Zhuma Hu

VEGF is a mediator of the renoprotective effects of multipotent marrow stromal cells in acute kidney injury

Adult stem cell treatment of complex disorders is a promising therapeutic approach and multipotent marrow stromal cells (MSCs) have been shown to be effective in various animal models of diseases. Acute kidney injury (AKI) is a common and serious problem in hospitalized patients and bone marrow derived multipotent MSCs have been shown to be effective in different models of AKI. The mechanism of action of MSCs is complex but involves paracrine actions including growth factor secretion. Knockdown of vascular enthothelial growth factor (VEGF) by siRNA reduced effectiveness of MSCs in the treatment of ischemic AKI in a rat model. Animals treated with MSCs had increased renal microvessel density compared to VEGF knockdown MSC‐treated and vehicle‐treated animals. These results show that VEGF is an important mediator of the early and late phase of renoprotective action after AKI in the context of stem cell treatment.

Bioluminescence imaging to monitor the in vivo distribution of administered mesenchymal stem cells in acute kidney injury.

Effective and targeted delivery of cells to injured organs is critical to the development of cell therapies. However, currently available in vivo cell tracking methods still lack sufficient sensitivity and specificity. We examined, therefore, whether a highly sensitive and specific bioluminescence method is suitable to noninvasively image the organ distribution of administered mesenchymal stem cells (MSCs) in vivo. MSCs were transfected with a luciferase/neomycin phosphotransferase construct (luc/neo-MSC). Bioluminescence of these cells was measured (charge-coupled device camera) after treatment with luciferin, showing a linear increase of photon emission with rising cell numbers. To track these cells in vivo, groups of mice were injected with 1 x 10(5) luc/neo-MSCs/animal and imaged with bioluminescence imaging at various time points. Injection of cells in the suprarenal aorta showed diffuse distribution of cells in normal animals, whereas distinct localization to the kidneys was observed in mice with ischemia- and reperfusion-induced acute kidney injury (AKI). Intrajugular infusion of MSCs demonstrated predominant accumulation of cells in both lungs. In animals with AKI, detectable cell numbers declined over time, as assessed by bioluminescence imaging and confirmed by PCR, a process that was associated with low apoptosis levels of intrarenally located MSCs. In conclusion, the described bioluminescence technology provides a sensitive and safe tool for the repeated in vivo tracking of infused luc/neo-MSCs in all major organs. This method will be of substantial utility in the preclinical testing and design of cell therapeutic strategies in kidney and other diseases.

Amelioration of Acute Renal Failure by Stem Cell Therapy—Paracrine Secretion Versus Transdifferentiation into Resident Cells Administered Mesenchymal Stem Cells Protect against Ischemic Acute Renal Failure through Differentiation-Independent Mechanisms. Am J Physiol Renal Physiol E-pub February 15, 2005

Although the kidney has been suspected, though not definitively proven, to contain organ-specific pluripotent stem cells, their role in regeneration after renal injury is uncertain ([1][1]–[3][2]). Recently, however, several investigators found evidence for a renoprotective role of non–organ-

Carbamylated Erythropoietin Outperforms Erythropoietin in the Treatment of AKI-on-CKD and Other AKI Models.

Erythropoietin (EPO) may be a beneficial tissue-protective cytokine. However, high doses of EPO are associate with adverse effects, including thrombosis, tumor growth, and hypertension. Carbamylated erythropoietin (CEPO) lacks both erythropoietic and vasoconstrictive actions. In this study, we compared the renoprotective, hemodynamic, and hematologic activities and survival effects of identical EPO and CEPO doses in rat models of clinically relevant AKI presentations, including ischemia-reperfusion-induced AKI superimposed on CKD (5000 U/kg EPO or CEPO; three subcutaneous injections) and ischemia-reperfusion-induced AKI in old versus young animals and male versus female animals (1000 U/kg EPO or CEPO; three subcutaneous injections). Compared with EPO therapy, CEPO therapy induced greater improvements in renal function and body weight in AKI on CKD animals, with smaller increases in hematocrit levels and similarly improved survival. Compared with EPO therapy in the other AKI groups, CEPO therapy induced greater improvements in protection and recovery of renal function and survival, with smaller increases in systolic BP and hematocrit levels. Overall, old or male animals had more severe loss in kidney function and higher mortality rates than young or female animals, respectively. Notably, mRNA and protein expression analyses confirmed the renal expression of the heterodimeric EPO receptor/CD131 complex, which is required for the tissue-protective effects of CEPO signaling. In conclusion, CEPO improves renal function, body and kidney weight, and survival in AKI models without raising hematocrit levels and BP as substantially as EPO. Thus, CEPO therapy may be superior to EPO in improving outcomes in common forms of clinical AKI.

Durable Control of Autoimmune Diabetes in Mice Achieved by Intraperitoneal Transplantation of “Neo-Islets,” Three-Dimensional Aggregates of Allogeneic Islet and “Mesenchymal Stem Cells”

Novel interventions that reestablish endogenous insulin secretion and thereby halt progressive end‐organ damage and prolong survival of patients with autoimmune Type 1 diabetes mellitus (T1DM) are urgently needed. While this is currently accomplished with allogeneic pancreas or islet transplants, their utility is significantly limited by both the scarcity of organ donors and life‐long need for often‐toxic antirejection drugs. Coadministering islets with bone marrow‐derived mesenchymal stem cells (MSCs) that exert robust immune‐modulating, anti‐inflammatory, anti‐apoptotic, and angiogenic actions, improves intrahepatic islet survival and function. Encapsulation of insulin‐producing cells to prevent immune destruction has shown both promise and failures. Recently, stem cell‐derived insulin secreting β‐like cells induced euglycemia in diabetic animals, although their clinical use would still require encapsulation or anti‐rejection drugs. Instead of focusing on further improvements in islet transplantation, we demonstrate here that the intraperitoneal administration of islet‐sized “Neo‐Islets” (NIs), generated by in vitro coaggregation of allogeneic, culture‐expanded islet cells with high numbers of immuno‐protective and cyto‐protective MSCs, resulted in their omental engraftment in immune‐competent, spontaneously diabetic nonobese diabetic (NOD) mice. This achieved long‐term glycemic control without immunosuppression and without hypoglycemia. In preparation for an Food and Drug Administration‐approved clinical trial in dogs with T1DM, we show that treatment of streptozotocin‐diabetic NOD/severe combined immunodeficiency mice with identically formed canine NIs produced durable euglycemia, exclusively mediated by dog‐specific insulin. We conclude that this novel technology has significant translational relevance for canine and potentially clinical T1DM as it effectively addresses both the organ donor scarcity (>80 therapeutic NI doses/donor pancreas can be generated) and completely eliminates the need for immunosuppression. Stem Cells Translational Medicine 2017;6:1631–1643

INHIBITION OF STROMA-DERIVED FACTOR 1 INACTIVATION POTENTIATES STEM CELL HOMING TO THE ACUTELY INJURED KIDNEY.: 315

Stroma derived factor 1 (SDF-1) is a chemokine that plays important roles in developmental neurobiology, inflammation, stem cell traffic to the bone marrow, and injured organs, such as the brain, heart, liver, and kidneys. Recently, we demonstrated that renal tubular expression and plasma levels of SDF-1 are markedly up-regulated following induction of acute ischemia/reperfusion kidney injury (AKI) in mice, and this increase in SDF-1 specifically boosted the homing of CD34 and SDF-1 receptor CXCR4 coexpressing bone marrow stem cells to the kidney (Kidney Int 2005;67:1772-8). Furthermore, we demonstrated that mesenchymal stem cell (MSC) administration protects against AKI through complex paracrine mechanisms (such as anti-inflammatory, antiapoptotic, and mitogenic) (Am J Physiol 2005;289:F31-42). Here we investigated whether by inhibiting dipeptidylpeptidase IV (CD26), which inactivates SDF-1, SDF-1-mediated homing of CXCR4 expressing MSCs to the kidney with AKI is increased and renal protection and repair are further improved. To test this hypothesis, we used bone marrow-derived MSCs from Fisher 344 rats for in vitro experiments. First, using FACS, we demonstrate that about 35% of cultured MSCs express both CD26 and CXCR4 proteins on their surface. In addition, cultured MSCs express functional CD26 and can effectively inactivate SDF-1. Finally, MSCs migration toward SDF-1 in a transwell culture system can be significantly increased by preincubation of MSCs with Diprotin A, a CD26 inhibitor, or decreased by preincubation with AMD 3100, a CXCR4 inhibitor. Conclusion These results indicate that inhibiting CD26 augments the SDF-1-CXCR4-mediated chemotactic activity of cultured MSCs. It is therefore highly likely that pretreatment of MSCs with a CD26 inhibitor or preinfusion of such an inhibitor in vivo may augment the homing and renal protective functions of administered MSCs in experimental AKI. These promising stem cell therapeutic and organ repair strategies are currently under investigation.

CELL THERAPY USING MULTIPOTENT MARROW STROMAL CELLS IS A PROMISING NEW TREATMENT APPROACH FOR ACUTE KIDNEY INJURY.: 314

Acute kidney injury (AKI) is a serious clinical entity, and no specific treatment is currently available. Stem cell therapy has been shown to be a promising multimodal approach directed at different pathophysiologic aspects of AKI. Bone marrow-derived mesenchymal stem cells provide a promising therapeutic perspective due to their immunomodulatory and antiapoptotic properties and the secretion of various growth factors. However, the optimal dosing regimen and source of multipotent marrow stromal cells (MSCs) are currently unknown. Here we tested both the early renoprotective potential and the long-term contribution of various doses (0.5/2/5 × 106) of allogeneic and syngeneic MSCs in a rat model of ischemic AKI. MSC from human placental alkaline phosphatase (hPAP) transgenic Fischer rats were used in an allogeneic and syngeneic setting. Postreflow administration of hPAP + MSCs from Fischer to Sprague-Dawley rats with severe AKI improved both recovery of renal function on days 1 and 3 after AKI, with the lowest dose being the most protective. In an autologous setting, MSCs improved recovery after AKI in a dose-dependent fashion, but there was no difference between 2 and 5 × 106 MSCs administered. After 3 months creatinine clearance was better preserved in MSC-treated animals (2.1) versus controls (1.5 mL/min), whereas there were no differences in blood pressure, hematocrit, and weight. Compared with controls, MSC-treated animals had a lower renal fibrosis score (1.8 vs 2.3), and renal TGF-β and PAI-1 mRNA levels were significantly down-regulated. hPAP + MSCs could not be detected in kidneys, brain, liver, lung, and spleen by highly sensitive PCR, whereas the bone marrow in 3 of 6 animals was positive for hPAP, suggesting low-level engraftment. We conclude that allogeneic MSCs enhance early recovery from AKI as well as isogeneic cells and appear safe in the long term. They do not contribute to significant replacement of renal cell types, while ameliorating both long-term fibrotic changes and loss in renal function after AKI.

346 RENOPROTECTIVE ACTIONS OF BONE MARROW-DERIVED MESENCHYMAL STEM CELLS ARE A POTENTIAL NEW TREATMENT FOR ACUTE RENAL FAILURE.

Since acute renal failure (ARF) remains a common clinical syndrome with high mortality rates and limited treatment options, fundamentally new interventions are needed. We reported recently (Am J Physiol and Kidney Int 2005) that the infusion of mesenchymal stem cells (MSCs) is strikingly renoprotective in rats with severe ischemic ARF. Because essentially no transdifferentiation of small numbers of MSCs in the kidney was detected, we investigated alternative renoprotective mechanisms. We found striking down-regulation of proinflammatory TNF-α, IL-1β, and IFN-γ, and up-regulation of anti-inflammatory IL-10 and antiapoptotic Bcl-2, while leukocyte infiltration was similar. MSC treatment induced tubular up-regulation of bFGF and TGF-α. Renal artery and cortical blood flows were not different in MSC vs control animals. We next tested the effect of MSC conditioned medium (MSC-CM) on endothelial cells (ECs). MSC-CM acted mitogenically and inhibited apoptosis, a response also observed in tubular cells. Because growth factor (GF) expression by MSCs (express VEGF, HGF, and IGF-I, all act renoprotectively in ARF) is another potential mediator of renoprotection and since pO2 in the ischemic kidney is low, we found that VEGF expression in MSCs cultured at 5% pO2 was markedly upregulated. Our data show that the renoprotective actions of MSCs in ARF are mediated primarily through paracrine mechanisms. These elicit powerful anti-inflammatory, antiapoptotic, and mitogenic responses in renal cells that together result from the intrarenal delivery of GFs by MSCs and from their induction in the kidney of additional protective growth factors. In conclusion, we posit that our data provide the basis for the development of an MSC-based therapy for clinical ARF.