Sitz, Rhys

Cardiovascular-kidney-metabolic (CKM) syndrome is a complex disease that affects up to 90% of US adults. This work characterized the renal transcriptomic landscape of a novel model of CKM in aging swine. The cardiovascular, renal, and metabolic parameters in pigs are very similar to those in humans, and our work may guide future studies aimed at modulating specific age-related renal abnormalities in CKM to slow disease progression and reduce mortality.

Background: Cardiovascular-Kidney-Metabolic (CKM) syndrome entails pathophysiological interactions leading to multiorgan dysfunction and a high rate of cardiovascular morbidity and mortality, largely driven by the development of heart failure (HF). Mechanisms leading to HF in CKM syndrome are unknown. We created a model of CKM syndrome in swine and showed that cardiac abnormalities associate with cardiac microvascular (MV) rarefaction and decreased expression and availability of VEGF. Emerging research focuses on micro-RNAs (miRNAs) and their role in the pathophysiology of HF, but whether they play a role in the development of cardiac injury in CKM syndrome via VEGF modulation has not been investigated. We aimed to identify specific miRNAs that can target VEGF to test the hypothesis that their direct cardiac inhibition will rescue cardiac VEGF, improve angiogenic signaling, and cardiac hemodynamics in CKM syndrome. Methods: CKM (bilateral renal artery stenosis+high-cholesterol/high-carbohydrate feeding) and normal pigs (n=4 each, 2 males/2 females) were studied for 14 weeks. Unbiased micro-RNA sequencing identified 26 miRNAs differentially expressed in CKM pig hearts, of which only miR-452-5p can target VEGFA, which was confirmed by pulldown analysis and qPCR. In addition, CKM (n=4, 2 males/2 females) were treated after 6 weeks with a single intracoronary (IC) administration of a miR-452-5p inhibitor (antagomiR). Cardiac hemodynamics (echocardiography), MV architecture (3D micro-CT) and cardiac expression of VEGF and miR-452-5p (RT-qPCR) were quantified before IC miR-452-5p inhibition, and again at the end of the study (14 weeks). Results: Pigs with CKM syndrome showed blunted diastolic relaxation, increased cardiac remodeling, and reduced subendocardial MV density and cardiac VEGF expression, accompanied by higher cardiac expression of miR-452-5p compared to normal pigs. Notably, most of these parameters were improved after in vivo IC miR-452-5p inhibition (single dose). Blood pressure was unchanged ( Table 1 ). Conclusions: Our study supports a role for VEGF in preserving cardiac MV integrity in CKM syndrome. Also, it identifies a targeted post-transcriptional regulation of VEGF via miR-452-5p and supports the feasibility and efficacy of its mechanistic modulation in vivo. The predictive power of our model conceptually supports the potential of a novel targeted intervention to protect the heart in CKM syndrome. [Table: see text] Support: NIH AG084154 (ARC), and DK129240 (AE) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Background and Aims Almost 36 million US adults (1 in 7, 14%) suffer from chronic kidney disease (CKD), and precision strategies to offset the progression to end-stage kidney disease are in dire need. We took advantage of a novel preclinical translational model of CKD in swine and single nucleus RNA sequencing (snRNA-seq) to identify potential therapeutic targets via characterization of the renal cell-specific transcriptomic landscape. Method Normal and CKD pigs were euthanized after 14 weeks (n = 3/group), kidneys were harvested, nuclei isolated, libraries prepared, and snRNA-seq performed. UMAP plots were generated with Seurat (v4.4.0) using default parameters. The expression profile of marker genes across single nuclei clusters was visualized to define renal cell types and ranked based on the number of differentially expressed genes (DEGs). The top cell type was subjected to subsequent analysis and DEG sorted based on their involvement in angiogenesis, inflammation, and fibrosis, which are prominent injury pathways in the pathophysiology of CKD. To mechanistically study the role of top overlapping candidate DEGs and support their potential as therapeutic targets, in vitro studies in primary renal vascular endothelial cells (RECs) were performed. Results A total of 39,935 nuclei were analyzed. Thirty clusters were identified and filtered by canonical gene markers, revealing 18 unique renal cell types (Fig. 1A). Endothelial cells exhibited the highest number of DEGs (Fig. 1B). Volcano plots (Fig. 1C) and Venn diagram (Fig. 1D) analysis of endothelial cells identified unique DEGs among three main renal injury pathways in CKD (angiogenesis, inflammation, fibrosis), with 2 overlapping DEGs (VWF, LAMA3) upregulated and participating in inflammation and fibrosis. Targeted silencing of VWF and LAMA3 (randomly selected) in RECs using siRNA ameliorated C5b9 and TGF-ẞ immunoreactivity, suggesting attenuated inflammatory and fibrogenic activity, respectively (Fig. 1E). Conclusion Our work characterized the single-nuclear renal cell transcriptomic landscape of a translational model of CKD and identified endothelial cells as the cluster with the largest number of DEGs. Our observations support the candidacy of VWF and LAMA3 as potential targets to decrease renal endothelial cell inflammation and fibrosis, which could pave the way for new therapeutic strategies in patients with CKD.

Abstract Background and Aims Almost 36 million US adults (1 in 7, 14%) suffer from chronic kidney disease (CKD), which is more prevalent in the elderly, affecting up to 34% of adults ≥ over 65. CKD has limited therapeutic options, partly due to the lack of large animal models that allow the translational study of the impact of age on CKD. This work reports the first preclinical model of CKD in naturally aged swine. Method CKD was induced in naturally aged (6-9 years old) and young (3 months old) domestic pigs (bilateral renal artery stenosis + high-cholesterol/high-carbohydrate diet), followed for 14 weeks, and compared to normal controls (n = 4–5/group). Renal hemodynamics were quantified in vivo by multidetector CT. Metabolic parameters, renal microvascular (MV) architecture (3D-micro-CT), and morphometric analysis (staining) were investigated ex vivo. Results Both young and old pigs developed CKD, but the renal and metabolic phenotype was accentuated in aging animals. We found that superimposition of aging on CKD resulted in higher triglycerides, glucose levels, insulin resistance, and adipose tissue accumulation (subcutaneous, visceral, perirenal) (Fig. 1A and B), accompanied by a more severe renal dysfunction (C), cortical MV rarefaction, and reduced peritubular capillary density (D and E), and more severe structural damage disclosed by increased glomerulosclerosis, tubule-interstitial fibrosis, and tubular injury (F). Notably, the severity of renal MV rarefaction in aging CKD correlated with functional and structural abnormalities (simple linear regression analysis, G). Conclusion Our study establishes the first large animal model of aging CKD, allowing the investigation of age as a biological variable in kidney-metabolic diseases. Age exacerbates metabolic and renal abnormalities, and our data support the critical role of renal MV disease in aging CKD and suggest that its exacerbation is a potential key player in its progression. This novel translational platform could foster new studies toward the elucidation of novel mechanisms of age-related deterioration of renal function in CKD and for developing therapeutic interventions.

Key Points We characterized the renal transcriptional profile at the single-nuclear level in a preclinical swine model of CKD. Our study reveals that renal endothelial cells represent the cluster with the highest number of differentially expressed genes related to inflammatory and fibrotic activity. These results offer new insights into CKD-induced transcriptional changes in renal endothelial cells at the single-nuclear level. Background The prevalence of CKD is on the rise, and precision strategies to offset the progression to ESKD are needed. We used a well-established preclinical translational model of CKD in swine and single-nucleus RNA sequencing (snRNA-seq) to identify potential therapeutic targets through characterization of the renal cell-specific transcriptomic landscape. Methods Normal and CKD pigs were studied in vivo and ex vivo after 14 weeks ( n =6/group). In randomly selected pigs ( n =3/group), kidneys were harvested, nuclei isolated, libraries prepared, and snRNA-seq performed. Protein expression of candidate differentially expressed genes (DEGs, log2FC >0.25, adjusted P value < 0.05) was determined by immunohistochemistry, and their expression in primary normal and CKD renal vascular endothelial cells was modulated (small interfering RNA) in vitro . Results A total of 52,213 nuclei were analyzed. Thirty clusters were identified and filtered by canonical gene markers, revealing 16 unique renal cell types. Endothelial cells were the top cell type exhibiting the highest number of DEGs, which were subsequently filtered by angiogenesis, inflammation, and fibrosis (major injurious pathways altered in CKD kidneys). Venn diagram analysis identified five unique overlapping DEGs in endothelial cells: vWF , LAMA3 , and KDR upregulated and PTGIS and ICAM1 downregulated in CKD versus normal kidneys. Venn diagram analysis indicates that vWF , LAMA3 , and ICAM1 participate in inflammatory and fibrotic signaling. Renal protein expression of these DEGs matched snRNA-seq findings. Furthermore, in vitro silencing of vWF and LAMA3 ameliorated endothelial cell inflammatory and fibrotic signaling. Conclusions Our work characterized the single-nuclear renal cell transcriptomic landscape of a translational model of CKD and singled out genes implicated in major renal injury pathways. These genes could serve as potential targets to pave the way for new therapeutic strategies in patients with CKD.

Abstract Background and Aims Patients with chronic kidney disease (CKD) have a higher risk of developing heart failure with preserved ejection fraction (HFpEF). We developed a model of CKD-HFpEF and showed that renal-cardio pathophysiology is associated with decreased expression and availability of vascular endothelial growth factor in both organs. We showed that renal VEGF therapy improved renal function in our model. We hypothesize that cardiac VEGF therapy will improve cardiac function and will investigate whether cardiac protection in CKD also carries recovery of renal function. Method CKD and normal pigs (n = 4 each, 2 males/2 females) were studied for 14 weeks. In addition, CKD-HFpEF (n = 4, 2 males/2 females) were treated after 6 weeks of disease with a single intracoronary (IC) administration of VEGF attached to a drug-delivery vector (ELP-VEGF) that increases cardiac targeting, and effects quantified 8 weeks later. Renal (multi-detector CT) and cardiac (echocardiography, PV loops) morphology and function were quantified in vivo, microvascular (MV) density (3D micro-CT) ex vivo, and cardiac expression of VEGF was quantified by qPCR. Results Cardiac function, VEGF expression, and MV density were improved after IC ELP-VEGF therapy compared to placebo. Notably, renal hemodynamics (expressed as % change) were significantly improved compared to pre-treatment values and to time-matched untreated CKD-HFpEF pigs (Figure). Conclusion Our study suggests strong crosstalk between the heart and the kidney in a pre-clinical model of CKD-HFpEF. Renal and cardiac hemodynamics were significantly improved after cardiac VEGF therapy, conceptually supporting a cardio-renal axis in CKD and showing the extent of beneficial effects of a novel targeted intervention in a translational fashion.

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- (&gt;1.4 FC, p&lt;0.05) and down-regulated (&lt;0.7 FC, p&lt;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.