McCarthy, Elizabeth Anne

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.