Eirin, Alfonso

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.

Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells that repair other damaged kidney cells. This study shows that renovascular disease (RVD) induces site-specific 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) modifications in DNA associated with transcriptional changes and impaired proliferative capacity of swine STCs, which could be mitigated in RVD-STCs treated with an epigenetic modulator. Therefore, our observations may contribute to the development of novel approaches to preserve the reparative capacity of STCs in individuals with RVD.

We provide the first longitudinal and most comprehensive analysis of the intrarenal microvasculature in a slowly progressive model of autosomal dominant polycystic kidney disease (ADPKD) and integrate the findings with studies in a young cohort of individuals with ADPKD. We identified vasculature-related pathways that could be targeted for therapeutic interventions and contribute promising, noninvasive biomarkers in patients with ADPKD. Alterations of the intrarenal microcirculation may affect drug delivery; a better understanding of its longitudinal changes may aid in treatment management.

Key Points Experimental autosomal dominant polycystic kidney disease is associated with longitudinal changes in the kidney micro-RNA and mRNA profiles. Alterations in cellular transport, a feature of CKD, and a common biological process within polycystic kidney disease models and human autosomal dominant polycystic kidney disease, is present from early stages. Early versus late micro-RNA expression changes in autosomal dominant polycystic kidney disease could be targeted for a more tailored therapeutic intervention in the disease. Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development and enlargement of bilateral kidney cysts, which often leads to kidney failure. This study comprehensively characterized the expression profile of micro-RNAs (miRNAs) and their target genes in Pkd1 RC/RC mice and further compared them with other murine models of polycystic kidney disease (PKD) and a model of CKD and individuals with ADPKD. Methods Pkd1 RC/RC and wild-type (WT) mice ( n =10 each, five males and five females) were studied at 1, 6, and 12 months. At each time point, kidney volume was determined in vivo (magnetic resonance imaging), followed by ex vivo histomorphometric analyses. In randomly selected Pkd1 RC/RC and WT mouse kidneys ( n =5/genotype, at 1 and 12 months), miRNA-sequencing (seq) was performed, followed by mRNA-seq, integrated (miRNA-seq/mRNA-seq analysis), and functional analysis of miRNA target genes. Venn diagrams were constructed to identify overlapping and novel differentially expressed (DE) miRNAs in Pkd1 RC/RC and other commonly used murine models of PKD, diabetic kidney disease, and individuals with ADPKD. Results miRNA-seq analysis identified 41 and 181 miRNAs DE in Pkd1 RC/RC versus WT kidneys at 1 and 12 months, respectively, which were confirmed by quantitative PCR. Target genes of miRNAs DE in Pkd1 RC/RC at early stages encoded for proteins mainly implicated in cell proliferation and α -ketoglutarate ( α -KG) transport, whereas those DE at late stages encoded for transport proteins involved in proinflammatory and metabolic processes. Urine α -KG concentration ( 1 H nuclear magnetic resonance spectroscopy), its fractional excretion, and tissue levels were higher in Pkd1 RC/RC during the entire course of the disease and associated with decreased protein expression of α -KG transporters sodium-coupled dicarboxylate transporter 3 and organic anion transporter 1. We further identified common DE miRNAs among murine models of PKD, diabetic kidney disease, and previous reports in individuals with ADPKD, as well as several novel miRNAs DE in early and late Pkd1 RC/RC kidneys, which have not been previously reported in either other murine models of PKD or patients with ADPKD. Conclusions Our study demonstrates that the renal miRNA expression profile changes longitudinally with the progression of the disease and might suggest that the post-transcriptional regulation of α -KG transport could represent a novel early feature of the disease.

Key Points The kidney microvasculature was quantified reliably in human autosomal dominant polycystic kidney disease early on, using super-resolution ultrasound localization microscopy. Microvascular parameters correlated with structural and functional indices, constituting promising biomarkers to evaluate noncystic parenchyma. Microvascular parameters may serve to assess disease severity and monitor progression from the early stages of the disease. Background In vitro studies in individuals with autosomal dominant polycystic kidney disease (ADPKD) have shown extensive intrarenal microvascular remodeling. However, analyses have been limited to the time of kidney failure because of the invasive nature of current methodologies to assess the intrarenal microvasculature. We hypothesized that super-resolution ultrasound microvascular imaging, known as ultrasound localization microscopy, enabled by high-framerate ultrasound imaging and microbubble localization and tracking postprocessing techniques, would safely, reliably, and reproducibly assess the intrarenal microvasculature in individuals with ADPKD at an early stage of the disease. Methods We prospectively enrolled young (<40 years of age), well-characterized, early-stage (eGFR >90 ml/min per 1.73 m 2 ) individuals with ADPKD, and sex–age 1:1 matched controls ( n =17 each) and assessed the intrarenal microvasculature using ultrasound localization microscopy. We determined the intersonographer reliability, intraobserver repeatability, and interobserver reproducibility of microvascular parameters in both groups, and the day-to-day variability in the control group. Microvascular parameters were correlated with structural (total kidney volume) and functional (eGFR) markers in individuals with ADPKD. Results Ultrasound localization microscopy provided a super-resolved map of the intrarenal microvasculature in ADPKD and matched controls. Intrarenal microvascular parameters ( e.g ., vessel density) were quantified in a safe, reliable (intraclass correlation coefficients, 0.96; 95% confidence interval, 0.91 to 0.98), reproducible (intraclass correlation coefficient, 0.96; 95% confidence interval, 0.93 to 0.98), and repeatable (coefficient of variations, 3.6%) manner. In control individuals, the day-to-day variability of most parameters was low, with coefficient of variation ranging from 1.8% to 10.7%. Microvascular parameters differed in individuals with ADPKD compared with controls, despite preserved eGFR. Interestingly, cortical vessel density and its derived perfusion correlated directly with eGFR, whereas tortuosity indices presented an inverse correlation. Conclusions Ultrasound localization microscopy provided a safe and reliable in vivo assessment of the human intrarenal microvasculature.

Chronic kidney disease (CKD), heart failure with preserved ejection fraction (HFpEF), and the recently defined cardiovascular-kidney-metabolic (CKM) syndrome are important challenges for the health care system, associated with a staggering increase in morbidity and mortality rates. Unfortunately, the complex pathophysiology of these processes remains to be fully elucidated, imposing hurdles to prevent their development and decelerate their progression. Large animal models are critical for understanding cardio-renal pathophysiology and for the discovery of new therapies. Among them, pig models offer significant translational power to grasp cardio-renal pathophysiology and to test the feasibility and efficacy of new strategies. We recently developed and characterized swine models of CKD, HFpEF, and CKM syndrome that closely recapitulate human disease and allow for the study of the impact of biological variables, such as sex or age, on these conditions. Furthermore, data from these studies revealed key mechanisms implicated in the pathogenesis of CKD, HFpEF, and CKM syndrome and identified potential targets for intervention. This focused mini review highlights the main features of these novel swine models and discusses ongoing and future research aimed at developing novel therapies.

Abstract Importance Mesenchymal stromal cells (MSCs) possess therapeutic properties that mediate repair. Obesity impairs MSC functionality and therapeutic efficacy, possibly by eliciting dynamic modifications of epigenetic markers, like 5-hydroxymethylcytosine (5hmC). Objective We hypothesized that human obesity alters the 5hmC landscape and anti-inflammatory capacity of adipose tissue-derived MSCs to activate the prominent inflammatory signaling mediator Interleukin (IL)-1β. Design, Setting, Participants, Intervention Adipose tissue samples were collected from obese and lean individuals (body mass index ≥30 or &lt;30 kg/m2, respectively, n = 11 each) during weight-loss or kidney donation surgery. Main Outcomes and Measures MSCs were harvested and analyzed for 5hmC profiles (MeDIP-seq) and mRNA expression (RNA-seq) (n = 5 each). Subsequently, MSCs or a vehicle were injected into mice, (n = 6 each) and two-weeks later, kidneys were evaluated using in-vivo magnetic resonance imaging and ex vivo studies. The role of IL-1β was then studied in-vitro in MSC-induced immunomodulation using siRNA in macrophages. Results Compared to MSC from lean patients, obese-MSC genes showed 2087 differential 5hmC modifications and 175 differential mRNA expression. Among them, 14 genes with overlapping alterations were involved in regulation of cytokine production, prominently IL-1β. Injecting obese MSCs elevated renal expression of IL-1β and M1 macrophage count but lowered kidney perfusion. Silencing IL-1β in obese-MSCs in vitro reduced M1 phenotype switching in co-incubated macrophages. Conclusions and Relevance Obesity induces epigenetic and gene expression changes in MSCs, particularly in IL-1β, associated with impaired anti-inflammatory functionality of MSCs. Targeting IL-1β could be a useful therapeutic approach to modulate the decline in MSC functionality resulting from obesity.

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 Renal artery stenosis (RAS) is the leading cause of secondary hypertension worldwide. However, current medical and surgical treatment modalities provide minimal benefits for kidney injury. Recent preclinical RAS models have demonstrated promising potential of human mesenchymal stem cells (MSC) and their daughter extracellular vesicles (EV) in improving murine renal function and attenuating inflammation. However, the extent and mechanisms underlying immune rejection of xenogeneic MSCs or EVs are yet undetermined. Therefore, adipose tissue was harvested from adult healthy patients. Adipose-derived MSCs were extracted and cultured, and EVs were isolated from their supernatants via ultra-centrifugation. Then, mice randomly assigned to RAS or sham surgery were divided into 6 groups: sham surgery, RAS, sham + MSC, RAS + MSC, sham + EV, and RAS + EV. Two weeks after intra-aortic injection of MSCs (5 × 105) or EVs (20 µg protein), we compared the intrarenal T-cell and macrophage accumulation, splenic B-cell numbers, circulating cytokines and anti-human antibodies levels among the groups. MSCs and EVs did not influence intrarenal immune cell infiltrations. However, MSCs significantly increased circulating anti-human antibodies. In the spleen, RAS + EV mice showed higher memory IgM+ B-cells but reduced CD19+ B-cells compared to RAS + MSC. In vitro T-cell recall assay showed that both MSCs and EVs exhibited reduced IFN-γ release upon re-stimulation, indicating an immunosuppressive effect. Therefore, xenogeneic MSCs induced a greater humoral response in mice, while EVs triggered a splenic cellular response, but neither elicits discernible kidney rejection. Our results provide key insights into the immunomodulatory mechanisms of MSCs and EVs and immune mechanisms underlying xenograft rejection.

The intrinsic reparative capacity of the adult mammalian kidney is restricted to the ability of scattered tubular-like cells (STCs) to repair damaged kidney cells. Our study provides evidence that chronic renal ischemia alters the mRNA/miRNA profile of angiogenic/vascular development genes of swine STCs, limiting their potential to repair injured tubular cells. Our observations may assist in developing new therapies to improve renal repair in individuals with chronic renal ischemia.