The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.

Elucidating the cell penetrating properties of self-assembling ß-peptides.

Self-assembling lipopeptide hydrogels have been widely developed for the delivery of therapeutics due to their rapid gelation, injectability, and highly controlled physicochemical properties. Lipopeptides are also known for their membrane-associating and cell penetrating properties, which may impact on their application in cell-encapsulation. Self-assembling lipidated-ß3-peptide materials developed in our laboratory have previously been used in cell culture as 2D substrates, thus as a continuation of this work we aimed to encapsulate cells in 3D by forming a hydrogel. We therefore assessed the self-assembling lipidated-ß3-peptides for cell-penetrating properties in mesenchymal stems cells (MSC) using fluorescence microscopy and membrane association with surface plasmon resonance spectroscopy (SPR). The results demonstrated that lipidated ß3-peptides penetrate the MSC plasma membrane and localise to the mitochondrial network. While self-assembling lipopeptide hydrogels have shown tremendous potential for delivery of therapeutics, further optimisation may be required to minimise the membrane uptake of the lipidated-ß3-peptides for cell encapsulation applications.

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.

A comparison of fixation methods for SEM analysis of self-assembling peptide hydrogel nanoarchitecture.

Determining the porosity of hydrogels is an important component of material characterisation. While scanning electron microscopy (SEM) is a widely used method to study hydrogel nanoarchitecture, it is well-established that SEM sample preparation methods can alter the structure of hydrogels. Herein we describe the impact of sample preparation on the SEM analysis of self-assembling ß-peptide hydrogels. Three methods of hydrogel preparation for SEM were compared, and each method preserved distinctly different nanoarchitecture, specifically, different levels of fibre alignment and porosity. Comparison of conventional SEM preparation and our hybrid method, which comprises high pressure freezing, freeze substitution without fixative and critical point drying, showed a high degree of similarity at the nanometre scale and diverging architecture at the micron scale. This study quantified the impact of chemical fixation versus high pressure freezing on self-assembling ß3-peptide hydrogels, demonstrated the effect of sample preparation on fibre alignment and porosity, and presents a novel hybrid preparation method where chemical fixation can be avoided when conventional SEM is desired.

Simultaneous late-gadolinium enhancement and T1 mapping of fibrosis and a novel cell-based combination therapy in hypertensive mice.

Fibrosis is a hallmark of chronic hypertension and disrupts the viability of human bone marrow-derived mesenchymal stromal cells (BM-MSCs) post-transplantation. This study thus, determined whether the anti-fibrotic drug, serelaxin (RLX), could enhance the therapeutic effects of BM-MSCs or BM-MSC-derived exosomes (BM-MSC-EXO) in hypertensive mice. Left ventricular (LV) fibrosis in particular was assessed using conventional histological staining and non-invasive cardiac magnetic resonance imaging (CMRI). CMRI was employed using a novel magnetisation prepared 2 rapid acquisition gradient echo (MP2RAGE) sequence to simultaneously perform late gadolinium enhancement imaging and T1 mapping. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and saline to drink (1 K/DOCA/salt) for 21 days, whilst control mice were given normal drinking water for the same time-period. On day 14 post-injury, subgroups of 1 K/DOCA/salt-hypertensive mice were treated with RLX alone or in combination with BM-MSCs or BM-MSC-EXO; or the mineralocorticoid receptor antagonist, spironolactone. At day 21 post-injury, LV and kidney histopathology was assessed, whilst LV fibrosis and function were additionally analysed by CMRI and echocardiography. 1 K/DOCA/salt-hypertensive mice developed kidney tubular injury, inflammation, fibrosis, and more moderate LV hypertrophy, fibrosis and diastolic dysfunction. RLX and BM-MSCs combined provided optimal protection against these pathologies and significantly reduced picrosirius red-stained organ fibrosis and MP2RAGE analysis of LV fibrosis. A significant correlation between MP2RAGE analysis and histologically-stained interstitial LV fibrosis was detected. It was concluded that the MP2RAGE sequence enhanced the non-invasive CMRI detection of LV fibrosis. Furthermore, combining RLX and BM-MSCs may represent a promising treatment option for hypertensive cardiorenal syndrome.

Enhancing the Therapeutic Potential of Mesenchymal Stromal Cell-Based Therapies with an Anti-Fibrotic Agent for the Treatment of Chronic Kidney Disease.

Chronic kidney disease (CKD) affects 1 in 10 members of the general population, placing these patients at an increasingly high risk of kidney failure. Despite the significant burden of CKD on various healthcare systems, there are no effective cures that reverse or even halt its progression. In recent years, human bone-marrow-derived mesenchymal stromal cells (BM-MSCs) have been recognised as a novel therapy for CKDs, owing to their well-established immunomodulatory and tissue-reparative properties in preclinical settings, and their promising safety profile that has been demonstrated in patients with CKDs from several clinical trials. However, renal fibrosis (scarring), a hallmark of CKD, has been shown to impair the viability and functionality of BM-MSCs post-transplantation. This has suggested that BM-MSCs might require a pre-treatment or adjunct therapy that can enhance the viability and therapeutic efficacy of these stromal cells in chronic disease settings. To address this, recent studies that have combined BM-MSCs with the anti-fibrotic drug serelaxin (RLX), have demonstrated the enhanced therapeutic potential of this combination therapy in normotensive and hypertensive preclinical models of CKD. In this review, a critical appraisal of the preclinical data available on the anti-fibrotic and renoprotective actions of BM-MSCs or RLX alone and when combined, as a treatment option for normotensive vs. hypertensive CKD, is discussed.

The Placental NLRP3 Inflammasome and Its Downstream Targets, Caspase-1 and Interleukin-6, Are Increased in Human Fetal Growth Restriction: Implications for Aberrant Inflammation-Induced Trophoblast Dysfunction.

Fetal growth restriction (FGR) is commonly associated with placental insufficiency and inflammation. Nonetheless, the role played by inflammasomes in the pathogenesis of FGR is poorly understood. We hypothesised that placental inflammasomes are differentially expressed and contribute to the aberrant trophoblast function. Inflammasome gene expression profiles were characterised by real-time PCR on human placental tissues collected from third trimester FGR and gestation-matched control pregnancies (n = 25/group). The functional significance of a candidate inflammasome was then investigated using lipopolysaccharide (LPS)-induced models of inflammation in human trophoblast organoids, BeWo cells in vitro, and a murine model of FGR in vivo. Placental mRNA expression of NLRP3, caspases 1, 3, and 8, and interleukin 6 increased (>2-fold), while that of the anti-inflammatory cytokine, IL-10, decreased (<2-fold) in FGR compared with control pregnancies. LPS treatment increased NLRP3 and caspase-1 expression (>2-fold) in trophoblast organoids and BeWo cell cultures in vitro, and in the spongiotrophoblast and labyrinth in the murine model of FGR. However, the LPS-induced rise in NLRP3 was attenuated by its siRNA-induced down-regulation in BeWo cell cultures, which correlated with reduced activity of the apoptotic markers, caspase-3 and 8, compared to the control siRNA-treated cells. Our findings support the role of the NLRP3 inflammasome in the inflammation-induced aberrant trophoblast function, which may contribute to FGR.

A Novel Approach to Enhance the Regenerative Potential of Circulating Endothelial Progenitor Cells in Patients with End-Stage Kidney Disease.

Circulating bone marrow-derived endothelial progenitor cells (EPCs) facilitate vascular repair in several organs including the kidney but are progressively diminished in end-stage kidney disease (ESKD) patients, which correlates with cardiovascular outcomes and related mortality. We thus determined if enhancing the tissue-reparative effects of human bone marrow-derived mesenchymal stromal cells (BM-MSCs) with the vasculogenic effects of recombinant human relaxin (RLX) could promote EPC proliferation and function. CD34+ EPCs were isolated from the blood of healthy and ESKD patients, cultured until late EPCs had formed, then stimulated with BM-MSC-derived condition media (CM; 25%), RLX (1 or 10 ng/mL), or both treatments combined. Whilst RLX alone stimulated EPC proliferation, capillary tube formation and wound healing in vitro, these measures were more rapidly and markedly enhanced by the combined effects of BM-MSC-derived CM and RLX in EPCs derived from both healthy and ESKD patients. These findings have important clinical implications, having identified a novel combination therapy that can restore and enhance EPC number and function in ESKD patients.

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.

Comparing the renoprotective effects of BM-MSCs versus BM-MSC-exosomes, when combined with an anti-fibrotic drug, in hypertensive mice.

Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow derived-mesenchymal stromal cells (BM-MSCs) post-transplantation. To address this, we demonstrated that combining BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), enhanced BM-MSC-induced renoprotection in preclinical CKD models. Given the increased interest and manufacturing advantages to using stem cell-derived exosomes (EXO) as therapeutics, this study determined whether RLX could enhance the therapeutic efficacy of BM-MSC-EXO, and compared the renoprotective effects of RLX and BM-MSC-EXO versus RLX and BM-MSCs in mice with hypertensive CKD. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and given saline to drink (1K/DOCA/salt) for 21 days. Control mice were uninephrectomised and given normal drinking water for the same time-period. Subgroups of 1K/DOCA/salt-hypertensive mice were then treated with either RLX (0.5 mg/kg/day) or BM-MSC-EXO (25 µg/mouse; equivalent to 1-2 × 106 BM-MSCs/mouse) alone; combinations of RLX and BM-MSC-EXO or BM-MSCs (1 × 106/mouse); or the mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day), from days 14-21. 1K/DOCA/salt-hypertensive mice developed kidney tubular damage, inflammation and fibrosis, and impaired kidney function 21 days post-injury. Whilst RLX alone attenuated the 1K/DOCA/salt-induced fibrosis, BM-MSC-EXO alone only diminished measures of tissue inflammation post-treatment. Comparatively, the combined effects of RLX and BM-MSC-EXO or BM-MSCs demonstrated similar anti-fibrotic efficacy, but RLX and BM-MSCs offered broader renoprotection over RLX and/or BM-MSC-EXO, and comparable effects to spironolactone. Only RLX and BM-MSCs, but not RLX and/or BM-MSC-EXO, also attenuated the 1K/DOCA/salt-induced hypertension. Hence, although RLX improved the renoprotective effects of BM-MSC-EXO, combining RLX with BM-MSCs provided a better therapeutic option for hypertensive CKD.

Combining mesenchymal stem cells with serelaxin provides enhanced renoprotection against 1K/DOCA/salt-induced hypertension.

BACKGROUND AND PURPOSE: Fibrosis is a hallmark of chronic kidney disease (CKD) that significantly contributes to renal dysfunction, and impairs the efficacy of stem cell-based therapies. This study determined whether combining bone marrow-derived mesenchymal stem cells (BM-MSCs) with the renoprotective effects of recombinant human relaxin (serelaxin) could therapeutically reduce renal fibrosis in mice with one kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)-induced hypertension, compared with the effects of the ACE inhibitor, perindopril. EXPERIMENTAL APPROACH: Adult male C57BL/6 mice were uni-nephrectomised and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt) for 21 days. Control mice were uni-nephrectomised but received water over the same time period. Sub-groups of 1K/DOCA/salt-injured mice (n = 5-8 per group) were treated with either serelaxin (0.5 mg·kg-1 ·day-1 ) or BM-MSCs (1 × 106 per mouse) alone; both treatments combined (with 0.5 × 106 or 1 × 106 BM-MSCs per mouse); or perindopril (2 mg·kg-1 ·day-1 ) from days 14-21. KEY RESULTS: 1K/DOCA/salt-injured mice developed elevated BP and hypertension-induced renal damage, inflammation and fibrosis. BM-MSCs alone reduced the injury-induced fibrosis and attenuated BP to a similar extent as perindopril. Serelaxin alone modestly reduced renal fibrosis and effectively reduced tubular injury. Strikingly, the combined effects of BM-MSCs (at both doses) with serelaxin significantly inhibited renal fibrosis and proximal tubular epithelial injury while restoring renal architecture, to a greater extent than either therapy alone, and over the effects of perindopril. CONCLUSION AND IMPLICATIONS: Combining BM-MSCs and serelaxin provided broader renoprotection over either therapy alone or perindopril and might represent a novel treatment for hypertensive CKD.

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.

mTOR-mediated podocyte hypertrophy regulates glomerular integrity in mice and humans.

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.

The Use of Live Cell Imaging and Automated Image Analysis to Assist With Determining Optimal Parameters for Angiogenic Assay in vitro.

Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.

The use of hydrogels for cell-based treatment of chronic kidney disease.

Chronic kidney disease (CKD) is a major and growing public health concern with increasing incidence and prevalence worldwide. The therapeutic potential of stem cell therapy, including mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) holds great promise for treatment of CKD. However, there are significant bottlenecks in the clinical translation due to the reduced number of transplanted cells and the duration of their presence at the site of tissue damage. Bioengineered hydrogels may provide a route of cell delivery to enhance treatment efficacy and optimise the targeting effectiveness while minimising any loss of cell function. In this review, we highlight the advances in stem cell therapy targeting kidney disease and discuss the emerging role of hydrogel delivery systems to fully realise the potential of adult stem cells as a regenerative therapy for CKD in humans. MSCs and EPCs mediate kidney repair through distinct paracrine effects. As a delivery system, hydrogels can prolong these paracrine effects by improving retention at the site of injury and protecting the transplanted cells from the harsh inflammatory microenvironment. We also discuss the features of a hydrogel, which may be tuned to optimise the therapeutic potential of encapsulated stem cells, including cell-adhesive epitopes, material stiffness, nanotopography, modes of gelation and degradation and the inclusion of bioactive molecules. This review concludes with a discussion of the challenges to be met for the widespread clinical use of hydrogel delivery system of stem cell therapy for CKD.

Induced Pluripotent Stem Cell-Derived Podocyte-Like Cells as Models for Assessing Mechanisms Underlying Heritable Disease Phenotype: Initial Studies Using Two Alport Syndrome Patient Lines Indicate Impaired Potassium Channel Activity.

Renal podocyte survival depends upon the dynamic regulation of a complex cell architecture that links the glomerular basement membrane to integrins, ion channels, and receptors. Alport syndrome is a heritable chronic kidney disease where mutations in α3, α4, or α5 collagen genes promote podocyte death. In rodent models of renal failure, activation of the calcium-sensing receptor (CaSR) can protect podocytes from stress-related death. In this study, we assessed CaSR function in podocyte-like cells derived from induced-pluripotent stem cells from two patients with Alport Syndrome (AS1 & AS2) and a renal disease free individual [normal human mesangial cell (NHMC)], as well as a human immortalized podocyte-like (HIP) cell line. Extracellular calcium elicited concentration-dependent elevations of intracellular calcium in all podocyte-like cells. NHMC and HIP, but not AS1 or AS2 podocyte-like cells, also showed acute reductions in intracellular calcium prior to elevation. In NHMC podocyte-like cells this acute reduction was blocked by the large-conductance potassium channel (KCNMA1) inhibitors iberiotoxin (10 nM) and tetraethylammonium (5 mM), as well as the focal adhesion kinase inhibitor PF562271 (N-methyl-N-(3-((2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino)-methyl)-pyridin-2-yl)-methanesulfonamide, 10 nM). Quantitative polymerase chain reaction (qPCR) and immunolabeling showed the presence of KCNMA1 transcript and protein in all podocyte-like cells tested. Cultivation of AS1 podocytes on decellularized plates of NHMC podocyte-like cells partially restored acute reductions in intracellular calcium in response to extracellular calcium. We conclude that the AS patient-derived podocyte-like cells used in this study showed dysfunctional integrin signaling and potassium channel function, which may contribute to podocyte death seen in Alport syndrome.

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.