Chade, Alejandro

Abstract Background: Scattered tubular-like cells (STCs) are differentiated renal tubular cells that during recovery from ischemic injury dedifferentiate to repair other injured renal cells. Renal artery stenosis (RAS), often associated with chronic inflammatory injury, compromises the integrity and function of STCs, but the underlying mechanisms remain unknown. We hypothesized that RAS alters the transcriptomic and epigenetic profile of inflammatory genes in swine STCs. Methods: STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each). STC mRNA profiles of inflammatory genes were analyzed using high-throughput mRNA-sequencing (seq) and their DNA methylation (5mC) and hydroxymethylation (5hmC) profiles by DNA immunoprecipitation and next-generation sequencing (MeDIP-seq) (n=3 each), followed by an integrated (mRNA-seq/MeDIP-seq) analysis. STC protein expression of candidate differentially expressed (DE) genes and common proinflammatory proteins were subsequently assessed in vitro before and after epigenetic (Bobcat339) modulation. Results: mRNA-seq identified 57 inflammatory genes up-regulated in RAS-STCs versus Normal-STCs (>1.4 or <0.7-fold, P<0.05), of which 14% exhibited lower 5mC and 5% higher 5hmC levels in RAS-STCs versus Normal-STCs, respectively. Inflammatory gene and protein expression was higher in RAS-STCs compared with Normal-STCs but normalized after epigenetic modulation. Conclusions: These observations highlight a novel modulatory mechanism of this renal endogenous repair system and support development of epigenetic or anti-inflammatory therapies to preserve the reparative capacity of STCs in individuals with RAS.

Abstract Background and Aims Chronic kidney disease (CKD) is a progressive disorder that affects over 15% of adults worldwide. A major component of CKD pathophysiology is renal microvascular rarefaction and loss that pairs the progression of the disease and complications. We developed a translational swine model of CKD and showed that loss of renal function associates with a significant decrease in renal vascular endothelial growth factor (VEGF) signaling and progressive microvascular (MV) rarefaction. VEGF is a key endogenous cytokine for MV proliferation and repair, but underlying mechanisms of renal VEGF downregulation in CKD are unknown. We hypothesized that micro-RNA (miRNA)-mediated processes contribute to the downregulation of renal VEGF signaling in CKD. Method Unbiased renal micro-RNA (miRNA)-seq was performed in a swine model of CKD stage 3 (14 weeks of bilateral renal artery stenosis + atherogenic diet) and normal controls (n = 5/group). Micro-RNAs up- (>1.4 FC, p<0.05) and down-regulated (<0.7 FC, p<0.05) were identified, followed by target prediction analysis (miRwalk 3.0, Target Scan 7.2). Renal gene and protein expression of VEGF was measured by qPCR and western blot, respectively, and renal MV architecture was studied by 3D micro-CT. Results Unbiased miRNA-seq and target prediction analysis identified four miRs upregulated in the kidney of CKD pigs vs. controls, of which miR-451b (validated by qPCR), broadly conserved among species, was capable of targeting VEGFA (FC = 4.46, p = 0.04). Renal gene and protein expression of VEGF was downregulated in CKD vs. controls, associated with significant cortical and medullary microvascular rarefaction (Figure 1). Conclusion Our data suggest that post-transcriptional modulation of renal VEGF signaling may serve as a prominent mechanism of VEGF downregulation, which leads to MV rarefaction and disease progression in CKD. Furthermore, this work in progress identified a novel candidate (miR-451b) and may set the stage for its targeted modulation to preserve VEGF signaling as a potential strategy to protect the renal microvasculature in CKD.

Background: Chronic kidney disease (CKD), an independent risk factor for heart failure with preserved ejection fraction (HFpEF), is present in over 50% of CKD patients and accounts for over 50% of hospital admissions for HF. We showed in a novel swine model of CKD-HFpEF that cardiac abnormalities associate with upregulation of pro-inflammatory nuclear factor kappa B (NF-kB). NF-κB activation is controlled by its natural inhibitor IkBα, a protein encoded by the NFKBIA gene that prevents its nuclear translocation, but the exact mechanisms remain unknown. We hypothesize that micro-RNAs (miRNAs), small noncoding RNAs that regulate gene expression, downregulate cardiac NFKBIA and activates NF-κB contributing to cardiomyocyte injury in CKD-HFpEF. Methods: Unbiased cardiac miRNA-sequencing was performed in normal and CKD-HFpEF pigs (n=4/group, 2 males/2 females). Differentially expressed (fold change ≥2 and false discovery rate (FDR)≤0.05) miRNAs capable of targeting NFKBIA were identified using miRWalk 3.0 and Target Scan 8.0, further validated by qPCR. To study the impact of CKD-HFpEF on cardiac tissue, cardiomyocites (CM) were exposed for 48 hours to plasma from normal and CKD-HFpEF pigs (n=3 each) and cardiac injury (LDH release) evaluated, and selected miRNAs modulated (mimic, antagomiR). Results: All CKD-HFpEF pigs showed a significant reduction in renal hemodynamics and function compared to controls accompannied by left ventricular hypertrophy (LVH), abnormal diastolic relaxation and LV strain, and increased circulating ANP and BNP levels, underscoring successful developing of CKD-HFpEF.Unbiased miRNA-seq identified 402 miRNAs targeting NFKBIA, of which 13 were broadly conserved among vertebrates and only 4 expressed in pig’s cardiac tissue. Among them, only miR-532-3p was upregulated in myocardium of CKD-HFpEF pigs.Expression of miR-532-3p was also higher in CM exposed to CKD-LVDD plasma or miR-532-3p mimic, associated with down-regulation of NFKBIA and LDH release. Treatment with anti-miR-532-3p decreased miR-532-3p, increased NFKBIA expression, and reduced CM injury. Conclusions: These observations support a role of miR-532-3p in cardiac NF-kB activation in CKD-HFpEF. In turn, these proof-of-concept studies may assist to identify novel therapeutic targets and develop new interventions to protect the heart in CKD. NIH HL095638 (ARC), DK129240 (AE) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Autologous mesenchymal stem/stromal cells (MSCs) have demonstrated important therapeutic effects in several diseases. Cardiovascular risk factors may impair MSC mitochondrial structure and function, but the underlying mechanisms remain unknown. We hypothesized that metabolic syndrome (MetS) induces epigenetic alterations in mitochondria-related genes in swine MSCs. Pigs were fed a Lean or MetS diet (n = 6 each) for 16 weeks. MSCs were collected from subcutaneous abdominal fat, and DNA hydroxymethylation (5 hmC) profiles of mitochondria-related genes (MitoCarta-2.0) were analyzed by hydroxymethylated DNA immunoprecipitation and next-generation sequencing (hMeDIP-seq) in Lean- and MetS-MSCs untreated or treated with the epigenetic modulator vitamin (Vit)-C (n = 3 each). Functional analysis of genes with differential 5 hmC regions was performed using DAVID6.8. Mitochondrial structure (electron microscopy), oxidative stress, and membrane potential were assessed. hMeDIP-seq identified 172 peaks (associated with 103 mitochondrial genes) with higher and 416 peaks (associated with 165 mitochondrial genes) with lower 5 hmC levels in MetS-MSCs versus Lean-MSCs (≥2-fold, p < 0.05). Genes with higher 5 hmC levels in MetS + MSCs were primarily implicated in fatty acid metabolism, whereas those with lower 5 hmC levels were associated with electron transport chain activity. Vit-C increased 5 hmC levels in mitochondrial antioxidant genes, improved mitochondrial structure and membrane potential, and decreased oxidative stress. MetS alters 5 hmC levels of mitochondria-related genes in swine MSCs. Vit-C modulated 5 hmC levels in these genes and preserved mitochondrial structure and function in MetS-MSCs. These observations may contribute to development of strategies to overcome the deleterious effects of MetS on MSCs.

Chronic kidney disease (CKD) often leads to left ventricular diastolic dysfunction (LVDD) and heart failure. Using a novel translational swine model of CKD-LVDD, we characterize the cardiac epigenetic landscape, identifying site-specific 5-methylcytosine changes in vascular endothelial growth factor (VEGF)-related genes associated with impaired angiogenesis and cardiac microvascular rarefaction. These observations shed light on the mechanisms of cardiac microvascular damage in CKD-LVDD and may assist in developing novel therapies for these patients.

Chronic kidney disease (CKD) is a progressive disorder in which more than 50% of deaths are attributed to cardiovascular disease. Using a swine model of CKD that develops left ventricular dysfunction (CKD-LVDD), we characterize the cardiac micro-RNA and transcriptomic profile, identifying dysregulated genes associated with cardiac remodeling and fatty acid metabolism that might be post-transcriptionally regulated early in the disease. These findings pinpointed pathological pathways that may open new avenues toward therapeutic research to reduce cardiovascular morbidity in CKD.

Background: Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells endowed with progenitor-like characteristics to repair injured parenchymal cells. STCs may be damaged and rendered ineffective by renal artery stenosis (RAS), but the underlying processes remain unclear. We hypothesized that RAS alters the epigenetic landscape on DNA and the ensuing gene transcriptional profile of swine STCs. Methods: CD24+/CD133+ STCs were isolated from pig kidneys after 10 weeks of RAS or sham (n = 3 each) and their whole 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles were examined by 5mC and 5hmC immunoprecipitation sequencing (MeDIP-/hMeDIP-seq, respectively). A subsequent integrated (MeDIP/hMeDIP-seq/mRNA-seq) analysis was performed by comparing all online available gene sets using Gene Set Enrichment Analysis. Apoptosis, proteolysis, and mitochondrial structure and function were subsequently evaluated in vitro. Results: Differential expression (DE) analysis revealed 239 genes with higher and 236 with lower 5mC levels and 275 genes with higher and 315 with lower 5hmC levels in RAS-STCs compared to Normal-STCs (fold change ≥1.4 or ≤0.7, p ≤ 0.05). Integrated MeDIP-/hMeDIP-seq/mRNA-seq analysis identified several overlapping (DE-5mC/mRNA and DE-5hmC/mRNA levels) genes primarily implicated in apoptosis, proteolysis, and mitochondrial functions. Furthermore, RAS-STCs exhibited decreased apoptosis, mitochondrial matrix density, and ATP production, and increased intracellular amino acid concentration and ubiquitin expression. Conclusions: Renal ischemia induces epigenetic changes in apoptosis-, proteolysis-, and mitochondria-related genes, which correlate with alterations in the transcriptomic profile and corresponding function of swine STCs. These observations may contribute to developing novel targeted interventions to preserve the reparative potency of STCs in renal disease.

Abstract BACKGROUND AND AIMS Patients with chronic kidney disease (CKD) have a higher risk to develop heart failure with preserved ejection fraction (HFpEF). We showed in a swine model of CKD with diastolic dysfunction and cardiac remodelling that these cardiac features are associated with blunted cardiac mRNA and protein expression of vascular endothelial growth factor (VEGF), a pro-angiogenic cytokine that plays important roles in cardiac vascular development, morphogenesis and contractility. Hence, we aim to identify potential underlying mechanisms of cardiac VEGF downregulation in CKD. METHOD CKD and normal pigs (n = 3 each) were studied for 14 weeks. Cardiac morphology and function (echocardiography) were quantified in vivo and microvascular density (3D micro-CT) ex vivo. Then, cardiac micro-RNA (miRNA) and 5-hydroxymethylcytosine (5-hmC) profiles of VEGFA gene were examined by hydroxymethylated DNA immunoprecipitation and miRNA sequencing (hMeDIP-seq and miRNA-seq, respectively) and validated by qPCR and Western blotting. RESULTS Blunted renal function in CKD pigs associated with left ventricular (LV) hypertrophy, and abnormal LV strain and diastolic dysfunction with pEF (Figure 1A) and reduced subendocardial microvascular density (Figure 1B) compared to normal. miRNA-seq showed that mir-183 was the only miRNA capable of targeting VEGFA and upregulated in CKD hearts (Figure 1C), accompanied by downregulation of VEGFA gene and VEGF protein (Figure 1D), whereas hMeDIP-seq revealed reduced VEGFA 5hmC levels in the hearts of CKD versus normal pigs (Figure 1E). CONCLUSION Our study suggests that miRNA-modulation and cardiac epigenetic changes in VEGF might contribute to cardiac damage and the development of HF in CKD. These observations may assist in developing novel therapies, like miRNA antagonists or epigenetic modulators, to preserve VEGF signalling and protect the heart in patients with CKD.

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) is an independent risk factor for heart failure with preserved ejection fraction (HFpEF), a progressive condition that affects ˃50% of patients in dialysis. We developed a swine model of CKD-HFpEF and showed that renal inflammatory signaling plays a key role in HFpEF pathophysiology via cardiac activation of nuclear factor-kappa B (NF-kB). However, the mechanisms of CKD-induced cardiac NF-κB activation remain unclear. We hypothesized that micro-RNA (miRNA)-mediated processes contribute to cardiac NF-kB activation in CKD-HFpEF. METHOD Using an in-silico approach (miRWalk3.0 and TargetScan 7.2), we identified all miRNAs capable of targeting the NFKB Inhibitor Alpha (NFKBIA) gene. We then identified those expressed in swine myocardium and measured their levels in normal and CKD pigs by qPCR. In a biomimetic heart culture system that mimics the cardiac CKD-HFpEF microenvironment, pig heart slices were exposed to plasma from normal or CKD pigs, and cardiac kinetics, NF-kB signaling, and miRNA expression levels were investigated in vitro. RESULTS We identified 5 miRNAs expressed in swine hearts capable of targeting NFKBIA, of which only miR-1271–3p showed higher expression in the myocardium of CKD-HFpEF pigs compared to normal pigs. miR-1271–3p upregulation was mirrored in pig heart slices exposed to plasma from CKD pigs, associated with blunted cardiac contraction/relaxation kinetics, downregulation of NFKBIA, and increased protein expression of NF-κB (Figure). CONCLUSION Our data support a novel pathophysiological mechanism for cardiac activation of NF-κB in CKD via post-transcriptional modulation of NFKBIA partly through miR-1271–3p. This miRNA might represent a novel therapeutic target to ameliorate the deleterious effects that renal inflammatory signaling in CKD imposes on the heart and may lead to HFpEF.

Background Chronic kidney disease (CKD) carries a higher cardiovascular mortality and is a prominent risk factor for development of HFpEF. However, the pathophysiology of HFpEF in CKD has not been established. We have recently shown that experimental CKD‐HFpEF is associated with increased renal release of inflammatory cytokines, particularly tumor necrosis factor (TNF)‐α and interleukin (IL)‐6, to the systemic circulation. Using a unique physiological biomimetic heart culture system, we test whether inflammatory cytokines of kidney origin contribute to the development of HFpEF in CKD. Methods Pigs (4 each) were assigned to sham or CKD (bilateral renovascular disease and dyslipidemia) groups and followed for 14 weeks. Circulating levels of TNF‐α and IL‐6 were quantified (ELISA). In a biomimetic heart culture system, pig heart slices were exposed to plasma from normal or CKD pigs, or from normal pigs enriched with TNF‐α and IL‐6 (matching CKD concentration) with or without cytokine neutralizing antibodies (NA). Contractility and relaxation kinetics in vitro were determined in a dose/time dependent manner. Results CKD‐HFpEF pigs exhibited elevated systemic levels of TNF‐α and IL‐6 (Figure 1A), associated with renal dysfunction, cardiac hypertrophy and fibrosis, and diastolic dysfunction. Plasma was collected from all animals. Then, pig heart slices were exposed to CKD‐HFpEF, normal, or normal plasma enriched with cytokines, showing impaired contractility and speed of relaxation that improved after TNF‐α and IL‐6 neutralization (Figure 1B). Conclusions Our observations implicate circulating inflammatory mediators released by dysfunctional kidneys in imposing damage to the remote myocardium, supporting a role of inflammatory cytokines of renal origin in renal‐cardio pathophysiology in CKD‐HFpEF.