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.

WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway.

Inflammation is a pathological feature of kidney injury and its progression correlates with the development of kidney fibrosis which can lead to kidney function impairment. This project investigated the regulatory function of WNT1-inducible signaling pathway protein 1 (WISP1) in kidney inflammation. Administration of recombinant WISP1 protein to healthy mice induced kidney inflammation (macrophage accrual and production of tumor necrosis factor α (TNF-α), CCL2 and IL-6), which could be prevented by inhibition of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). Furthermore, inhibition of WISP1, by gene knockdown or neutralising antibody, could inhibit cultured macrophages producing inflammatory cytokines following stimulation with lipopolysaccharides (LPSs) and kidney fibroblasts proliferating in response to TNFα, which both involved NF-κB signaling. Kidney expression of WISP1 was found to be increased in mouse models of progressive kidney inflammation-unilateral ureter obstruction (UUO) and streptozotocin (STZ)-induced diabetic nephropathy (DN). Treatment of UUO mice with WISP1 antibody reduced the kidney inflammation in these mice. Therefore, pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting inflammation in kidney disease.

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.

WNT1-inducible-signaling pathway protein 1 regulates the development of kidney fibrosis through the TGF-ß1 pathway.

Fibrosis is a pathological feature of chronic kidney disease and its progression correlates with declining renal function. Kidney fibrosis is driven by multiple profibrotic factors. This project examined the regulatory function of WNT1-inducible-signaling pathway protein 1 (WISP1) in the development of kidney fibrosis. Induction of WISP1 by transforming growth factor beta 1 (TGF-ß1), and the role of WISP1 in TGF-ß1/Smad signaling and fibrotic responses, was examined in multiple kidney cells. Kidney expression of WISP1 was examined in mouse models of unilateral ureter obstruction (UUO) and streptozotocin-induced diabetic nephropathy. WISP1 antibody was administered to UUO mice during the induction of kidney injury and the impact on kidney fibrosis was examined. WISP1 expression was upregulated in both mouse models. TGF-ß1-induced expression of WISP1 and profibrotic genes in cultured kidney cells via TGF-ßR1. Recombinant WISP1-induced expression of TGF-ßR1 in kidney cells. Suppression of WISP1 by shRNA or neutralizing antibody reduced TGF-ß1-mediated activation of Smad3, fibrotic gene expression, and fibroblast proliferation. Treatment with WISP1 antibody inhibited the development of kidney fibrosis in UUO mice. WISP1 mediates the profibrotic effects of TGF-ß1 in kidney cells and in kidney disease. Pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting kidney fibrosis.

Inpp5e suppresses polycystic kidney disease via inhibition of PI3K/Akt-dependent mTORC1 signaling.

Polycystic kidney disease (PKD) is a common cause of renal failure with few effective treatments. INPP5E is an inositol polyphosphate 5-phosphatase that dephosphorylates phosphoinositide 3-kinase (PI3K)-generated PI(3,4,5)P3 and is mutated in ciliopathy syndromes. Germline Inpp5e deletion is embryonically lethal, attributed to cilia stability defects, and is associated with polycystic kidneys. However, the molecular mechanisms responsible for PKD development upon Inpp5e loss remain unknown. Here, we show conditional inactivation of Inpp5e in mouse kidney epithelium results in severe PKD and renal failure, associated with a partial reduction in cilia number and hyperactivation of PI3K/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment with an mTORC1 inhibitor improved kidney morphology and function, but did not affect cilia number or length. Therefore, we identify Inpp5e as an essential inhibitor of the PI3K/Akt/mTORC1 signaling axis in renal epithelial cells, and demonstrate a critical role for Inpp5e-dependent mTORC1 regulation in PKD suppression.

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.

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.

Gene editing of stem cells for kidney disease modelling and therapeutic intervention.

Recent developments in targeted gene editing have paved the way for the wide adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nucleases (Cas9) as an RNA-guided molecular tool to modify the genome of eukaryotic cells of animals. Theoretically, the translation of CRISPR-Cas9 can be applied to the treatment of inherited or acquired kidney disease, kidney transplantation and genetic corrections of somatic cells from kidneys with inherited mutations, such as polycystic kidney disease. Human pluripotent stem cells have been used to generate an unlimited source of kidney progenitor cells or, when spontaneously differentiated into three-dimensional kidney organoids, to model kidney organogenesis or the pathogenesis of disease. Gene editing now allows for the tagging and selection of specific kidney cell types or disease-specific gene knock in/out, which enables more precise understanding of kidney organogenesis and genetic diseases. This review discusses the mechanisms of action, in addition to the advantages and disadvantages, of the three major gene editing technologies, namely, CRISPR-Cas9, zinc finger nucleases and transcription activator-like effector nucleases. The implications of using gene editing to better understand kidney disease is reviewed in detail. In addition, the ethical issues of gene editing, which could be easily neglected in the modern, fast-paced research environment, are highlighted.

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.

Serelaxin improves the therapeutic efficacy of RXFP1-expressing human amnion epithelial cells in experimental allergic airway disease.

Current asthma therapies primarily target airway inflammation (AI) and suppress episodes of airway hyperresponsiveness (AHR) but fail to treat airway remodelling (AWR), which can develop independently of AI and contribute to irreversible airway obstruction. The present study compared the anti-remodelling and therapeutic efficacy of human bone marrow-derived mesenchymal stem cells (MSCs) to that of human amnion epithelial stem cells (AECs) in the setting of chronic allergic airways disease (AAD), in the absence or presence of an anti-fibrotic (serelaxin; RLX). Female Balb/c mice subjected to the 9-week model of ovalbumin (OVA)-induced chronic AAD, were either vehicle-treated (OVA alone) or treated with MSCs or AECs alone [intranasally (i.n.)-administered with 1×106 cells once weekly], RLX alone (i.n.-administered with 0.8 mg/ml daily) or a combination of MSCs or AECs and RLX from weeks 9-11 (n=6/group). Measures of AI, AWR and AHR were then assessed. OVA alone exacerbated AI, epithelial damage/thickness, sub-epithelial extracellular matrix (ECM) and total collagen deposition, markers of collagen turnover and AHR compared with that in saline-treated counterparts (all P<0.01 compared with saline-treated controls). RLX or AECs (but not MSCs) alone normalized epithelial thickness and partially diminished the OVA-induced fibrosis and AHR by ∼40-50% (all P<0.05 compared with OVA alone). Furthermore, the combination treatments normalized epithelial thickness, measures of fibrosis and AHR to that in normal mice, and significantly decreased AI. Although AECs alone demonstrated greater protection against the AAD-induced AI, AWR and AHR, compared with that of MSCs alone, combining RLX with MSCs or AECs reversed airway fibrosis and AHR to an even greater extent.

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.

Renal cellular hypoxia in adenine-induced chronic kidney disease.

We determined whether adenine-induced chronic kidney disease (CKD) in rats is associated with renal tissue hypoxia. Adenine (100 mg) or its vehicle was administered to male Sprague-Dawley rats, daily by oral gavage, over a 15-day period. Renal function was assessed before, and 7 and 14 days after, adenine treatment commenced, by collection of a 24-hour urine sample and a blood sample from the tail vein. On day 15, arterial pressure was measured in conscious rats via the tail artery. Renal tissue hypoxia was then assessed by pimonidazole adduct immunohistochemistry and fibrosis was assessed by staining tissue with picrosirius red and Masson's trichrome. CKD was evident within 7 days of commencing adenine treatment, as demonstrated by increased urinary albumin to creatinine ratio (30 ± 12-fold). By day 14 of adenine treatment plasma creatinine concentration was more than 7-fold greater, and plasma urea more than 5-fold greater, than their baseline levels. On day 15, adenine-treated rats had slightly elevated mean arterial pressure (8 mmHg), anaemia and renomegaly. Kidneys of adenine-treated rats were characterised by the presence of tubular casts, dilated tubules, expansion of the interstitial space, accumulation of collagen, and tubulointerstitial hypoxia. Pimonidazole staining (hypoxia) co-localised with fibrosis and was present in both patent and occluded tubules. We conclude that renal tissue hypoxia develops rapidly in adenine-induced CKD. This model, therefore, should prove useful for examination of the temporal and spatial relationships between tubulointerstitial hypoxia and the development of CKD, and thus the testing of the 'chronic hypoxia hypothesis'.

Mesenchymal stem cells as novel micro-ribonucleic acid delivery vehicles in kidney disease.

MicroRNAs (miRNAs) are short single strands of RNA responsible for post-transcriptional regulation of gene expression and have been implicated in the pathogenesis of chronic kidney disease (CKD). Emerging evidence reports that miRNAs can reduce kidney fibrosis through regulation of targets associated with collagen and extracellular matrix accumulation. However, the development of miRNA therapies has been hampered by the lack of targeted and sustainable methods of systemic miRNA delivery. Mesenchymal stem cells (MSCs) provide a promising miRNA delivery platform to overcome toxicity, the potential for insertional mutations and the low efficiency of previous methods. MSCs are endogenously immunoprivileged and home to sites of inflammation. They also release trophic growth factors to modulate the immune system, alter the polarization of macrophages and provide renal protection and repair. The potential to engineer MSCs to express or overexpress miRNAs, released by exosomes, may enhance their natural functions. Clinical studies are already being conducted individually for the use of miRNAs in cancer and MSCs in diseases associated with CKD. Hence, the combination of miRNAs and MSCs may provide an unparalleled cell-based therapy for treating CKD.

M2 macrophage accumulation in the aortic wall during angiotensin II infusion in mice is associated with fibrosis, elastin loss, and elevated blood pressure.

Macrophages accumulate in blood vessels during hypertension. However, their contribution to vessel remodeling is unknown. In the present study, we examined the polarization state of macrophages (M1/M2) in aortas of mice during hypertension and investigated whether antagonism of chemokine receptors involved in macrophage accumulation reduces vessel remodeling and blood pressure (BP). Mice treated with ANG II (0.7 mg·kg(-1)·day(-1), 14 days) had elevated systolic BP (158 ± 3 mmHg) compared with saline-treated animals (122 ± 3 mmHg). Flow cytometry revealed that ANG II infusion increased numbers of CD45(+)CD11b(+)Ly6C(hi) monocytes and CD45(+)CD11b(+)F4/80(+) macrophages by 10- and 2-fold, respectively. The majority of macrophages were positive for the M2 marker CD206 but negative for the M1 marker inducible nitric oxide synthase. Expression of other M2 genes (arginase-1, Fc receptor-like S scavenger receptor, and receptor-1) was elevated in aortas from ANG II-treated mice, whereas M1 genes [TNF and chemokine (C-X-C motif) ligand 2] were unaltered. A PCR array to identify chemokine receptor targets for intervention revealed chemokine (C-C motif) receptor 2 (CCR2) to be upregulated in aortas from ANG II-treated mice, while flow cytometry identified Ly6C(hi) monocytes as the main CCR2-expressing cell type. Intervention with a CCR2 antagonist (INCB3344; 30 mg·kg(-1)·day(-1)), 7 days after the commencement of ANG II infusion, reduced aortic macrophage numbers. INCB334 also reduced aortic collagen deposition, elastin loss, and BP in ANG II-treated mice. Thus, ANG II-dependent hypertension in mice is associated with Ly6C(hi) monocyte and M2 macrophage accumulation in the aorta. Inhibition of macrophage accumulation with a CCR2 antagonist prevents ANG II-induced vessel fibrosis and elevated BP, highlighting this as a promising approach for the future treatment of vessel remodeling/stiffening in hypertension.

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.

The effect of CSF-1 administration on lung maturation in a mouse model of neonatal hyperoxia exposure.

BACKGROUND: Lung immaturity due to preterm birth is a significant complication affecting neonatal health. Despite the detrimental effects of supplemental oxygen on alveolar formation, it remains an important treatment for infants with respiratory distress. Macrophages are traditionally associated with the propagation of inflammatory insults, however increased appreciation of their diversity has revealed essential functions in development and regeneration. METHODS: Macrophage regulatory cytokine Colony-Stimulating Factor-1 (CSF-1) was investigated in a model of neonatal hyperoxia exposure, with the aim of promoting macrophages associated with alveologenesis to protect/rescue lung development and function. Neonatal mice were exposed to normoxia (21% oxygen) or hyperoxia (Hyp; 65% oxygen); and administered CSF-1 (0.5 µg/g, daily × 5) or vehicle (PBS) in two treatment regimes; 1) after hyperoxia from postnatal day (P)7-11, or 2) concurrently with five days of hyperoxia from P1-5. Lung structure, function and macrophages were assessed using alveolar morphometry, barometric whole-body plethysmography and flow cytometry. RESULTS AND DISCUSSION: Seven days of hyperoxia resulted in an 18% decrease in body weight and perturbation of lung structure and function. In regime 1, growth restriction persisted in the Hyp + PBS and Hyp + CSF-1 groups, although perturbations in respiratory function were resolved by P35. CSF-1 increased CSF-1R+/F4/80+ macrophage number by 34% at P11 compared to Hyp + PBS, but was not associated with growth or lung structural rescue. In regime 2, five days of hyperoxia did not cause initial growth restriction in the Hyp + PBS and Hyp + CSF-1 groups, although body weight was decreased at P35 with CSF-1. CSF-1 was not associated with increased macrophages, or with functional perturbation in the adult. Overall, CSF-1 did not rescue the growth and lung defects associated with hyperoxia in this model; however, an increase in CSF-1R+ macrophages was not associated with an exacerbation of lung injury. The trophic functions of macrophages in lung development requires further elucidation in order to explore macrophage modulation as a strategy for promoting lung maturation.