Kidney-Derived c-Kit(+) Cells Possess Regenerative Potential

Kidney-derived c-Kit(+) cells exhibit progenitor/stem cell properties in vitro (self-renewal capacity, clonogenicity, and multipotentiality). These cells can regenerate epithelial tubular cells following ischemia-reperfusion injury and accelerate foot processes effacement reversal in a model of acute proteinuria in rats. Several mechanisms are involved in kidney regeneration by kidney-derived c-Kit(+ )cells, including cell engraftment and differentiation into renal-like structures, such as tubules, vessels, and podocytes. Moreover, paracrine mechanisms could also account for kidney regeneration, either by stimulating proliferation of surviving cells or modulating autophagy and podocyte cytoskeleton rearrangement through mTOR-Raptor and -Rictor signaling, which ultimately lead to morphological and functional improvement. To gain insights into the functional properties of c-Kit(+) cells during kidney development, homeostasis, and disease, studies on lineage tracing using transgenic mice will unveil their fate. The results obtained from these studies will set the basis for establishing further investigation on the therapeutic potential of c-Kit(+) cells for treatment of kidney disease in preclinical and clinical studies.Conselho Nacional em Pesquisa e Desenvolvimento (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)European Foundation for the Study of Diabetes (EFSD)Univ Sao Paulo, Renal Div, Lab Cellular Genet & Mol Nephrol, Sao Paulo, SP, BrazilUniv Miami, Leonard M Miller Sch Med, Interdisciplinary Stem Cell Inst, Miami, FL USAUniv Miami, Leonard M Miller Sch Med, Dept Mol & Cellular Pharmacol, Miami, FL USAUniv Miami, Div Cardiol, Leonard M Miller Sch Med, Miami, FL USAHosp Israelita Albert Einstein, Inst Israelita Ensino & Pesquisa Albert Einstein, Albert Einstein Ave,627-701 Bldg A, BR-05652900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Div Nephrol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Div Nephrol, Sao Paulo, SP, BrazilCNPq: 456959/2013-0FAPESP: 13/19560-6Web of Scienc

Mesenchymal stem cells as therapeutic candidates for halting the progression of diabetic nephropathy

Mesenchymal stem cells (MSCs) possess pleiotropic properties that include immunomodulation, inhibition of apoptosis, fibrosis and oxidative stress, secretion of trophic factors, and enhancement of angiogenesis. These properties provide a broad spectrum for their potential in a wide range of injuries and diseases, including diabetic nephropathy (DN). MSCs are characterized by adherence to plastic, expression of the surface molecules CD73, CD90, and CD105 in the absence of CD34, CD45, HLA-DR, and CD14 or CD11b and CD79a or CD19 surface molecules, and multidifferentiation capacity in vitro. MSCs can be derived from many tissue sources, consistent with their broad, possibly ubiquitous distribution. This article reviews the existing literature and knowledge of MSC therapy in DN, as well as the most appropriate rodent models to verify the therapeutic potential of MSCs in DN setting. Some preclinical relevant studies are highlighted and new perspectives of combined therapies for decreasing DN progression are discussed. Hence, improved comprehension and interpretation of experimental data will accelerate the progress towards clinical trials that should assess the feasibility and safety of this therapeutic approach in humans. Therefore, MSC-based therapies may bring substantial benefit for patients suffering from DN.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo/Sao Paulo Research Foundation) [2013/19560-6]CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico/National Counsel of Technological and Scientific Development) [456959/2013-0]EFSD (European Foundation for the Study of Diabetes)Sociedade Beneficente Albert Einstein, Albert Einstein Hospital, 05652 São Paulo, SP, Brazil[University of São Paulo, 01246 São Paulo, SP, BrazilFederal University of São Paulo, 04023 São Paulo, SP, BrazilFederal University of São Paulo, 04023 São Paulo, SP, BrazilFAPESP: 2013/19560-6CNPq: 456959/2013-0Web of Scienc

Progenitor/Stem Cell Delivery by Suprarenal Aorta Route in Acute Kidney Injury

Progenitor/stem cell-based kidney regenerative strategies are a key step towards the development of novel therapeutic regimens for kidney disease treatment. However, the route of cell delivery, e.g., intravenous, intra-arterial, or intra-parenchymal, may affect the efficiency for kidney repair in different models of acute and chronic injury. Here, we describe a protocol of intra-aorta progenitor/stem cell injection in rats following either acute ischemia-reperfusion injury or acute proteinuria induced by puromycin aminonucleoside (PAN) – the experimental prototype of human minimal change disease and early stages of focal and segmental glomerulosclerosis. Vascular clips were applied across both renal pedicles for 35 min, or a single dose of PAN was injected via intra-peritoneal route, respectively. Subsequently, 2 x 106 stem cells [green fluorescent protein (GFP)-labeled c-Kit+ progenitor/stem cells or GFP-mesenchymal stem cells] or saline were injected into the suprarenal aorta, above the renal arteries, after application of a vascular clip to the abdominal aorta below the renal arteries. This approach contributed to engraftment rates of ∼10% at day 8 post ischemia-reperfusion injury, when c-Kit+ progenitor/stem cells were injected, which accelerated kidney recovery. Similar rates of engraftment were found after PAN-induced podocyte damage at day 21. With practice and gentle surgical technique, 100% of the rats could be injected successfully, and, in the week following injection, ∼ 85% of the injected rats will recover completely. Given the similarities in mammals, much of the data obtained from intra-arterial delivery of progenitor/stem cells in rodents can be tested in translational research and clinical trials with endovascular catheters in humans

Mesenchymal Stem Cells as Therapeutic Candidates for Halting the Progression of Diabetic Nephropathy

Mesenchymal stem cells (MSCs) possess pleiotropic properties that include immunomodulation, inhibition of apoptosis, fibrosis and oxidative stress, secretion of trophic factors, and enhancement of angiogenesis. These properties provide a broad spectrum for their potential in a wide range of injuries and diseases, including diabetic nephropathy (DN). MSCs are characterized by adherence to plastic, expression of the surface molecules CD73, CD90, and CD105 in the absence of CD34, CD45, HLA-DR, and CD14 or CD11b and CD79a or CD19 surface molecules, and multidifferentiation capacity in vitro. MSCs can be derived from many tissue sources, consistent with their broad, possibly ubiquitous distribution. This article reviews the existing literature and knowledge of MSC therapy in DN, as well as the most appropriate rodent models to verify the therapeutic potential of MSCs in DN setting. Some preclinical relevant studies are highlighted and new perspectives of combined therapies for decreasing DN progression are discussed. Hence, improved comprehension and interpretation of experimental data will accelerate the progress towards clinical trials that should assess the feasibility and safety of this therapeutic approach in humans. Therefore, MSC-based therapies may bring substantial benefit for patients suffering from DN

Abstract 7: S-nitrosylation Affects Differentiation of Mesenchymal Stem Cells

Introduction: Bone marrow-derived mesenchymal stem cells (BMMSCs) appear to play an important role in the formation of atherosclerotic lesions. Circulating BMMSCs are attracted to arterial wall where they undergo adipogenic or osteogenic differentiation to form ectopic aggregates of fat and bone. The factors that determine the direction of BMMSC differentiation are poorly understood. We assessed the role of nitric oxide (NO), an inhibitor of lipid formation and vascular calcification, in BMMSC differentiation. Hypothesis: S-nitrosylation mediated-NO signaling controls the balance between adipogenic and osteogenic differentiation of BMMSCs. Methods: We isolated BMMSCs from wild type mice (WT) and mice deficient in S-nitrosoglutathione reductase (GSNOR -/- ), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation. Cells were cultured in either adipogenic or osteogenic differentiation media followed by functional and gene expression assays. Results: MSCs derived from GSNOR -/- mice had impaired fat droplet formation and lower expression of the adipogenic markers PPARγ (1329±415.3-fold increase in WT vs. 158±65.61-fold in GSNOR -/- , P<0.05) and FABP4 (11.06±3.29-fold in WT vs. 4.06±0.62-fold in GSNOR -/- , P<0.05). Conversely, GSNOR -/- MSCs exhibited enhanced osteogenic differentiation as indicated by greater calcium deposition and higher expression of the osteogenic marker Osteopontin (1.48±0.17-fold in WT vs. 16.18±5.26-fold in GSNOR -/- , P<0.05). Interestingly, GSNOR -/- cells had higher baseline expression of Osteopontin, Osteocalcin and Runx2. Treatment of GSNOR -/- MSCs with L-NAME, a nitric oxide synthase (NOS) inhibitor, resulted in decreased osteogenic differentiation without affecting adipogenic differentiation. Conclusion: Our results indicate that GSNOR enhances adipogenic differentiation and impairs osteogenic differentiation of BMMSCs in vitro and suggest that interaction of GSNOR- and NOS-mediated processes regulates the balance between adipogenic and osteogenic differentiation of BMMSCs

Abstract 239: Nitric Oxide Suppresses the Angiogeneic Potential of Mesenchymal Stem Cells

Introduction: Mesenchymal stem cells (MSCs) participate in blood vessel formation which is crucial for tissue regeneration. However, the mechanism involved in regulating MSC angiogenesis remains elusive. We examined the role of Nitric Oxide (NO) in this process by comparing angiogenesis by MSCs derived from wild type mice and mice lacking S-nitrosoglutathione reductase (GSNOR-/-), an enzyme that increases intracellular GSNO and enhances endogenous S-nitrosylation (a ubiquitous redox-related modification of cysteine thiol by NO) Hypothesis: Nitric oxide signaling pathways are mediated via small molecular thiols that regulate MSC angiogenesis. Method: Bone marrow-derived MSCs were isolated from wild type and GSNOR-/- mice and grown for 7 days in endothelial growth media followed by 24h in Matrigel (in vitro angiogenesis assay) in the presence of either vehicle, 15µM L-NAME (a nitric oxide synthase (NOS) inhibitor) or 500µM SNAP (a nitric oxide donor). Human (h)MSCs were similarly treated with vehicle, 15µM L-NAME and 100µM SNAP. MSC NO production and NOS expression were assessed. Results: Both types of MSCs exhibited similar levels of NO production and NOS expression, however, in the Matrigel Assay, MSCs from GSNOR-/- mice formed fewer (29.9±12.0 vs.. 50.0±15.4, p<0.001) and shorter (62.8 ± 34.6 µm vs. 105.9±57.0 µm, p<0.001) tubes than MSCs derived from wild type mice. L-NAME treatment normalized GSNOR-/- tube number (49.9 ± 14.8, p<0.001) and length (82.8±42.5 µm, p<0.001). Treatment of wild type MSCs with SNAP impaired tube formation (15.6±9.0, p<0.001) and length (57.1±24.9 µm, p<0.001). Similarly, hMSCs treated with SNAP exhibited impaired tube formation, both in number (25.0±4.9 vs. 49.2±8.1, p<0.001) and length (81±29.1 vs. 128.0±52.5 µm, p<0.001) compared to vehicle treated MSCs. L-NAME had no effect on hMSCs . Conclusion: These findings are consistent with a paradoxical negative influence of NO/GSNO on vascular formation by MSCs and reveal a novel mechanism whereby NO/GSNO inhibits MSC dependent angiogenesis. These findings have implications for disease states characterized by excessive or dysregulated angiogenesis; moreover NO deficient environments may trigger MSC mediated angiogenesis

Kidney-derived c-kit + progenitor/stem cells contribute to podocyte recovery in a model of acute proteinuria

Kidney-derived c-kit cells exhibit progenitor/stem cell properties and can regenerate epithelial tubular cells following ischemia-reperfusion injury in rats. We therefore investigated whether c-kit progenitor/stem cells contribute to podocyte repair in a rat model of acute proteinuria induced by puromycin aminonucleoside (PAN), the experimental prototype of human minimal change disease and early stages of focal and segmental glomerulosclerosis. We found that c-kit progenitor/stem cells accelerated kidney recovery by improving foot process effacement (foot process width was lower in c-kit group vs saline treated animals, P = 0.03). In particular, these cells engrafted in small quantity into tubules, vessels, and glomeruli, where they occasionally differentiated into podocyte-like cells. This effect was related to an up regulation of α-Actinin-4 and mTORC2-Rictor pathway. Activation of autophagy by c-kit progenitor/stem cells also contributed to kidney regeneration and intracellular homeostasis (autophagosomes and autophagolysosomes number and LC3A/B-I and LC3A/B-II expression were higher in the c-kit group vs saline treated animals, P = 0.0031 and P = 0.0009, respectively). Taken together, our findings suggest that kidney-derived c-kit progenitor/stem cells exert reparative effects on glomerular disease processes through paracrine effects, to a lesser extent differentiation into podocyte-like cells and contribution to maintenance of podocyte cytoskeleton after injury. These findings have clinical implications for cell therapy of glomerular pathobiology

Abstract P025: Modulatory Effect of Angiotensin II on Endothelial Differentiation and Vasculogenic Capacity of Human MSCs

Human bone marrow-derived mesenchymal stem cell (MSC)-based therapy holds great promise as a new approach for cardiovascular regeneration. In heart failure, there is activation of the renin-angiotensin system and increased formation of angiotensin (Ang) II. We tested the hypothesis that Ang II impairs endothelial differentiation and vasculogenic capacity of human MSCs. MSCs were cultured in endothelial growth media (EGM) and treated with vehicle, 1µM Ang II, or 10µM Ang II for 7 days to assess by real time RT-PCR the expression of two markers of endothelial differentiation, platelet endothelial cell adhesion molecule (PECAM) and von Willebrand factor (vWF). Expression of PECAM and vWF increased similarly in each treatment group at day 7, suggesting endothelial differentiation of MSCs. To assess vasculogenesis, MSCs were cultured in EGM for 7 days and then plated on growth factor reduced Matrigel for 5 hours under the following conditions: vehicle, Ang II (1µM), Ang II + type 1 receptor (AT1R) inhibitor (10µM losartan), or Ang II + type 2 receptor (AT2R) inhibitor (0.1µM PD123177). The vascular index (VI) was determined by multiplying the average number of endothelial tubes formed by the average tube length. MSCs treated for 5 hours with 1µM Ang II exhibited a reduced VI (84.5 ± 2.6% of vehicle treated cells, P<0.05). Treatment with Ang II+AT1R inhibitor increased VI (115 ± 4.9% of vehicle, P<0.05) whereas Ang II+AT2R inhibitor decreased VI (77.1 ± 4.1% of vehicle, P<0.05). Chronic treatment for 7 days in EGM with 1µM or 10µM Ang II also showed decreased VI compared to vehicle (75.4 ± 5.4% and 60.7 ± 2% of vehicle, respectively, P<0.01). These results reveal that both acute and chronic treatment with Ang II produces a negative effect on the vasculogenic potential of endothelial-differentiated human MSCs and suggests opposing effects of AT1R and AT2R on vasculogenesis. In conclusion, we propose that selective modulation of AT1R and AT2R mediated pathways may serve as a promising means for enhancing the regenerative capacity of human MSCs in cardiovascular disease

S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells

Although nitric oxide (NO) signaling promotes differentiation and maturation of endothelial progenitor cells, its role in the differentiation of mesenchymal stem cells (MSCs) into endothelial cells remains controversial. We tested the role of NO signaling in MSCs derived from WT mice and mice homozygous for a deletion of S-nitrosoglutathione reductase (GSNOR(-/-)), a denitrosylase that regulates S-nitrosylation. GSNOR(-/-) MSCs exhibited markedly diminished capacity for vasculogenesis in an in vitro Matrigel tube-forming assay and in vivo relative to WT MSCs. This decrease was associated with down-regulation of the PDGF receptorα (PDGFRα) in GSNOR(-/-) MSCs, a receptor essential for VEGF-A action in MSCs. Pharmacologic inhibition of NO synthase with L-N(G)-nitroarginine methyl ester (L-NAME) and stimulation of growth hormone-releasing hormone receptor (GHRHR) with GHRH agonists augmented VEGF-A production and normalized tube formation in GSNOR(-/-) MSCs, whereas NO donors or PDGFR antagonist reduced tube formation ∼50% by murine and human MSCs. The antagonist also blocked the rescue of tube formation in GSNOR(-/-) MSCs by L-NAME or the GHRH agonists JI-38, MR-409, and MR-356. Therefore, GSNOR(-/-) MSCs have a deficient capacity for endothelial differentiation due to downregulation of PDGFRα related to NO/GSNOR imbalance. These findings unravel important aspects of modulation of MSCs by VEGF-A activation of the PDGFR and illustrate a paradoxical inhibitory role of S-nitrosylation signaling in MSC vasculogenesis. Accordingly, disease states characterized by NO deficiency may trigger MSC-mediated vasculogenesis. These findings have important implications for therapeutic application of GHRH agonists to ischemic disorders

Charcot Neuroarthropathy After Simultaneous Pancreas-Kidney Transplant

Background. Immunosuppressive regimen is associated with several metabolic adverse effects. Bone loss and fractures are frequent after transplantation and involve multifactorial mechanisms.Methods. A retrospective analysis of 130 patients submitted to simultaneous pancreas-kidney transplantation (SPKT) and an identification of risk factors involved in de novo Charcot neuroarthropathy by multivariate analysis were used; P<0.05 was considered significant.Results. Charcot neuroarthropathy was diagnosed in 4.6% of SPKT recipients during the first year. Cumulative glucocorticoid doses (daily dose plus methylprednisolone pulse) during the first 6 months both adjusted to body weight (978 mg/kg) and not adjusted to body weight were associated with Charcot neuroarthropathy (P=0.001 and P<0.0001, respectively). Age, gender, race, time on dialysis, time of diabetes history, and posttransplantation hyperparathyroidism were not related to Charcot neuroarthropathy after SPKT.Conclusions. Glucocorticoids are the main risk factors for de novo Charcot neuroarthropathy after SPKT. Protocols including glucocorticoid avoidance or minimization should be considered.Universidade Federal de São Paulo, Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Div Endocrinol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Surg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Div Endocrinol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Surg, BR-04023900 São Paulo, BrazilWeb of Scienc