GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals.

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.

The protective effects of a novel AT2 receptor agonist, ß-Pro7Ang III in ischemia-reperfusion kidney injury.

BACKGROUND AND PURPOSE: This study investigated the reno-protective effects of a highly selective AT2R agonist peptide, ß-Pro7Ang III in a mouse model of acute kidney injury (AKI). METHODS: C57BL/6 J mice underwent either sham surgery or unilateral kidney ischemia-reperfusion injury (IRI) for 40 min. IRI mice were treated with either ß-Pro7Ang III or perindopril and at 7 days post-surgery the kidneys analysed for histopathology and the development of fibrosis and matrix metalloproteinase (MMP)-2 and -9 activity. The association of the therapeutic effects of ß-Pro7Ang III with macrophage number and phenotype was determined in vivo and in vitro. KEY RESULTS: Decreased kidney tubular injury, interstitial matrix expansion and reduced interstitial immune cell infiltration in IRI mice receiving ß-Pro7Ang III treatment was observed at day 7, compared to IRI mice without treatment. This correlated to reduced collagen accumulation and MMP-2 activity in IRI mice following ß-Pro7Ang III treatment. FACS analysis showed a reduced number and proportion of CD45+CD11b+F4/80+ macrophages in IRI kidneys in response to ß-Pro7Ang III, correlating with a significant increase in M2 macrophage markers and decreased M1 markers at day 3 and 7 post-IR injury, respectively. In vitro analysis of cultured THP-1 cells showed that ß-Pro7Ang III attenuated lipopolysaccharide (LPS)-induced tumour necrosis factor-α (TNF-α) and interleukin (IL)- 6 production but increased IL-10 secretion, compared to LPS alone. CONCLUSION: Administration of ß-Pro7Ang III via mini-pump improved kidney structure and reduced interstitial collagen accumulation, in parallel with an alteration of macrophage phenotype and anti-inflammatory cytokine release, therefore mitigating the downstream progression of ischemic AKI.

The Differentiation of Human Induced Pluripotent Stem Cells into Podocytes In Vitro.

Induced pluripotent stem cells (iPSCs) hold enormous potential in the field of regenerative medicine due to their pluripotent properties, where they can give rise to all cell types in the body. Here we describe a detailed 20-day culture and differentiation protocol to generate iPSC-derived podocytes grown as a monolayer. These iPSC-derived podocytes appear arborised by morphology and express podocyte-specific markers. Also described is a detailed immunofluorescence staining protocol to confirm successful differentiation using the podocyte-specific markers, Wilms' tumor protein (WT1) and podocin.

Percutaneous intrarenal transplantation of differentiated induced pluripotent stem cells into newborn mice.

The in vivo engraftment of induced pluripotent stem cell (iPSC)-derived podocytes following allogeneic transplantation into host kidneys remains a challenge. Here we investigate the survival and engraftment of human dermal fibroblasts-derived differentiated iPSCs using a newborn mouse model, which represents a receptive immunoprivileged host environment. iPSCs were generated from skin biopsies of patients using Sendai virus reprogramming. Differentiation of nephrin (NPHS1)-green fluorescent protein (GFP) iPSCs into kidney podocytes (iPSC-PODs) was performed by the addition of Activin A, bone morphogenetic protein 7 (BMP7), and retinoic acid over 10 days of culture. To assess the in vivo incorporation of cells, undifferentiated iPSCs or day 10 iPSC-PODs, were labeled with either carboxyfluorescein succinimidyl ester (CFSE) or Qdot nanocrystals (Q705). Thereafter, 1 × 105 differentiated iPSC-PODs were injected directly into the kidneys of mouse pups at postnatal day one (P1). Using co-expression analysis of glomerular and podocyte-specific markers, Day 10 differentiated iPSC-PODs that were positive for podocin, were detected following direct kidney injection into newborn mice up to 1 week after transplantation. Undifferentiated iPSC-PODs were not detected at the same timepoint. The transplanted cells were viable and located in the outer nephrogenic zone where they were found to colocalize with, or sit adjacent to, cells positive for glomerular-specific markers including podocin, synaptopodin, and Wilms' tumor 1 (WT1). This study provides proof-of-principle that transplanted iPSC-POD can survive in recipient newborn mouse kidneys due to the immature and immunoprivileged nature of the developing postnatal kidneys.

Renal epithelial cells retain primary cilia during human acute renal allograft rejection injury.

OBJECTIVES: Primary cilia are sensory organelles which co-ordinate several developmental/repair pathways including hedgehog signalling. Studies of human renal allografts suffering acute tubular necrosis have shown that length of primary cilia borne by epithelial cells doubles throughout the nephron and collecting duct, and then normalises as renal function returns. Conversely the loss of primary cilia has been reported in chronic allograft rejection and linked to defective hedgehog signalling. We investigated the fate of primary cilia in renal allografts suffering acute rejection. RESULTS: Here we observed that in renal allografts undergoing acute rejection, primary cilia were retained, with their length increasing 1 week after transplantation and remaining elevated. We used a mouse model of acute renal injury to demonstrate that elongated renal primary cilia in the injured renal tubule show evidence of smoothened accumulation, a biomarker for activation of hedgehog signalling. We conclude that primary cilium-mediated activation of hedgehog signalling is still possible during the acute phase of renal allograft rejection.

The therapeutic effect of mesenchymal stem cells on pulmonary myeloid cells following neonatal hyperoxic lung injury in mice.

BACKGROUND: Exposure to high levels of oxygen (hyperoxia) after birth leads to lung injury. Our aims were to investigate the modulation of myeloid cell sub-populations and the reduction of fibrosis in the lungs following administration of human mesenchymal stem cells (hMSC) to neonatal mice exposed to hyperoxia. METHOD: Newborn mice were exposed to 90% O2 (hyperoxia) or 21% O2 (normoxia) from postnatal days 0-4. A sub-group of hyperoxia mice were injected intratracheally with 2.5X105 hMSCs. Using flow cytometry we assessed pulmonary immune cells at postnatal days 0, 4, 7 and 14. The following markers were chosen to identify these cells: CD45+ (leukocytes), Ly6C+Ly6G+ (granulocytes), CD11b+CD11c+ (macrophages); macrophage polarisation was assessed by F4/80 and CD206 expression. hMSCs expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (fluc) were administered via the trachea at day 4. Lung macrophages in all groups were profiled using next generation sequencing (NGS) to assess alterations in macrophage phenotype. Pulmonary collagen deposition and morphometry were assessed at days 14 and 56 respectively. RESULTS: At day 4, hyperoxia increased the number of pulmonary Ly6C+Ly6G+ granulocytes and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. At days 7 and 14, hyperoxia increased numbers of CD45+ leukocytes, CD11b+CD11c+ alveolar macrophages and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. hMSCs administration ameliorated these effects of hyperoxia, notably reducing numbers of CD11b+CD11c+ and F4/80lowCD206low macrophages; in contrast, F4/80highCD206high macrophages were increased. Genes characteristic of anti-inflammatory 'M2' macrophages (Arg1, Stat6, Retnla, Mrc1, Il27ra, Chil3, and Il12b) were up-regulated, and pro-inflammatory 'M1' macrophages (Cd86, Stat1, Socs3, Slamf1, Tnf, Fcgr1, Il12b, Il6, Il1b, and Il27ra) were downregulated in isolated lung macrophages from hyperoxia-exposed mice administered hMSCs, compared to mice without hMSCs. Hydroxyproline assay at day 14 showed that the 2-fold increase in lung collagen following hyperoxia was reduced to control levels in mice administered hMSCs. By day 56 (early adulthood), hMSC administration had attenuated structural changes in hyperoxia-exposed lungs. CONCLUSIONS: Our findings suggest that hMSCs reduce neonatal lung injury caused by hyperoxia by modulation of macrophage phenotype. Not only did our cell-based therapy using hMSC induce structural repair, it limited the progression of pulmonary fibrosis.

Phenotype and influx kinetics of leukocytes and inflammatory cytokine production in kidney ischemia/reperfusion injury.

BACKGROUND AND AIM: Kidney ischemia/reperfusion (IR) injury is characterized by tubular epithelial cell (TEC) death and an inflammatory response involving cytokine production and immune cell infiltration. In various kidney diseases, increased macrophage numbers correlate with injury severity and poor prognosis. However, macrophage plasticity enables a diverse range of functions, including wound healing, making them a key target for novel therapies. This study aimed to comprehensively characterize the changes in myeloid and epithelial cells and the production of cytokines throughout the experimental IR model of acute kidney injury to aid in the identification of targets to promote and enhance kidney regeneration and repair. METHODS: Flow cytometric analysis of murine unilateral IR injury was used to assess TEC and myeloid cell subpopulations in conjunction with histological analysis and cytokine production at 6 h, 1, 3, 5 and 7 days post IR injury, spanning the initial inflammatory phase and the following reparative phase. RESULTS: IR injury resulted in a rapid infiltration of Ly6Chigh monocytes and neutrophils with a steady rise in F4/80high MHCIIhigh macrophages over the injury time. The production of the inflammatory cytokines IL-6, MCP-1 and TNF coincided with an increase in IL-10 production. CONCLUSION: This characterization will provide a reference point for future studies designed to manipulate immune cell phenotype and function in order to promote endogenous repair of damaged kidneys.

miR-378 reduces mesangial hypertrophy and kidney tubular fibrosis via MAPK signalling.

The regulatory role of a novel miRNA, miR-378, was determined in the development of fibrosis through repression of the MAPK1 pathway, miR-378 and fibrotic gene expression was examined in streptozotocin (STZ)-induced diabetic mice at 18 weeks or in unilateral ureteral obstruction (UUO) mice at 7 days. miR-378 transfection of proximal tubular epithelial cells, NRK52E and mesangial cells was assessed with/without endogenous miR-378 knockdown using the locked nucleic acid (LNA) inhibitor. NRK52E cells were co-transfected with the mothers against decapentaplegic homolog 3 (SMAD3) CAGA reporter and miR-378 in the presence of transforming growth factor-ß (TGF-ß1) was assessed. Quantitative polymerase chain reaction (qPCR) showed a significant reduction in miR-378 (P<0.05) corresponding with up-regulated type I collagen, type IV collagen and α-smooth muscle actin (SMA) in kidneys of STZ or UUO mice, compared with controls. TGF-ß1 significantly increased mRNA expression of type I collagen (P<0.05), type IV collagen (P<0.05) and α-SMA (P<0.05) in NRK52E cells, which was significantly reduced (P<0.05) following miR-378 transfection and reversed following addition of the LNA inhibitor of endogenous miR-378 Overexpression of miR-378 inhibited mesangial cell expansion and proliferation in response to TGF-ß1, with LNA-miR-378 transfection reversing this protective effect, associated with cell morphological alterations. The protective function of MAPK1 on miR-378 was shown in kidney cells treated with the MAPK1 inhibitor, selumetinib, which inhibited mesangial cell hypertrophy in response to TGF-ß1. Taken together, these results suggest that miR-378 acts via regulation of the MAPK1 pathway. These studies demonstrate the protective function of MAPK1, regulated by miR-378, in the induction of kidney cell fibrosis and mesangial hypertrophy.

Mesenchymal Stem Cells Deliver Exogenous MicroRNA-let7c via Exosomes to Attenuate Renal Fibrosis.

The advancement of microRNA (miRNA) therapies has been hampered by difficulties in delivering miRNA to the injured kidney in a robust and sustainable manner. Using bioluminescence imaging in mice with unilateral ureteral obstruction (UUO), we report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c (miR-let7c-MSCs), selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared with nontargeting control (NTC)-MSCs. miR-let7c-MSC therapy attenuated kidney injury and significantly downregulated collagen IVα1, metalloproteinase-9, transforming growth factor (TGF)-ß1, and TGF-ß type 1 receptor (TGF-ßR1) in UUO kidneys, compared with controls. In vitro analysis confirmed that the transfer of miR-let7c from miR-let7c-MSCs occurred via secreted exosomal uptake, visualized in NRK52E cells using cyc3-labeled pre-miRNA-transfected MSCs with/without the exosomal inhibitor, GW4869. The upregulated expression of fibrotic genes in NRK52E cells induced by TGF-ß1 was repressed following the addition of isolated exosomes or indirect coculture of miR-let7c-MSCs, compared with NTC-MSCs. Furthermore, the cotransfection of NRK52E cells using the 3'UTR of TGF-ßR1 confirmed that miR-let7c attenuates TGF-ß1-driven TGF-ßR1 gene expression. Taken together, the effective antifibrotic function of engineered MSCs is able to selectively transfer miR-let7c to damaged kidney cells and will pave the way for the use of MSCs for therapeutic delivery of miRNA targeted at kidney disease.

Human mesenchymal stem cells alter the gene profile of monocytes from patients with Type 2 diabetes and end-stage renal disease.

AIM: Macrophage infiltration contributes to the pathogenesis of Type 2 diabetes. Mesenchymal stem cells (MSCs) possess immunomodulatory properties, making them an ideal candidate for therapeutic intervention. This study investigated whether MSCs can modulate the phenotype of monocytes isolated from Type 2 diabetic patients with end-stage renal disease. MATERIALS & METHODS: Monocytes from control (n = 4) and Type 2 diabetic patients with end-stage renal disease (n = 5) were assessed using flow cytometry and microarray profiling, following 48 h of co-culture with MSCs. RESULTS: Control subjects had a greater proportion of CD14(++)CD16(-) monocytes while diabetic patients had a higher proportion of CD14(++)CD16(+) and CD14(+)CD16(++) monocytes. MSCs promoted the proliferation of monocytes isolated from diabetic patients, reduced HLA-DR expression in both groups and promoted the expression of anti-inflammatory genes. CONCLUSION: MSC-derived factors alter the polarization of monocytes isolated from healthy and diabetic subjects toward an M2 phenotype.

Combination therapy of mesenchymal stem cells and serelaxin effectively attenuates renal fibrosis in obstructive nephropathy.

Chronic kidney disease (CKD) results from the development of fibrosis, ultimately leading to end-stage renal disease (ESRD). Although human bone marrow-derived mesenchymal stem cells (MSCs) can accelerate renal repair following acute injury, the establishment of fibrosis during CKD may affect their potential to influence regeneration capacity. Here we tested the novel combination of MSCs with the antifibrotic serelaxin to repair and protect the kidney 7 d post-unilateral ureteral obstruction (UUO), when fibrosis is established. Male C57BL6 mice were sham-operated or UUO-inured (n = 4-6) and received vehicle, MSCs (1 × 10(6)), serelaxin (0.5 mg/kg per d), or the combination of both. In vivo tracing studies with luciferin/enhanced green fluorescent protein (eGFP)-tagged MSCs showed specific localization in the obstructed kidney where they remained for 36 h. Combination therapy conferred significant protection from UUO-induced fibrosis, as indicated by hydroxyproline analysis (P < 0.001 vs. vehicle, P < 0.05 vs. MSC or serelaxin alone). This was accompanied by preserved structural architecture, decreased tubular epithelial injury (P < 0.01 vs. MSCs alone), macrophage infiltration, and myofibroblast localization in the kidney (both P < 0.01 vs. vehicle). Combination therapy also stimulated matrix metalloproteinase (MMP)-2 activity over either treatment alone (P < 0.05 vs. either treatment alone). These results suggest that the presence of an antifibrotic in conjunction with MSCs ameliorates established kidney fibrosis and augments tissue repair to a greater extent than either treatment alone.

Human mesenchymal stem cells alter macrophage phenotype and promote regeneration via homing to the kidney following ischemia-reperfusion injury.

Mesenchymal stem cells (MSCs) ameliorate injury and accelerate repair in many organs, including the kidney, although the reparative mechanisms and interaction with macrophages have not been elucidated. This study investigated the reparative potential of human bone marrow-derived MSCs and traced their homing patterns following administration to mice with ischemia-reperfusion (IR) injury using whole body bioluminescence imaging. The effect of MSCs on macrophage phenotype following direct and indirect coculture was assessed using qPCR. Human cytokine production was measured using multiplex arrays. After IR, MSCs homed to injured kidneys where they afforded protection indicated by decreased proximal tubule kidney injury molecule-1 expression, blood urea nitrogen, and serum creatinine levels. SDS-PAGE and immunofluorescence labeling revealed MSCs reduced collagen α1(I) and IV by day 7 post-IR. Gelatin zymography confirmed that MSC treatment significantly increased matrix metalloproteinase-9 activity in IR kidneys, which contributed to a reduction in total collagen. Following direct and indirect coculture, macrophages expressed genes indicative of an anti-inflammatory "M2" phenotype. MSC-derived human GM-CSF, EGF, CXCL1, IL-6, IL-8, MCP-1, PDGF-AA, and CCL5 were identified in culture supernatants. In conclusion, MSCs home to injured kidneys and promote repair, which may be mediated by their ability to promote M2 macrophage polarization.

Establishing the flow cytometric assessment of myeloid cells in kidney ischemia/reperfusion injury.

Polychromatic flow cytometry is a powerful tool for assessing populations of cells in the kidney through times of homeostasis, disease and tissue remodeling. In particular, macrophages have been identified as having central roles in these three settings. However, because of the plasticity of myeloid cells it has been difficult to define a specific immunophenotype for these cells in the kidney. This study developed a gating strategy for identifying and assessing monocyte and macrophage subpopulations, along with neutrophils and epithelial cells in the healthy kidney and following ischemia/reperfusion (IR) injury in mice, using antibodies against CD45, CD11b, CD11c, Ly6C, Ly6G, F4/80, CSF-1R (CD115), MHC class II, mannose receptor (MR or CD206), an alternatively activated macrophage marker, and the epithelial cell adhesion marker (EpCAM or CD326). Backgating analysis and assessment of autofluorescence was used to extend the knowledge of various cell types and the changes that occur in the kidney at various time-points post-IR injury. In addition, the impact of enzymatic digestion of kidneys on cell surface markers and cell viability was assessed. Comparisons of kidney myeloid populations were also made with those in the spleen. These results provide a useful reference for future analyses of therapies aimed at modulating inflammation and enhancing endogenous remodeling following kidney injury.

Mesenchymal stem cells in kidney inflammation and repair.

Mesenchymal stem cells are a heterogeneous population of fibroblast-like stromal cells that have been isolated from the bone marrow and a number of organs and tissues including the kidney. They have multipotent and self-renewing properties and can differentiate into cells of the mesodermal lineage. Following their administration in vivo, mesenchymal stem cells migrate to damaged kidney tissue where they produce an array of anti-inflammatory cytokines and chemokines that can alter the course of injury. Mesenchymal stem cells are thought to elicit repair through paracrine and/or endocrine mechanisms that modulate the immune response resulting in tissue repair and cellular replacement. This review will discuss the features of mesenchymal stem cells and the factors they release that protect against kidney injury; the mechanisms of homing and engraftment to sites of inflammation; and further elucidate the immunomodulatory effect of mesenchymal stem cells and their ability to alter macrophage phenotype in a setting of kidney damage and repair.