Effects of obesity on reparative function of human adipose tissue-derived mesenchymal stem cells on ischemic murine kidneys.

INTRODUCTION: Obesity is a health burden that impairs cellular processes. Mesenchymal stem/stromal cells (MSCs) are endowed with reparative properties and can ameliorate renal injury. Obesity impairs human MSC function in-vitro, but its effect on their in-vivo reparative potency remains unknown. SUBJECTS AND METHODS: Abdominal adipose tissue-derived MSC were harvested from patients without ('lean') or with obesity ('obese') (body mass index <30 or ≥30 kg/m2, respectively) during kidney donation or bariatric surgery, respectively. MSC (5 × 105/200 µL) or vehicle were then injected into 129S1 mice 2 weeks after renal artery stenosis (RAS) or sham surgery (n = 8/group). Two weeks later, mice underwent magnetic resonance imaging to assess renal perfusion and oxygenation in-vivo, and kidneys then harvested for ex-vivo studies. RESULTS: Similar numbers of lean and obese-MSCs engrafted in stenotic mouse kidneys. Vehicle-treated RAS mice had reduced stenotic-kidney cortical and medullary perfusion and oxygenation. Lean (but not obese) MSC normalized ischemic kidney cortical perfusion, whereas both effectively mitigated renal hypoxia. Serum creatinine and blood pressure were elevated in RAS mice and lowered only by lean-MSC. Both types of MSCs alleviated stenotic-kidney fibrosis, but lean-MSC more effectively than obese-MSC. MSC senescence-associated beta-gal activity, and gene expression of p16, p21, and vascular endothelial growth factor correlated with recipient kidney perfusion and tissue injury, linking MSC characteristics with their in-vivo reparative capacity. DISCUSSION: Human obesity impairs the reparative properties of adipose-tissue-derived MSCs, possibly by inducing cellular senescence. Dysfunction and senescence of the endogenous MSC repair system in patients with obesity may warrant targeting interventions to restore MSC vitality.

Microvascular remodeling and altered angiogenic signaling in human kidneys distal to occlusive atherosclerotic renal artery stenosis.

BACKGROUND: Renal artery stenosis (RAS) is an important cause of chronic kidney disease and secondary hypertension. In animal models, renal ischemia leads to downregulation of growth factor expression and loss of intrarenal microcirculation. However, little is known about the sequelae of large-vessel occlusive disease on the microcirculation within human kidneys. METHOD: This study included five patients who underwent nephrectomy due to renovascular occlusion and seven nonstenotic discarded donor kidneys (four deceased donors). Micro-computed tomography was performed to assess microvascular spatial densities and tortuosity, an index of microvascular immaturity. Renal protein expression, gene expression and histology were studied in vitro using immunoblotting, polymerase chain reaction and staining. RESULTS: RAS demonstrated a loss of medium-sized vessels (0.2-0.3 mm) compared with donor kidneys (P = 0.037) and increased microvascular tortuosity. RAS kidneys had greater protein expression of angiopoietin-1, hypoxia-inducible factor-1α and thrombospondin-1 but lower protein expression of vascular endothelial growth factor (VEGF) than donor kidneys. Renal fibrosis, loss of peritubular capillaries (PTCs) and pericyte detachment were greater in RAS, yet they had more newly formed PTCs than donor kidneys. Therefore, our study quantified significant microvascular remodeling in the poststenotic human kidney. RAS induced renal microvascular loss, vascular remodeling and fibrosis. Despite downregulated VEGF, stenotic kidneys upregulated compensatory angiogenic pathways related to angiopoietin-1. CONCLUSIONS: These observations underscore the nature of human RAS as a microvascular disease distal to main vessel stenosis and support therapeutic strategies directly targeting the poststenotic kidney microcirculation in patients with RAS.

Impaired immunomodulatory capacity in adipose tissue-derived mesenchymal stem/stromal cells isolated from obese patients.

Immune-modulatory properties of adipose tissue-derived mesenchymal stem/stromal cells (MSCs) might be susceptible to metabolic disturbances. We hypothesized that the immune-modulatory function of MSCs might be blunted in obese human subjects. MSCs were collected from abdominal subcutaneous fat of obese and lean subjects during bariatric or kidney donation surgeries, respectively. MSCs were co-cultured in vitro for 24 h with M1 macrophages, which were determined as M1or M2 phenotypes by flow cytometry, and cytokines measured in conditioned media. In vivo, lean or obese MSCs (5 × 105 ), or PBS, were injected into mice two weeks after unilateral renal artery stenosis (RAS) or sham surgeries (n = 6 each). Fourteen days later, kidneys were harvested and stained with M1 or M2 markers. Lean MSCs decreased macrophages M1 marker intensity, which remained elevated in macrophages co-cultured with obese MSCs. TNF-α levels were four-fold higher in conditioned media collected from obese than from lean MSCs. RAS mouse kidneys were shrunk and showed increased M1 macrophage numbers and inflammatory cytokine expression compared with normal kidneys. Lean MSCs decreased M1 macrophages, M1/M2 ratio and inflammation in RAS kidneys, whereas obese MSCs did not. MSCs isolated from lean human subjects decrease inflammatory M1 macrophages both in vivo and in vitro, an immune-modulatory function which is blunted in MSCs isolated from obese subjects.

Renovascular disease induces mitochondrial damage in swine scattered tubular cells.

Scattered tubular-like cells (STCs) contribute to repair neighboring injured renal tubular cells. Mitochondria mediate STC biology and function but might be injured by the ambient milieu. We hypothesized that the microenviroment induced by the ischemic and metabolic components of renovascular disease impairs STC mitochondrial structure and function in swine, which can be attenuated with mitoprotection. CD24+/CD133+ STCs were quantified in pig kidneys after 16 wk of metabolic syndrome (MetS) or lean diet (Lean) with or without concurrent renal artery stenosis (RAS) (n = 6 each). Pig STCs were isolated and characterized, and mitochondrial structure, membrane potential, and oxidative stress were assessed in cells untreated or incubated with the mitoprotective drug elamipretide (1 nM for 6 h). STC-protective effects were assessed in vitro by their capacity to proliferate and improve viability of injured pig tubular epithelial cells. The percentage of STCs was higher in MetS, Lean + RAS, and MetS + RAS kidneys compared with Lean kidneys. STCs isolated from Lean + RAS and MetS + RAS pigs showed mitochondrial swelling and decreased matrix density, which were both restored by mitoprotection. In addition, mitochondrial membrane potential and ATP production were reduced and production of reactive oxygen species elevated in MetS, Lean + RAS, and MetS + RAS STCs. Importantly, mitoprotection improved mitochondrial structure and function as well as the capacity of MetS + RAS STCs to repair injured tubular cells in vitro. Renovascular disease in swine is associated with a higher prevalence of STCs but induces structural and functional alterations in STC mitochondria, which impair their reparative potency. These observations suggest a key role for mitochondria in the renal reparative capacity of STCs.

Rac1 GTPase Inhibition Blocked Podocyte Injury and Glomerular Sclerosis during Hyperhomocysteinemia via Suppression of Nucleotide-Binding Oligomerization Domain-Like Receptor Containing Pyrin Domain 3 Inflammasome Activation.

Elevated homocysteine (Hcy) levels have been shown to activate nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome leading to podocyte dysfunction and glomerular injury. However, it remains unclear how this inflammasome activation in podocytes is a therapeutic target for reversal of glomerular injury and ultimate sclerosis. The present study tested whether inhibition of Rac1 GTPase activity suppresses NLRP3 inflammation activation and thereby blocks podocyte injury induced by elevated Hcy. In cultured podocytes, we found that L-Hcy (the active Hcy form) stimulated the NLRP3 inflammasome formation, as shown by increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1, which was accompanied by increased interleukin-1ß production and caspase-1 activity, indicating NLRP3 inflammasome activation. Rac1 activator, uridine triphosphate (UTP), mimicked L-Hcy-induced NLRP3 inflammasome activation, while Rac1 inhibitor NSC23766 blocked it. This Rac1 inhibition also prevented L-Hcy-induced podocyte dysfunction. All these effects were shown to be mediated via lipid raft redox signaling platforms with nicotinamide adenine dinucleotide phosphate oxidase subunits and consequent O2- production. In animal studies, hyperhomocysteinemia (hHcy) induced by folate-free diet was shown to induce NLRP3 inflammasome formation and activation in glomeruli, which was also mimicked by UTP and inhibited by NSC23766 to a comparable level seen in Nlrp3 gene knockout mice. These results together suggest that Rac1 inhibition protects the kidney from hHcy-induced podocyte injury and glomerular sclerosis due to its action to suppress NLRP3 inflammasome activation in podocytes.

NLRP3 inflammasome as a novel target for docosahexaenoic acid metabolites to abrogate glomerular injury.

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis during hyperhomocysteinemia (hHcys). However, it remains unclear whether the NLRP3 inflammasome can be a therapeutic target for treatment of hHcys-induced kidney injury. Given that DHA metabolites-resolvins have potent anti-inflammatory effects, the present study tested whether the prototype, resolvin D1 (RvD1), and 17S-hydroxy DHA (17S-HDHA), an intermediate product, abrogate hHcys-induced podocyte injury by targeting the NLRP3 inflammasome. In vitro, confocal microscopy demonstrated that 17S-HDHA (100 nM) and RvD1 (60 nM) prevented Hcys-induced formation of NLRP3 inflammasomes, as shown by reduced colocalization of NLRP3 with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) or caspase-1. Both DHA metabolites inhibited Hcys-induced caspase-1 activation and interleukin-1ß production. However, DHA had no significant effect on these Hcys-induced changes in podocytes. In vivo, DHA lipoxygenase metabolites substantially inhibited podocyte NLRP3 inflammasome formation and activation and consequent glomerular sclerosis in mice with hHcys. Mechanistically, RvD1 and 17S-HDHA were shown to suppress Hcys-induced formation of lipid raft redox signaling platforms and subsequent O2·- production in podocytes. It is concluded that inhibition of NLRP3 inflammasome activation is one of the important mechanisms mediating the beneficial action of RvD1 and 17S-HDHA on Hcys-induced podocyte injury and glomerular sclerosis.

Nod-like receptor protein 3 (NLRP3) inflammasome activation and podocyte injury via thioredoxin-interacting protein (TXNIP) during hyperhomocysteinemia.

NADPH oxidase-derived reactive oxygen species (ROS) have been reported to activate NLRP3 inflammasomes resulting in podocyte and glomerular injury during hyperhomocysteinemia (hHcys). However, the mechanism by which the inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by siRNA or verapamil prevented Hcys-induced TXNIP protein recruitment to form NLRP3 inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1ß production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers, podocin and desmin. In vivo, adult C57BL/6J male mice were fed a folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water) or by local microbubble-ultrasound TXNIP shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP shRNA-transfected mice or those receiving verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1ß production in glomeruli of mice with hHcys. Correspondingly, TXNIP shRNA transfection and verapamil attenuated hHcys-induced proteinuria, albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3 inflammasome formation and activation and subsequent glomerular injury.

Inhibition of hyperhomocysteinemia-induced inflammasome activation and glomerular sclerosis by NLRP3 gene deletion.

BACKGROUND/AIMS: Hyperhomocysteinemia (hHcys) has been reported to initiate Nod-like receptor protein 3 (NLRP3) inflammasome formation and activation in podocytes, leading to glomerular dysfunction and sclerosis. However, it remains unknown whether Nlrp3 gene is critical for the formation and activation of inflammasomes in glomeruli of hHcys mice. METHODS: Plasma homocysteine concentration was estimated utilizing HPLC, inflammasome formation and immunofluorescence expression from confocal microscopy, IL-1ß production from ELISA. RESULTS: Uninephrectomized Nlrp3 knockout (Nlrp3(-/-)) and wild type (Nlrp3(+/+)) and intra renal Nlrp3 shRNA-transfected wild type mice (Nlrp3 shRNA) were fed a folate free (FF) diet or normal chow (ND) for 4 weeks to produce hHcys. The plasma Hcys levels were significantly elevated in both Nlrp3(-/-) and Nlrp3(+/+) mice fed a FF diet compared to ND fed mice. The FF diet significantly increased the colocalization of Nlrp3 with apoptosis-associated speck-like protein (ASC) or caspase-1, caspase-1 activity and IL-1ß production in glomeruli of Nlrp3(+/+), but not in Nlrp3(-/-) mice and local Nlrp3 shRNA transfected mice. Correspondingly, the glomerular damage index (GDI) and urinary protein excretion were significantly higher in Nlrp3(+/+) mice compared to ND fed mice. However, the hHcys-induced increase in GDI and proteinuria were significantly lower in Nlrp3(-/-) and local Nlrp3 shRNA transfected mice than in Nlrp3(+/+) mice. Immunocytochemical analysis showed that hHcys decreased expression of podocin and nephrin, but increased desmin expression in glomeruli of Nlrp3(+/+) mice compared to Nlrp3(-/-) mice. CONCLUSION: Nlrp3 gene is an essential component of Nlrp3 inflammasomes and that targeting Nlrp3 may be important therapeutic strategy to prevent inflammasome activation and thereby protect podocytes and glomeruli from hHcys-induced injury.

Efficacy of Human Embryonic Stem Cells Compared to Adipose Tissue-Derived Human Mesenchymal Stem/Stromal Cells for Repair of Murine Post-Stenotic Kidneys.

Clinical translation of mesenchymal stem/stromal cell (MSC) therapy has been impeded by the heterogenous nature and limited replicative potential of adult-derived MSCs. Human embryonic stem cell-derived MSCs (hESC-MSCs) that differentiate from immortal cell lines are phenotypically uniform and have shown promise in-vitro and in many disease models. Similarly, adipose tissue-derived MSCs (MSC(AT)) possess potent reparative properties. How these two cell types compare in efficacy, however, remains unknown. We randomly assigned mice to six groups (n = 7-8 each) that underwent unilateral RAS or a sham procedure (3 groups each). Two weeks post-operation, each mouse was administered either vehicle, MSC(AT)s, or hESC-MSCs (5 × 105 cells) into the aorta. Mice were scanned with micro-MRI to determine renal hemodynamics two weeks later and kidneys then harvested. hESC-MSCs and MSC(AT)s were similarly effective at lowering systolic blood pressure. However, MSC(AT)s more robustly increased renal perfusion, oxygenation, and glomerular filtration rate in the post-stenotic kidney, and more effectively mitigated tubular injury, fibrosis, and vascular remodeling. These observations suggest that MSC(AT) are more effective than hESC-MSC in ameliorating kidney dysfunction and tissue injury distal to RAS. Our findings highlight the importance of tissue source in selection of MSCs for therapeutic purposes and underscore the utility of cell-based therapy for kidney disease.

Human Obesity Attenuates Cardioprotection Conferred by Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells.

To explore the impact of obesity on reparative potency of adipose tissue-derived mesenchymal stromal/stem cells (A-MSC) in hypertensive cardiomyopathy, A-MSC were harvested from subcutaneous fat of obese and age-matched non-obese human subjects during bariatric or kidney donation surgeries, and then injected into mice 2 weeks after inducing renovascular hypertension (RVH) or sham surgery. Two weeks later, left ventricular (LV) function and deformation were estimated in vivo by micro-magnetic resonance imaging and myocardial damage ex vivo. Blood pressure and myocardial wall thickening were elevated in RVH + Vehicle and normalized only by lean-A-MSC. Both A-MSC types reduced LV mass and normalized the reduced LV peak strain radial in RVH, yet obese-A-MSC also impaired LV systolic function. A-MSC alleviated myocardial tissue damage in RVH, but lean-A-MSC decreased oxidative stress more effectively. Obese-A-MSC also showed increased cellular inflammation in vitro. Therefore, obese-A-MSC are less effective than lean-A-MSC in blunting hypertensive cardiomyopathy in mice with RVH.

Diabetic kidney disease induces transcriptome alterations associated with angiogenesis activity in human mesenchymal stromal cells.

BACKGROUND: Therapeutic interventions that optimize angiogenic activities may reduce rates of end-stage kidney disease, critical limb ischemia, and lower extremity amputations in individuals with diabetic kidney disease (DKD). Infusion of autologous mesenchymal stromal cells (MSC) is a promising novel therapy to rejuvenate vascular integrity. However, DKD-related factors, including hyperglycemia and uremia, might alter MSC angiogenic repair capacity in an autologous treatment approach. METHODS: To explore the angiogenic activity of MSC in DKD, the transcriptome of adipose tissue-derived MSC obtained from DKD subjects was compared to age-matched controls without diabetes or kidney impairment. Next-generation RNA sequencing (RNA-seq) was performed on MSC (DKD n = 29; Controls n = 9) to identify differentially expressed (DE; adjusted p < 0.05, |log2fold change|> 1) messenger RNA (mRNA) and microRNA (miRNA) involved in angiogenesis (GeneCards). Paracrine-mediated angiogenic repair capacity of MSC conditioned medium (MSCcm) was assessed in vitro using human umbilical vein endothelial cells incubated in high glucose and indoxyl sulfate for a hyperglycemic, uremic state. RESULTS: RNA-seq analyses revealed 133 DE mRNAs (77 upregulated and 56 down-regulated) and 208 DE miRNAs (119 up- and 89 down-regulated) in DKD-MSC versus Control-MSC. Interestingly, miRNA let-7a-5p, which regulates angiogenesis and participates in DKD pathogenesis, interacted with 5 angiogenesis-associated mRNAs (transgelin/TAGLN, thrombospondin 1/THBS1, lysyl oxidase-like 4/LOXL4, collagen 4A1/COL4A1 and collagen 8A1/COL8A1). DKD-MSCcm incubation with injured endothelial cells improved tube formation capacity, enhanced migration, reduced adhesion molecules E-selectin, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 mRNA expression in endothelial cells. Moreover, angiogenic repair effects did not differ between treatment groups (DKD-MSCcm vs. Control-MSCcm). CONCLUSIONS: MSC from individuals with DKD show angiogenic transcriptome alterations compared to age-matched controls. However, angiogenic repair potential may be preserved, supporting autologous MSC interventions to treat conditions requiring enhanced angiogenic activities such as DKD, diabetic foot ulcers, and critical limb ischemia.

Comparable in vitro Function of Human Liver-Derived and Adipose Tissue-Derived Mesenchymal Stromal Cells: Implications for Cell-Based Therapy.

Mesenchymal stem/stromal cells (MSCs) have been investigated extensively for their immunotherapeutic and regenerative properties, which may differ by cell source. In MSCs harvested from donors matched for sex, age, and body mass index, we compared the proliferative and migration functions of liver-derived MSCs (L-MSCs) and adipose tissue-derived MSCs (A-MSCs) (n = 6 donors each). Cellular senescence was evaluated by senescence-associated beta-galactosidase enzyme activity and expression of senescence-associated secretory phenotype (SASP) factors using real-time quantitative polymerase chain and by western blot assay. The pro-angiogenic and reparative potency of MSCs was compared by co-culturing MSCs with injured human umbilical vein endothelial cells (HUVEC). The proliferation and migration properties were similar in L-MSCs and A-MSCs. Although cell cycle arrest and SASP genes were similarly expressed in both MSCs, tumor necrosis factor alpha gene and protein expression were significantly downregulated in L-MSCs. In co-cultured injured HUVEC, A-MSCs restored significantly more tubes and tube connections than L-MSCs. Therefore, despite many functional similarities between L-MSCs and A-MSCs, L-MSCs have enhanced immunomodulatory properties, while A-MSCs appear to have better pro-angiogenic and vascular reparative potency. Availability of a broad range of cellular options might enable selecting cell-based therapy appropriate for the specific underlying disease.

A systematic review and meta-analysis of cell-based interventions in experimental diabetic kidney disease.

Regenerative, cell-based therapy is a promising treatment option for diabetic kidney disease (DKD), which has no cure. To prepare for clinical translation, this systematic review and meta-analysis summarized the effect of cell-based interventions in DKD animal models and treatment-related factors modifying outcomes. Electronic databases were searched for original investigations applying cell-based therapy in diabetic animals with kidney endpoints (January 1998-May 2019). Weighted or standardized mean differences were estimated for kidney outcomes and pooled using random-effects models. Subgroup analyses tested treatment-related factor effects for outcomes (creatinine, urea, urine protein, fibrosis, and inflammation). In 40 studies (992 diabetic rodents), therapy included mesenchymal stem/stromal cells (MSC; 61%), umbilical cord/amniotic fluid cells (UC/AF; 15%), non-MSC (15%), and cell-derived products (13%). Tissue sources included bone marrow (BM; 65%), UC/AF (15%), adipose (9%), and others (11%). Cell-based therapy significantly improved kidney function while reducing injury markers (proteinuria, histology, fibrosis, inflammation, apoptosis, epithelial-mesenchymal-transition, oxidative stress). Preconditioning, xenotransplantation, and disease-source approaches were effective. MSC and UC/AF cells had greater effect on kidney function while cell products improved fibrosis. BM and UC/AF tissue sources more effectively improved kidney function and proteinuria vs adipose or other tissues. Cell dose, frequency, and administration route also imparted different benefits. In conclusion, cell-based interventions in diabetic animals improved kidney function and reduced injury with treatment-related factors modifying these effects. These findings may aid in development of optimal repair strategies through selective use of cells/products, tissue sources, and dose administrations to allow for successful adaptation of this novel therapeutic in human DKD.

Global epigenetic alterations of mesenchymal stem cells in obesity: the role of vitamin C reprogramming.

Obesity promotes dysfunction and impairs the reparative capacity of mesenchymal stem/stromal cells (MSCs), and alters their transcription, protein content, and paracrine function. Whether these adverse effects are mediated by chromatin-modifying epigenetic changes remains unclear. We tested the hypothesis that obesity imposes global DNA hydroxymethylation and histone tri-methylation alterations in obese swine abdominal adipose tissue-derived MSCs compared to lean pig MSCs. MSCs from female lean (n = 7) and high-fat-diet fed obese (n = 7) domestic pigs were assessed using global epigenetic assays, before and after in-vitro co-incubation with the epigenetic modulator vitamin-C (VIT-C) (50 µg/ml). Dot blotting was used to measure across the whole genome 5-hydroxyemthycytosine (5hmC) residues, and Western blotting to quantify in genomic histone-3 protein tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. MSC migration and proliferation were studied in-vitro. Obese MSCs displayed reduced global 5hmC and H3K4m3 levels, but comparable H3K9me3 and H3K27me3, compared to lean MSCs. Global 5hmC, H3K4me3, and HK9me3 marks correlated with MSC migration and reduced proliferation, as well as clinical and metabolic characteristics of obesity. Co-incubation of obese MSCs with VIT-C enhanced 5hmC marks, and reduced their global levels of H3K9me3 and H3K27me3. Contrarily, VIT-C did not affect 5hmC, and decreased H3K4me3 in lean MSCs. Obesity induces global genomic epigenetic alterations in swine MSCs, involving primarily genomic transcriptional repression, which are associated with MSC function and clinical features of obesity. Some of these alterations might be reversible using the epigenetic modulator VIT-C, suggesting epigenetic modifications as therapeutic targets in obesity.

Hypoxic preconditioning induces epigenetic changes and modifies swine mesenchymal stem cell angiogenesis and senescence in experimental atherosclerotic renal artery stenosis.

BACKGROUND: Atherosclerotic renal artery stenosis (ARAS) is a risk factor for ischemic and hypertensive kidney disease (HKD) for which autologous mesenchymal stem cell (MSC) appears to be a promising therapy. However, MSCs from ARAS patients exhibit impaired function, senescence, and DNA damage, possibly due to epigenetic mechanisms. Hypoxia preconditioning (HPC) exerts beneficial effects on cellular proliferation, differentiation, and gene and protein expression. We hypothesized that HPC could influence MSC function and senescence in ARAS by epigenetic mechanisms and modulating gene expression of chromatin-modifying enzymes. METHODS: Adipose-derived MSC harvested from healthy control (N = 8) and ARAS (N = 8) pigs were cultured under normoxia (20%O2) or hypoxia (1%O2) conditions. MSC function was assessed by migration, proliferation, and cytokine release in conditioned media. MSC senescence was evaluated by SA-ß-gal activity. Specific pro-angiogenic and senescence genes were assessed by reverse transcription polymerase chain reaction (RT-PCR). Dot blotting was used to measure global genome 5-hydroxymethylcytosine (5hmC) levels on DNA and Western blotting of modified histone 3 (H3) proteins to quantify tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. RESULTS: Specific pro-angiogenic genes in ARAS assessed by RT-PCR were lower at baseline but increased under HPC, while pro-senescence genes were higher in ARAS at baseline as compared healthy MSCs. ARAS MSCs under basal conditions, displayed higher H3K4me3, H3K27me3, and 5hmC levels compared to healthy MSCs. During HPC, global 5hmC levels were decreased while no appreciable changes occurred in histone H3 tri-methylation. ARAS MSCs cultured under HPC had higher migratory and proliferative capacity as well as increased vascular endothelial growth factor and epidermal growth factor expression compared to normoxia, and SA-ß-gal activity decreased in both animal groups. CONCLUSIONS: These data demonstrate that swine ARAS MSCs have decreased angiogenesis and increased senescence compared to healthy MSCs and that HPC mitigates MSC dysfunction, senescence, and DNA hydroxymethylation in ARAS MSC. Thus, HPC for MSCs may be considered for their optimization to improve autologous cell therapy in patients with nephropathies.

Diabetic Kidney Disease Alters the Transcriptome and Function of Human Adipose-Derived Mesenchymal Stromal Cells but Maintains Immunomodulatory and Paracrine Activities Important for Renal Repair.

Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell-derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-ß-exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A1c, kidney function, and urine albumin excretion. However, senescence-associated ß-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials.

Mesenchymal Stem/Stromal Cells and their Extracellular Vesicle Progeny Decrease Injury in Poststenotic Swine Kidney Through Different Mechanisms.

Novel therapies are needed to address the increasing prevalence of chronic kidney disease. Mesenchymal stem/stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) augment tissue repair. We tested the hypothesis that EVs are as effective as MSCs in protecting the stenotic kidney, but target different injury pathways. Pigs were studied after 16 weeks of renal injury achieved by diet-induced metabolic syndrome (MetS) and renal artery stenosis (RAS). Pigs were untreated or treated 4 weeks earlier with intrarenal delivery of autologous adipose tissue-derived MSCs (107) or their EVs (1011). Lean pigs and sham RAS served as controls (n = 6 each). Stenotic-kidney function was studied in vivo using computed tomography and magnetic resonance imaging. Histopathology and expression of necroptosis markers [receptor-interacting protein kinase (RIPK)-1 and RIPK-3], inflammatory, and growth factors (angiopoietin-1 and vascular endothelial growth factor) were studied ex vivo. Stenotic-kidney glomerular filtration rate and blood flow in MetS + RAS were both lower than Lean and increased in both MetS + RAS + MSC and MetS + RAS + EV. Both MSCs and EV improved renal function and decreased renal hypoxia, fibrosis, and apoptosis. MSCs were slightly more effective in preserving microvascular (0.02-0.2 mm diameters) density and prominently attenuated renal inflammation. However, EV more significantly upregulated growth factor expression and decreased necroptosis. In conclusion, adipose tissue-derived MSCs and their EV both improve stenotic kidney function and decrease tissue injury in MetS + RAS by slightly different mechanisms. MSCs more effectively preserved the microcirculation, while EV bestowed better preservation of renal cellular integrity. These findings encourage further exploration of this novel approach to attenuate renal injury.

Human Obesity Induces Dysfunction and Early Senescence in Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells.

BACKGROUND: Chronic inflammatory conditions like obesity may adversely impact the biological functions underlying the regenerative potential of mesenchymal stromal/stem cells (MSC). Obesity can impair MSC function by inducing cellular senescence, a growth-arrest program that transitions cells to a pro-inflammatory state. However, the effect of obesity on adipose tissue-derived MSC in human subjects remains unclear. We tested the hypothesis that obesity induces senescence and dysfunction in human MSC. METHODS: MSC were harvested from abdominal subcutaneous fat collected from obese and age-matched non-obese subjects (n = 40) during bariatric or kidney donation surgeries, respectively. MSC were characterized, their migration and proliferation assessed, and cellular senescence evaluated by gene expression of cell-cycle arrest and senescence-associated secretory phenotype markers. In vitro studies tested MSC effect on injured human umbilical vein endothelial cells (HUVEC) function. RESULTS: Mean age was 59 ± 8 years, 66% were females. Obese subjects had higher body-mass index (BMI) than non-obese. MSC from obese subjects exhibited lower proliferative capacities than non-obese-MSC, suggesting decreased function, whereas their migration remained unchanged. Senescent cell burden and phenotype, manifested as p16, p53, IL-6, and MCP-1 gene expression, were significantly upregulated in obese subjects' MSC. BMI correlated directly with expression of p16, p21, and IL-6. Furthermore, co-incubation with non-obese, but not with obese-MSC, restored VEGF expression and tube formation that were blunted in injured HUVEC. CONCLUSION: Human obesity triggers an early senescence program in adipose tissue-derived MSC. Thus, obesity-induced cellular injury may alter efficacy of this endogenous repair system and hamper the feasibility of autologous transplantation in obese individuals.

Metabolic Syndrome Modulates Protein Import into the Mitochondria of Porcine Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are currently being tested in several clinical trials. Mitochondria regulate many aspects of MSC function. Mitochondrial preproteins are rapidly translated and trafficked into the mitochondrion for assembly in their final destination, but whether coexisting cardiovascular risk factors modulate this process is unknown. We hypothesized that metabolic syndrome (MetS) modulates mitochondrial protein import in porcine MSCs. MSCs were isolated from porcine abdominal adipose tissue after 16 weeks of Lean or MetS diet (n = 5 each). RNA-sequencing was performed and differentially expressed mitochondrial mRNAs and microRNAs were identified and validated. Protein expression of transporters of mitochondrial proteins (presequences and precursors) and their respective substrates were measured. Mitochondrial homeostasis was assessed by Western blot and function by cytochrome-c oxidase-IV activity. Forty-five mitochondrial mRNAs were upregulated and 25 downregulated in MetS-MSCs compared to Lean-MSCs. mRNAs upregulated in MetS-MSCs encoded for precursor proteins, whereas those downregulated encoded for presequences. Micro-RNAs upregulated in MetS-MSCs primarily target mRNAs encoding for presequences. Transporters of precursor proteins and their substrates were also upregulated, associated with changes in mitochondrial homeostasis and dysfunction. MetS interferes with mitochondrial protein import, favoring upregulation of precursor proteins, which might be linked to post-transcriptional regulation of presequences. This in turn alters mitochondrial homeostasis and impairs energy production. Our observations highlight the importance of mitochondria in MSC function and provide a molecular framework for optimization of cell-based strategies as we move towards their clinical application.

Metabolic Syndrome Induces Release of Smaller Extracellular Vesicles from Porcine Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) belong to the endogenous cellular reparative system, and can be used exogenously in cell-based therapy. MSCs release extracellular vesicles (EVs), including exosomes and microvesicles, which mediate some of their therapeutic activity through intercellular communication. We have previously demonstrated that metabolic syndrome (MetS) modifies the cargo packed within swine EV, but whether it influences their phenotypical characteristics remains unclear. This study tested the hypothesis that MetS shifts the size distribution of MSC-derived EVs. Adipose tissue-derived MSC-EV subpopulations from Lean (n = 6) and MetS (n = 6) pigs were characterized for number and size using nanoparticle-tracking analysis, flow cytometry, and transmission electron microscopy. Expression of exosomal genes was determined using next-generation RNA-sequencing (RNA-seq). The number of EV released from Lean and MetS pig MSCs was similar, yet MetS-MSCs yielded a higher proportion of small-size EVs (202.4 ± 17.7 nm vs. 280.3 ± 15.1 nm), consistent with exosomes. RNA-seq showed that their EVs were enriched with exosomal markers. Lysosomal activity remained unaltered in MetS-MSCs. Therefore, MetS alters the size distribution of MSC-derived EVs in favor of exosome release. These observations may reflect MSC injury and membrane recycling in MetS or increased expulsion of waste products, and may have important implications for development of adequate cell-based treatments.