Eirin, Alfonso

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.

Abstract Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs.

Autophagy eliminates excessive nutrients and maintains homeostasis. Obesity and metabolic syndrome (MetS) dysregulate autophagy, possibly partly due to mitochondria injury and inflammation. Elamipretide (ELAM) improves mitochondrial function. We hypothesized that MetS blunts kidney autophagy, which ELAM would restore. Domestic pigs were fed a control or MetS-inducing diet for 16 weeks. During the 4 last weeks, MetS pigs received subcutaneous injections of ELAM (0.1 mg/kg/day, MetS + ELAM) or vehicle (MetS), and kidneys were then harvested to measure protein expression of autophagy mediators and apoptosis. Systemic and renal venous levels of inflammatory cytokines were measured to calculate renal release. The function of isolated mitochondria was assessed by oxidative stress, energy production, and pro-apoptotic activity. MetS slightly downregulated renal expression of autophagy mediators including p62, ATG5-12, mTOR, and AMPK vs. control. Increased mitochondrial H2O2 production accompanied decreased ATP production, elevated apoptosis, and renal fibrosis. In MetS + ELAM, mito-protection restored autophagic protein expression, improved mitochondrial energetics, and blunted renal cytokine release and fibrosis. In vitro, mitoprotection restored mitochondrial membrane potential and reduced oxidative stress in injured proximal tubular epithelial cells. Our study suggests that swine MetS mildly affects renal autophagy, possibly secondary to mitochondrial damage, and may contribute to kidney structural damage in MetS.

Our study demonstrates a novel pericyte-based therapy for the injured kidney. The beneficial effect of pericyte delivery appears to be mediated by ameliorating oxidative stress, inflammation, cellular apoptosis, and senescence in the stenotic kidney and improved tissue hypoxia, vascular loss, fibrosis, and tubular atrophy. Our data may form the basis for pericyte-based therapy, and additional research studies are needed to gain further insight into their role in improving renal function.

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.