Cinzia Rota

Human Bone Marrow Mesenchymal Stem Cells Accelerate Recovery of Acute Renal Injury and Prolong Survival in Mice

Transplantation of bone marrow mesenchymal stem cells (BM‐MSC) or stromal cells from rodents has been identified as a strategy for renal repair in experimental models of acute kidney injury (AKI), a highly life‐threatening clinical setting. The therapeutic potential of BM‐MSC of human origin has not been reported so far. Here, we investigated whether human BM‐MSC treatment could prevent AKI induced by cisplatin and prolong survival in an immunodeficient mouse model. Results showed that human BM‐MSC infusion decreased proximal tubular epithelial cell injury and ameliorated the deficit in renal function, resulting in reduced recipient mortality. Infused BM‐MSC became localized predominantly in peritubular areas and acted to reduce renal cell apoptosis and to increase proliferation. BM‐MSC also induced protection against AKI‐related peritubular capillary changes consisting of endothelial cell abnormalities, leukocyte infiltration, and low endothelial cell and lumen volume density as assessed by morphometric analysis. These findings indicate that human MSC of bone marrow origin hold potential to prolong survival in AKI and should be considered for testing in a clinical trial.

Insulin-Like Growth Factor-1 Sustains Stem Cell–Mediated Renal Repair

In mice with cisplatin-induced acute kidney injury, administration of bone marrow-derived mesenchymal stem cells (MSC) restores renal tubular structure and improves renal function, but the underlying mechanism is unclear. Here, we examined the process of kidney cell repair in co-culture experiments with MSC and cisplatin-injured proximal tubular epithelial cells (PTEC). Exposure of PTEC to cisplatin markedly reduced cell viability at 4 days, but co-culture with MSC provided a protective effect by promoting tubular cell proliferation. This effect was mediated by insulin-like growth factor-1 (IGF-1), highly expressed by MSC as mRNA and protein, since blocking the growth factors function with a specific antibody attenuated cell proliferation of PTEC. Confirming this, knocking down IGF-1 expression in MSC by small interfering-RNA also resulted in a significant decrease in PTEC proliferation and increased apoptosis. Furthermore, in the murine model of cisplatin-induced kidney injury, administering IGF-1 gene-silenced MSC limited their protective effect on renal function and tubular structure. These findings indicate that MSC exert beneficial effects on tubular cell repair in acute kidney injury by producing the mitogenic and pro-survival factor IGF-1.

Transfer of Growth Factor Receptor mRNA Via Exosomes Unravels the Regenerative Effect of Mesenchymal Stem Cells

Bone marrow-mesenchymal stem cells (BM-MSC) ameliorate renal dysfunction and repair tubular damage of acute kidney injury by locally releasing growth factors, including the insulin-like growth factor-1 (IGF-1). The restricted homing of BM-MSC at the site of injury led us to investigate a possible gene-based communication mechanism between BM-MSC and tubular cells. Human BM-MSC (hBM-MSC) released microparticles and exosomes (Exo) enriched in mRNAs. A selected pattern of transcripts was detected in Exo versus parental cells. Exo expressed the IGF-1 receptor (IGF-1R), but not IGF-1 mRNA, while hBM-MSC contained both mRNAs. R- cells lacking IGF-1R exposed to hBM-MSC-derived Exo acquired the human IGF-1R transcript that was translated in the corresponding protein. Transfer of IGF-1R mRNA from Exo to cisplatin-damaged proximal tubular cells (proximal tubular epithelial cell [PTEC]) increased PTEC proliferation. Coincubation of damaged PTEC with Exo and soluble IGF-1 further enhanced cell proliferation. These findings suggest that horizontal transfer of the mRNA for IGF-1R to tubular cells through Exo potentiates tubular cell sensitivity to locally produced IGF-1 providing a new mechanism underlying the powerful renoprotection of few BM-MSC observed in vivo.

Life-Sparing Effect of Human Cord Blood-Mesenchymal Stem Cells in Experimental Acute Kidney Injury

In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)‐MSCs to cure mice with AKI. Infusion of CB‐MSCs in immunodeficient mice with cisplatin‐induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB‐MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno‐protective effect of CB‐MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB‐MSCs in vitro increased the production of growth factors and inhibited IL‐1β and TNFα synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti‐inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB‐MSCs as future cell therapy for testing in human AKI. STEM CELLS 2010;28:513–522

Localization of Mesenchymal Stromal Cells Dictates Their Immune or Proinflammatory Effects in Kidney Transplantation

Multipotent mesenchymal stromal cells (MSC) have recently emerged as promising candidates for cell‐based immunotherapy in solid‐organ transplantation. However, optimal conditions and settings for fully harnessing MSC tolerogenic properties need to be defined. We recently reported that autologous MSC given posttransplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Here, we moved back to a murine kidney transplant model with the aim to define the best timing of MSC infusion capable of promoting immune tolerance without negative effects on early graft function. We also investigated the mechanisms of the immunomodulatory and/or proinflammatory activities of MSC according to whether cells were given before or after transplant. Posttransplant MSC infusion in mice caused premature graft dysfunction and failed to prolong graft survival. In this setting, infused MSC localized mainly into the graft and associated with neutrophils and complement C3 deposition. By contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by a Treg‐dependent mechanism. MSC‐infused pretransplant localized into lymphoid organs where they promoted early expansion of Tregs. Thus, pretransplant MSC infusion may be a useful approach to fully exploit their immunomodulatory properties in kidney transplantation.

Bone Marrow-Derived Mesenchymal Stem Cells Improve Islet Graft Function in Diabetic Rats

Type 1 diabetes is associated with a progressive loss of beta cells and pancreatic islet transplantation could represent a cure for this disease. Herein we explored whether transplantation of bone marrow-derived mesenchymal stem cells (MSCs) allowed a reduced number of pancreatic islets to improve glycemic control in diabetic rats, by promoting islet vascularization. We transplanted 2000 syngenic islets alone or in combination with MSCs (10(6) cells) under the kidney capsules of diabetic Lewis rats. Animals transplanted with 2000 islets never reached normoglycemia. In contrast, rats transplanted with 2000 islets plus MSCs, showed a gradual fall in glycemia after transplantation, with normoglycemia maintained until killing. Comparable glycemic control was obtained with transplantation of 3000 islets alone. The MSC preparation used for in vivo experiments expressed high levels of vascular endothelial growth factor (VEGF(165)) and, at less extent, VEGF(189), as evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). In transplanted animals, vascularization was quantified by morphometric analysis of islet grafts with anti-RECA and anti-insulin antibodies. MSCs were stained with PKH-26. Mean capillary density was 1002 +/- 55 capillaries/mm(2) in islets transplanted alone. Co-infusion of MSCs with islets significantly increased the number of capillaries to 1459 +/- 66 capillaries/mm(2). In conclusion, our study indicated that co-transplantation of MSCs with pancreatic islets improved islet graft function by promoting graft vascularization.

Sirtuin 3–dependent mitochondrial dynamic improvements protect against acute kidney injury

Acute kidney injury (AKI) is a public health concern with an annual mortality rate that exceeds those of breast and prostate cancer, heart failure, and diabetes combined. Oxidative stress and mitochondrial damage are drivers of AKI-associated pathology; however, the pathways that mediate these events are poorly defined. Here, using a murine cisplatin-induced AKI model, we determined that both oxidative stress and mitochondrial damage are associated with reduced levels of renal sirtuin 3 (SIRT3). Treatment with the AMPK agonist AICAR or the antioxidant agent acetyl-l-carnitine (ALCAR) restored SIRT3 expression and activity, improved renal function, and decreased tubular injury in WT animals, but had no effect in Sirt3-/- mice. Moreover, Sirt3-deficient mice given cisplatin experienced more severe AKI than WT animals and died, and neither AICAR nor ALCAR treatment prevented death in Sirt3-/- AKI mice. In cultured human tubular cells, cisplatin reduced SIRT3, resulting in mitochondrial fragmentation, while restoration of SIRT3 with AICAR and ALCAR improved cisplatin-induced mitochondrial dysfunction. Together, our results indicate that SIRT3 is protective against AKI and suggest that enhancing SIRT3 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury.

Human Amniotic Fluid Stem Cell Preconditioning Improves Their Regenerative Potential

Human amniotic fluid stem (hAFS) cells, a novel class of broadly multipotent stem cells that share characteristics of both embryonic and adult stem cells, have been regarded as promising candidate for cell therapy. Taking advantage by the well-established murine model of acute kidney injury (AKI), we studied the proregenerative effect of hAFS cells in immunodeficient mice injected with the nephrotoxic drug cisplatin. Infusion of hAFS cells in cisplatin mice improved renal function and limited tubular damage, although not to control level, and prolonged animal survival. Human AFS cells engrafted injured kidney predominantly in peritubular region without acquiring tubular epithelial markers. Human AFS cells exerted antiapoptotic effect, activated Akt, and stimulated proliferation of tubular cells possibly via local release of factors, including interleukin-6, vascular endothelial growth factor, and stromal cell-derived factor-1, which we documented in vitro to be produced by hAFS cells. The therapeutic potential of hAFS cells was enhanced by cell pretreatment with glial cell line-derived neurotrophic factor (GDNF), which markedly ameliorated renal function and tubular injury by increasing stem cell homing to the tubulointerstitial compartment. By in vitro studies, GDNF increased hAFS cell production of growth factors, motility, and expression of receptors involved in cell homing and survival. These findings indicate that hAFS cells can promote functional recovery and contribute to renal regeneration in AKI mice via local production of mitogenic and prosurvival factors. The effects of hAFS cells can be remarkably enhanced by GDNF preconditioning.

Inhibiting Angiotensin-Converting Enzyme Promotes Renal Repair by Limiting Progenitor Cell Proliferation and Restoring the Glomerular Architecture

We previously reported that angiotensin-converting enzyme inhibitor (ACEi) renoprotection in Munich Wistar Frömter (MWF) rats, which develop progressive glomerular injury, was associated with podocyte repopulation and preservation of glomerular architecture. Here, we studied the time course of the lesions, their cellular components, and the effect of ACEi. Early glomerular lesions were synechiae, followed by extracapillary crescents and glomerulosclerosis. The majority of cells forming crescents were claudin1(+) parietal epithelial cells and, to a lesser extent, WT1(+) podocytes, both in active proliferation. In crescents, cells expressing the metanephric mesenchyme marker NCAM were also found. Three distinct populations of parietal epithelial cells were identified in the rat Bowmans capsule: NCAM(+)WT1(-) cells, also expressing progenitor cell marker CD24, and NCAM(+)WT1(+) and NCAM(-)WT1(+) cells, the latter population representing parietal podocytes. After exposure to inductive medium, cultured parietal epithelial cells that were obtained by capsulated glomeruli generated podocytes, documenting their progenitor nature. Mitotic activity of cultured renal progenitors was induced by angiotensin II through the down-regulation of cell cycle inhibitor C/EBPδ expression. Treatment with ACEi reduced number and extension of crescents and glomerulosclerosis in MWF rats. Renoprotection was accomplished through the limitation of NCAM(+) progenitor proliferation via the modulation of C/EBPδ. Thus, chaotic migration and proliferation of the Bowmans capsule progenitor cells pave the way to crescent formation and subsequent sclerosis. ACEi, by moderating progenitor cell activation, restores glomerular architecture and prevents renal disease progression.

Mesenchymal stem cell therapy promotes renal repair by limiting glomerular podocyte and progenitor cell dysfunction in adriamycin-induced nephropathy

We previously reported that in a model of spontaneously progressive glomerular injury with early podocyte loss, abnormal migration, and proliferation of glomerular parietal epithelial progenitor cells contributed to the formation of synechiae and crescentic lesions. Here we first investigated whether a similar sequence of events could be extended to rats with adriamycin (ADR)-induced nephropathy. As a second aim, the regenerative potential of therapy with bone marrow-derived mesenchymal stem cells (MSCs) on glomerular resident cells was evaluated. In ADR-treated rats, decrease of WT1(+) podocyte number due to apoptosis was associated with reduced glomerular expression of nephrin and CD2AP. As a consequence of podocyte injury, glomerular adhesions of the capillary tuft to the Bowmans capsule were observed, followed by crescent-like lesions and glomerulosclerosis. Cellular components of synechiae were either NCAM(+) parietal progenitor cells or nestin(+) podocytes. In ADR rats, repeated injections of MSCs limited podocyte loss and apoptosis and partially preserved nephrin and CD2AP. MSCs attenuated the formation of glomerular podocyte-parietal epithelial cell bridges and normalized the distribution of NCAM(+) progenitor cells along the Bowmans capsule, thereby reducing glomerulosclerosis. Finding that MSCs increased glomerular VEGF expression and limited microvascular rarefaction may explain the prosurvival effect by stem cell therapy. MSCs also displayed anti-inflammatory activity. Coculture of MSCs with ADR-damaged podocytes showed a functional role of stem cell-derived VEGF on prosurvival pathways. These data suggest that MSCs by virtue of their tropism for damaged kidney and ability to provide a local prosurvival environment may represent a useful strategy to preserve podocyte viability and reduce glomerular inflammation and sclerosis.