Zhu, Xiangyang

Metabolic syndrome (MetS) is associated with low-grade inflammation, which can be exacerbated by renal artery stenosis (RAS) and renovascular hypertension, potentially worsening outcomes through pro-inflammatory cytokines. This study investigated whether mesenchymal stem/stromal cells (MSCs) could reduce fat inflammation in pigs with MetS and RAS. Twenty-four pigs were divided into Lean (control), MetS, MetS + RAS, and MetS + RAS + MSCs. In the MSC-treated group, autologous adipose-derived MSCs (107 cells) were injected into the renal artery six weeks after RAS induction. After four weeks, fat volumes and inflammatory markers were assessed. MSC treatment reduced levels of pro-inflammatory cytokines (MCP-1, TNF-a, IL-6) in the renal vein blood and in perirenal fat. The MSCs also decreased fat fibrosis, restored adipocyte size, and altered adipogenesis-related gene expression, particularly in the perirenal fat. These effects were less pronounced in subcutaneous fat. The MSC therapy attenuated fat inflammation and improved metabolic outcomes in pigs with MetS + RAS, suggesting that adipose-derived MSCs may offer a promising therapeutic approach for metabolic disorders.

The single-cell platform mass cytometry (CyTOF) enables detection of significant changes in one million of renal cells, especially in subsets of immune cells in ischemic human kidneys distal to renal artery stenosis (RAS). We found that pro-repair cell types such as scattered tubular-progenitor-like cells, aged neutrophils, and regulatory B cells show a compensatory increase in RAS. Immune cell phenotype changes may reflect ongoing inflammation and impaired immune defense capability in the kidneys.

To address the ongoing shortage of organs available for replacement, xenotransplantation of hearts, corneas, skin, and kidneys has been attempted. However, a major obstacle facing xenotransplants is rejection due to a cycle of immune reactions to the graft. Both adaptive and innate immune systems contribute to this cycle, in which natural killer cells, macrophages, and T-cells play a significant role. While advancements in the field of genetic editing can circumvent some of these obstacles, biomarkers to identify and predict xenograft rejection remain to be standardized. Several T-cell markers, such as CD3, CD4, and CD8, are useful in both the diagnosis and prediction of xenograft rejection. Furthermore, an increase in the levels of various circulating DNA markers and microRNAs is also predictive of xenograft rejection. In this review, we summarize recent findings on the advancements in xenotransplantation, with a focus on pig-to-human, the role of immunity in xenograft rejection, and its biomarkers.

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.

Objectives Quantitative magnetization transfer (qMT) is useful for measurement of murine renal fibrosis at high and ultrahigh field strengths. However, its utility at clinical field strengths and in human-like kidneys remains unknown. We tested the hypothesis that qMT would successfully detect fibrosis in swine kidneys with unilateral renal artery stenosis (RAS) at 3.0 T. Methods The qMT protocol is composed of MT scans with variable flip angles and offset frequencies, and of B0 , B1 , and T1 mapping. Pigs were scanned 10 weeks after RAS or control. A 2-pool model was used to fit the bound pool fraction f of the renal cortex (CO) and outer medulla (OM). Then qMT-derived f in 5 normal and 10 RAS pigs was compared with histological fibrosis determined using Masson's trichrome staining and to renal perfusion assessed with computed tomography. Results The qMT 2-pool model provided accurate fittings of data collected on swine kidneys. Stenotic kidneys showed significantly elevated f in both the CO (9.8% ± 2.7% vs 6.4% ± 0.9%, P = 0.002) and OM (7.6% ± 2.2% vs 4.7% ± 1.1%, P = 0.002), as compared with normal kidneys. Histology-measured renal fibrosis and qMT-derived f correlated directly in both the cortex (Pearson correlation coefficient r = 0.93, P < 0.001) and OM (r = 0.84, P = 0.002), and inversely with stenotic kidney perfusion (r = 0.85, P = 0.002). Conclusions This study demonstrates the feasibility of qMT for measuring fibrosis in human-like swine kidneys, and the association between tissue macromolecule content and renal perfusion. Therefore, qMT may be useful as a tool for noninvasive assessment of renal fibrosis in subjects with RAS at clinical field strengths.

Background Mesenchymal stem/stromal cells (MSCs) release extracellular vesicles (EVs), which shuttle proteins to recipient cells, promoting cellular repair. We hypothesized that cardiovascular risk factors may alter the pattern of proteins packed within MSC‐derived EVs, blunting their capacity to preserve the post‐stenotic kidney in pigs with metabolic syndrome (MetS) and renal artery stenosis (RAS). Methods We compared the protein cargo of EVs to their parent MSCs obtained from pigs with MetS and Lean controls. Porcine MSCs were harvested from abdominal fat after 16 weeks of Lean‐ or MetS‐diet (n=5 each), and their EVs isolated. Following LC‐MS/MS proteomics, clustering analysis was performed to identify primary functional categories of proteins enriched in Lean‐ and MetS‐EVs. In addition, MetS pigs were studied after 16 weeks of RAS untreated or treated 4 weeks earlier with a single intrarenal delivery of allogeneic labeled Lean‐ or MetS‐EVs (2.5x10^5/Kg). Lean and MetS Sham served as controls (n=6 each). Single‐kidney volume, renal blood flow (RBF), and glomerular filtration rate (GFR) were studied in‐vivo (multi‐detector CT), and tubular injury (Periodic Acid Schiff) and fibrosis (Trichrome) ex‐vivo. Results Proteomics analysis identified 6,690 and 6,790 distinct proteins in Lean‐ and MetS‐EVs, respectively. Differential expression analysis revealed that 91 proteins were upregulated in Lean‐EVs and 130 proteins in MetS‐EVs compared to their parent MSCs (FDR<0.05 and log2 fold‐change EVs/MSCs>2). Proteins enriched exclusively in Lean‐EVs modulate pathways related to the MSC reparative capacity, including cell proliferation, differentiation, and activation, as well as transforming growth factor‐β signaling (Figure 1A). Contrarily, proteins enriched only in MetS‐EVs are linked to pro‐inflammatory pathways, including acute inflammatory response, leukocyte transendothelial migration, and cytokine production (Figure 1B). Blood pressure was elevated in all MetS groups and all RAS groups develop moderate, but significant stenosis of a similar degree (Table). EVs were detected in the stenotic‐kidney 4 weeks after injection (Figure 2A). Renal volume, RBF, and GFR, which increased in MetS compared to Lean (consistent with hyperfiltration), fell in MetS+RAS, but restored in pigs treated with Lean‐EVs. Lean‐EVs also attenuated tubular injury and renal fibrosis (Figure 2B). Yet, these renoprotective effects were blunted in pigs treated with MetS‐EVs. Conclusion MetS alters the protein cargo of porcine MSC‐derived EVs, selectively packaging specific pro‐inflammatory signatures. Intrarenal delivery of Lean‐EVs attenuated renal injury and fibrosis and improved function in pigs with MetS+RAS, whereas these beneficial effects were blunted in pigs treated with MetS‐EVs. Therefore, our observations suggest that approaches to improve the therapeutic efficacy of autologous MSC‐derived EVs might be useful for patients with MetS+RAS. Support or Funding Information This study was partly supported by the National Institutes of Health grant numbers: DK104273, HL123160, DK102325, and DK106427. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Transplantation of autologous mesenchymal stem cells (MSCs) may be a viable option for treatment of several diseases. MSCs efficacy depends on adequate function of their mitochondria, which might be impaired in a noxious milieu. We hypothesized that obesity compromises MSCs mitochondrial structure and function, possibly via micro‐RNA (miRNA)‐based mechanisms. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet (n = 7 each). Mitochondrial structure was assessed by electron microscopy and function by membrane potential and cytochrome‐c oxidase (COX)‐IV activity. Oxidative stress was assessed by Mito‐SOX and dihydroethidium staining. Next‐generation sequencing (RNA‐seq) was performed to identify miRNAs expression in MSCs, and predicted mitochondrial target genes were then identified (MitoCarta). Compared to Lean‐MSCs, mitochondria from Obese‐MSCs were smaller and showed cristae remodeling and loss. Mitochondrial membrane potential and COX‐IV activity decreased in Obese‐MSCs, associated with increased mitochondrial oxidative stress. RNA‐seq generated reads for 413 miRNAs, of which 5 miRNAs were upregulated in Obese‐MSCs (fold change >2, p < 0.05) and found to target 43 specific mitochondrial genes. Obesity impairs MSC mitochondrial structure and function, possibly mediated partly through miRNA‐induced mitochondrial gene regulation, leading to increased oxidative stress. Importantly, these alterations may limit the therapeutic use of autologous MSCs in subjects with obesity.

AbstractAutologous transplantation of mesenchymal stem cells (MSCs) is a viable option for the treatment of several diseases. Evidence indicates that MSCs release extracellular vesicles (EVs) and that EVs shuttle miRNAs to damaged parenchymal cells to activate an endogenous repair program. We hypothesize that comorbidities may interfere with the packaging of cargo in MSC‐derived EVs. Therefore, we examined whether metabolic syndrome (MetS) modulates the miRNA content packed within MSC‐derived EVs. MSCs were collected from swine abdominal adipose tissue after 16 weeks of lean or obese diet (n = 7 each). Next‐generation RNA sequencing of miRNAs (miRNA‐seq) was performed to identify miRNAs enriched in MSC‐derived EVs and their predicted target genes. Functional pathway analysis of the top 50 target genes of the top 4 miRNAs enriched in each group was performed using gene ontology analysis. Lean‐ and MetS‐EVs were enriched in, respectively, 14 and 8 distinct miRNAs. Target genes of miRNAs enriched in MetS‐EVs were implicated in the development of MetS and its complications, including diabetes‐related pathways, validated transcriptional targets of AP1 family members Fra1 and Fra2, Class A/1 (Rhodopsin‐like receptors), and Peptide ligand‐binding receptors. In contrast, miRNAs enriched in Lean EVs target primarily EphrinA‐EPHA and the Rho family of GTPases. MetS alters the miRNA content of EVs derived from porcine adipose tissue MSCs. These alterations could impair the efficacy and limit the therapeutic use of autologous MSCs in subjects with MetS. Our findings may assist in developing adequate regenerative strategies to preserve the reparative potency of MSCs in individuals with MetS. © 2017 International Society for Advancement of Cytometry

Background— Endothelial progenitor cells (EPCs) promote neovascularization and endothelial repair. Renal artery stenosis (RAS) may impair renal function by inducing intrarenal microvascular injury and remodeling. We investigated whether replenishment with EPCs would protect the renal microcirculation in chronic experimental renovascular disease. Methods and Results— Single-kidney hemodynamics and function were assessed with the use of multidetector computed tomography in vivo in pigs with RAS, pigs with RAS 4 weeks after intrarenal infusion of autologous EPCs, and controls. Renal microvascular remodeling and angiogenic pathways were investigated ex vivo with the use of micro–computed tomography, histology, and Western blotting. EPCs increased renal expression of angiogenic factors, stimulated proliferation and maturation of new vessels, and attenuated renal microvascular remodeling and fibrosis in RAS. Furthermore, EPCs normalized the blunted renal microvascular and filtration function. Conclusions— The present study shows that a single intrarenal infusion of autologous EPCs preserved microvascular architecture and function and decreased microvascular remodeling in experimental chronic RAS. It is likely that restoration of the angiogenic cascade by autologous EPCs involved not only generation of new vessels but also acceleration of their maturation and stabilization. This contributed to preserving the blood supply, hemodynamics, and function of the RAS kidney, supporting EPCs as a promising therapeutic intervention for preserving the kidney in renovascular disease.