‐β signaling (Figure 1A). Contrarily, proteins enriched only in MetS‐EVs are linked to pro‐inflammatory pathways, including acute inflammatory response, leukocyte transendothelial migration, and cytokine production (Figure 1B). Blood pressure was elevated in all MetS groups and all RAS groups develop moderate, but significant stenosis of a similar degree (Table). EVs were detected in the stenotic‐kidney 4 weeks after injection (Figure 2A). Renal volume, RBF, and GFR, which increased in MetS compared to Lean (consistent with hyperfiltration), fell in MetS+RAS, but restored in pigs treated with Lean‐EVs. Lean‐EVs also attenuated tubular injury and renal fibrosis (Figure 2B). Yet, these renoprotective effects were blunted in pigs treated with MetS‐EVs. Conclusion MetS alters the protein cargo of porcine MSC‐derived EVs, selectively packaging specific pro‐inflammatory signatures. Intrarenal delivery of Lean‐EVs attenuated renal injury and fibrosis and improved function in pigs with MetS+RAS, whereas these beneficial effects were blunted in pigs treated with MetS‐EVs. Therefore, our observations suggest that approaches to improve the therapeutic efficacy of autologous MSC‐derived EVs might be useful for patients with MetS+RAS. Support or Funding Information This study was partly supported by the National Institutes of Health grant numbers: DK104273, HL123160, DK102325, and DK106427. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background: The mechanisms responsible for cardiac injury in the early stage of metabolic syndrome (MetS) remain unknown. Mitochondria are intimately associated with myofibrils, where the cytoskeleton functions as a linkage coordinator. We hypothesized that early MetS is characterized by cytoskeletal-mitochondrial disorganization, and that mitoprotection with mitochondria-targeted peptides (MTP) would preserve cytoskeletal-mitochondrial structure and attenuate myocardial injury in early swine MetS. Methods: Pigs were studied after 16wks of diet-induced MetS, MetS treated for the last 4wks with the MTP Elamipretide (0.1mg/kg SC q.d), and Lean controls (n=6 each). Cardiac function (multidetector CT) was assessed in-vivo, and mitochondrial structure (electron microscopy), the mitochondrial inner membrane phospholipid cardiolipin (mass spectrometry), cytoskeletal proteins, oxidative stress, and apoptosis ex-vivo. Results: MetS pigs developed hypertension and insulin resistance, yet cardiac function was preserved. MetS induced loss of desmin and tubulin that was paralleled by mitochondrial disorganization, decreased 18:2 cardiolipin, and increased oxidative stress and apoptosis. MTP slightly improved cardiolipin species profile, restored mitochondrial organization, and upregulated cytoskeletal proteins, attenuating apoptosis and oxidative stress. Conclusion: Early MetS leads to disorganization of the cardiomyocyte cytoskeletal- mitochondrial architecture and cardiac injury. MTP may provide a novel therapeutic potential for improving cardiac injury in early MetS, and potentially preventing future deterioration in cardiac function.

MRE is a noninvasive novel MRI modality that detects changes in tissue viscoelastic properties by imaging shear wave propagation. Renal artery stenosis (RAS) induces kidney fibrosis, yet we have previously shown that concomitant reductions in renal perfusion pressure mask an increase in cortical stiffness. We hypothesized that due to its lower vascular volume and turgor, change es in medullary elasticity in RAS would be detectable by MRE. MRE 3D elastograms were obtained in pigs after 10 weeks of RAS or matched controls (n=6 per group), and contrast‐enhanced computed tomography images obtained in the same pigs guided selection of MRE medullary regions of interest (Fig 1). Trichrome staining was used for computer‐aided quantification of medullary fibrosis ex‐vivo. The renal medulla was significantly more fibrotic (5.96±0.19 vs. 1.82±0.49%, p<0.001) and stiffer (10.9±0.84 vs. 7.88±0.26 kPa, p<0.05) in RAS compared to controls (Fig 1 and 2). The histological degree of medullary fibrosis directly correlated with MRE‐derived stiffness (R 2 =0.55, p<0.05, Fig 3). This study therefore suggests that renal medullary MRE may be useful for detection of chronic kidney fibrosis in RAS. Grant Funding Source: NIH DK73608, HL77131 , HL085307 , and EB001981

Percutaneous transluminal renal angioplasty and stenting (PTRS) for RVH may not revert the secondary cardiac dysfunction and remodeling. In experimental RVH, infusion of the tetrapeptide Bendavia (that inhibits mitochondrial permeability transition pore opening) during PTRS attenuated stenotic kidney inflammatory injury and improved its function. We hypothesized that adjunct Bendavia during PTRS would also attenuate cardiac remodeling after reversal of RVH. RVH was induced in 18 pigs, of which 6 were sham treated (RVH) and 12 underwent PTRS plus a 4h IV infusion of Bendavia (0.05 mg/kg) or vehicle (n=6, each); 6 normal pigs served as controls. Cardiac oxygenation and function were assessed 4 wks later by blood oxygen level‐dependent (BOLD)‐MRI and CT, and tissue studies performed. PTRS normalized blood pressure (Table). Systolic function was unchanged. Apoptotic activity (caspase‐3), hypoxia (BOLD‐R2*), inflammation (TNF‐α, IL‐6), and fibrosis (trichrome) remained elevated and diastolic function (E/A) impaired in PTRS+Vehicle, but all improved in PTRS+Bendavia (Figure). Thus, novel cardioprotective properties of Bendavia enhance cardiac recovery after reversal of experimental RVH, and may provide a novel therapeutic option.

8005 Background: CARTITUDE-1 (NCT03548207) is a phase 1b/2 study evaluating ciltacabtagene autoleucel (cilta-cel; JNJ-68284528), a CAR T-cell therapy with two BCMA–targeting single-domain antibodies, in patients (pts) with R/R MM. Here, we report updated results in pts with a longer duration (median 12.4 months) of follow-up. Methods: Eligible pts had MM and received ≥3 prior regimens or were double refractory to a proteasome inhibitor (PI) and immunomodulatory drug (IMiD), and had received a PI, IMiD, and anti-CD38 antibody. After apheresis, bridging therapy was permitted. Pts received a single cilta-cel infusion (target dose: 0.75×106 CAR+ viable T cells/kg; range 0.5-1.0×106) 5–7 days (d) after lymphodepletion (300 mg/m2 cyclophosphamide, 30 mg/m2 fludarabine daily for 3 d). The primary objectives were to characterize cilta-cel safety, confirm the recommended phase 2 dose (RP2D; phase 1b), and evaluate efficacy (phase 2). Cytokine release syndrome (CRS) was graded by Lee et al (Blood 2014) and neurotoxicity by CTCAE, v5.0 (in phase 1b). CRS and ICANS were graded by ASTCT criteria (in phase 2). Here, Lee et al and CTCAE v5.0 were mapped to ASTCT for CRS and ICANS, respectively. Results: As of Sept 1, 2020, 97 pts with a median of 6 prior lines received cilta-cel. Overall response rate per independent review committee (primary endpoint) was 97% (95% CI, 91–99), with 67% achieving stringent complete response (sCR). Median time to first response was 1 month (range, 1–9), and median time to CR or better was 2 months (range, 1–15). Responses deepened over time, and median duration of response was not reached. Of 57 pts evaluable for minimal residual disease (MRD) assessment, 93% were MRD-negative at 10-5. The 12-month progression-free survival (PFS) and overall survival rates (95% CI) were 77% (66–84) and 89% (80–94), respectively; median PFS was not reached. Grade 3/4 hematologic AEs ≥20% included neutropenia (95%), anemia (68%), leukopenia (61%), thrombocytopenia (60%), and lymphopenia (50%). CRS occurred in 95% of pts (4% grade 3/4), with median time to onset of 7 d (range, 1–12), and median duration of 4 d (range, 1–14, excluding 1 pt with 97-d duration). CRS resolved in all but one with grade 5 CRS/haemophagocytic lymphohistiocytosis. CAR T-cell neurotoxicity occurred in 21% of pts (grade ≥3, 10%). Fourteen deaths occurred during the study after cilta-cel infusion: none within the first 30 days, 2 within 100 days; and 12 more than 100 days post infusion, of which 5 were due to disease progression, and 4 due to treatment-related AEs. Conclusions: A single infusion of cilta-cel yielded early, deep, and durable responses in heavily pretreated pts with MM, with a manageable safety profile at the RP2D. Cilta-cel is under further investigation in other MM populations in earlier lines of therapy and in outpatient settings. Clinical trial information: NCT03548207.

Introduction: Subjects with renovascular hypertension (RVH) often manifest with metabolic syndrome (MetS), increasing risk for heart failure, yet the mechanisms underlying cardiac injury remain unknown. Reduced mitophagy and accumulation of impaired mitochondria have been linked to progression of heart failure. Hypothesis: We hypothesized that superimposition of MetS on RVH in swine inhibits myocardial mitophagy, aggravating mitochondrial damage, cardiac injury and dysfunction. Methods: Pigs were studied after 16 weeks of RVH (renal artery stenosis) with or without diet-induced MetS, and Lean controls (n=6 each). Cardiac function was assessed in vivo (multidetector-CT), and cardiac mitochondrial structure, function, mitophagy, and myocardial fibrosis ex vivo. Results: MetS groups developed obesity, whereas RVH groups developed hypertension (Table). Mitochondrial matrix density and ATP production were lower and H2O2 production higher in both RVH groups vs. Lean and MetS (Fig. AB). However, MetS+RVH failed to activate mitophagy and downregulate myocardial expression of mitophagy-related microRNAs achieved by Lean+RVH (Fig. CD). Myocardial expression of the mitophagy marker PTEN-induced kinase (PINK)-1 inversely correlated with myocardial fibrosis (R 2 =0.195, p=0.031). Cardiac fibrosis, hypertrophy, and diastolic dysfunction (decreased E/A ratio) were greater in MetS+RVH vs. Lean+RVH (Table, Fig. E). Conclusions: MetS aggravates myocardial mitochondrial damage and dysfunction in swine RVH. MetS+RVH failed to activate mitophagy, resulting in greater cardiac remodeling, fibrosis, and diastolic dysfunction. Mitochondrial injury and impaired mitophagy may constitute important mechanisms and potential therapeutic targets to ameliorate cardiac structural damage and dysfunction in patients with coexisting MetS and RVH.

Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell–derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-β–exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A1c, kidney function, and urine albumin excretion. However, senescence-associated β-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials.

Abstract Background: Guidelines suggest the use of the Khorana score to select patients with solid cancer receiving chemotherapy for thromboprophylaxis to prevent venous thromboembolism (VTE), but its performance in different types of cancers remains uncertain. Methods: The present analysis includes individual patient data from seven randomized controlled trials that had compared prophylactic (ultra)-low-molecular-weight heparin (LMWH) with placebo or observation in patients with solid cancer. The analysis addresses the performance of the continuous and dichotomized Khorana score in predicting the 6-month risk of VTE in the trial control groups, overall and in types of cancer studies, as well as the efficacy and safety of LMWH among patients with a high-risk Khorana score. Random effects meta-analysis provided the basis for summary estimates. Findings: In the 3,403 patients from the control groups included in the analyses, the mean age was 61 years, 59% were male, and 58% had lung cancer. During 6-months of follow-up, 188 patients (5.5%) developed VTE. Overall, the 6-month VTE incidence was 9.8% among high-risk Khorana score patients and 6.4% among low-to-intermediate risk patients (OR 1.6; 95%-CI 1.1-2.2). The dichotomous Khorana score performed differently in lung cancer patients (OR 1.1; 95%-CI, 0.72-1.7) than in those with other types of cancer (OR 4.4; 95%-CI, 2.7-7.3; P interaction=0.002). Among high-risk patients, LMWH decreased the risk of VTE by 64% compared to placebo or observation (OR 0.36; 95%-CI, 0.22-0.58). In the group of patients with types of cancer other than lung cancer and a high-risk Khorana score (N=619), the 6-month VTE incidence was 3.3% (95% CI, 1.4 to 7.7) among LMWH recipients and 13% (95% CI, 6.8 to 24) among those not receiving LMWH (OR 0.23, 95% CI, 0.11 to 0.46; P &lt;0.001). There was no difference in major bleeding (OR 1.2, 95% CI, 0.56 to 2.5). Interpretation: The Khorana score performs poorly in differentiating between those at high and low risk of VTE in patients with lung cancer, but is associated with a 4-fold increased risk of VTE in those with other types of cancer. Thromboprophylaxis is effective and safe in patients with a high-risk Khorana score. Funding: Canadian Institutes of Health Research knowledge synthesis grant, KRS 126594 Registration: International Prospective Register for Systematic Reviews (PROSPERO), CRD42013003526. Disclosures Van Es: Pfizer: Employment, Other: Comment: Dr. van Es reports personal fees from Pfizer as a member of their advisory board. These fees are unrelated to this project.. Crowther: Alexion: Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Leo Pharma: Research Funding; Pfizer: Honoraria; Portola: Consultancy; Shinogi: Consultancy. Macbeth: Cancer Research UK: Research Funding; Pfizer: Other: Provision of Dalteparin for FRAGMATIC trial. Griffiths: Pfizer: Consultancy, Other: Comment: I run an academic clinical trials unit, have received educational/investigator intiated research grants from companies that make these heparin agents. As consultant &gt; 3 years ago, advised Pfizer on clinical trial designs unrelated to this study., Research Funding. Streiff: Roche: Research Funding; Portola: Research Funding; Janssen Scientific Affairs, LLC: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding. Ageno: Daiichi Sankyo: Consultancy, Honoraria; Bayer AG: Consultancy, Honoraria, Research Funding; BMS-Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria. Bozas: PharmaMar: Honoraria. Maraveyas: Bayer: Other: Personal fees and conference attendance; Bristol-Myers Squibb: Other: Grants and personal fees; Leo Pharma: Other: Grants, personal fees and conference attendance; Pfizer: Other: Personal fees. Loprinzi: Bristol Myers: Other: Grant - unrelated to this project; Janssen: Other: Grant - unrelated to this project. McBane: Bristol Myers Squibb: Other: Research grant for cancer associated VTE. Schünemann: Canadian Institutes of Health Research: Research Funding.