The administration of certain progenitor cells is protective in experimental acute kidney injury (AKI), and mechanisms may involve the release of paracrine factors. Endothelial colony-forming cells (ECFCs) are endothelial precursor cells with a high proliferative capacity and pro-angiogenic potential. We examined the effects of human umbilical cord blood-derived ECFCs and their extracellular vesicles in a mouse model of ischemic AKI and in cultured human umbilical vein endothelial cells subjected to hypoxia/reoxygenation. In mice with ischemic AKI, administration of ECFCs (i.v.) at the time of reperfusion significantly attenuated increases in plasma creatinine, tubular necrosis, macrophage infiltration, oxidative stress, and apoptosis, without cell persistence in the kidneys. In cultured human umbilical vein endothelial cells, hypoxia/reoxygenation stimulated apoptosis. This effect was inhibited by incubation with conditioned medium or exosomes (40- to 100-nm diameter) derived from ECFCs, but not by microparticles (100- to 1000-nm diameter) or vesicle-depleted conditioned medium. Administration of exosomes (i.v.) directly to mice with ischemic AKI attenuated renal injury, as assessed by plasma creatinine, tubular necrosis, and apoptosis. Taken together, these studies indicate protective effects of human cord blood-derived ECFCs in experimental AKI and suggest that ECFC-derived exosomes may mediate the protective response via inhibition of endothelial cell apoptosis.

Ubiquitin specific protease 4 (USP4) is a highly networked deubiquitinating enzyme with reported roles in cancer, innate immunity and RNA splicing. In mammals it has two dominant isoforms arising from inclusion or skipping of exon 7 (E7). We evaluated two plausible mechanisms for the generation of these isoforms: (A) E7 skipping due to a long upstream intron and (B) E7 skipping due to inefficient 5' splice sites (5'SS) and/or branchpoint sites (BPS). We then assessed whether E7 alternative splicing is maintained by selective pressure or arose from genetic drift. Both transcript variants were generated from a USP4-E7 minigene construct with short flanking introns, an observation consistent with the second mechanism whereby differential splice signal strengths are the basis of E7 skipping. Optimization of the downstream 5'SS eliminated E7 skipping. Experimental validation of the correlation between 5'SS identity and exon skipping in vertebrates pinpointed the +6 site as the key splicing determinant. Therian mammals invariably display a 5'SS configuration favouring alternative splicing and the resulting isoforms have distinct subcellular localizations. We conclude that alternative splicing of mammalian USP4 is under selective maintenance and that long and short USP4 isoforms may target substrates in various cellular compartments.

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.

Chronic kidney disease (CKD) is a worldwide health burden with increases risk of end-stage renal function if left untreated. CKD induced in the context of metabolic syndrome (MS) increases risks of hypertension, hyperglycemia, excess body fat and dyslipidemia. To test if combining a high-fat diet (HFD) regimen onto the hypertensive/ diabetic phenotype would mimic features of MS induced-CKD in mice, hyperglycemia was induced in genetically hypertensive mice (Lin), followed by HFD regimen. For that, 8-week-old male were subjected to streptozotocin (STZ) intraperitoneal (i.p.) injections (50 mg/kg, 5 days consecutive). LinSTZ were fed a 60% kCal HFD for 8 weeks. Lin mice treated with STZ developed polydipsia, became hypertensive and hyperglycemic. HFD induced weight gain, protected against glomerular hypertrophy, scarring, and albuminuria at endpoint compared to regular diet fed LinSTZ. On the other hand, HFD induced steatosis, liver fibrosis, inflammation, and increase in AST/ALT ratio, characteristics of non-alcoholic liver disease. Taken together, our results show that LinSTZ mice fed a HFD did not lead to a more robust model of MS-induced CKD, protected against kidney injury, but inducing liver damage. More studies are necessary to understand the kidney protective mechanisms of HFD when superimposed with hypertension and type 1 diabetes.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer. As few tools exist to study ILC metastasis, we isolated ILC cells with increased invasive properties to establish a spontaneously metastasising xenograft model.

Progress in the development of therapeutic interventions to treat or slow the progression of Alzheimer's disease has been hampered by lack of efficacy and unforeseen side effects in human clinical trials. This setback highlights the need for new approaches for pre-clinical testing of possible interventions. Systems modelling is becoming increasingly recognised as a valuable tool for investigating molecular and cellular mechanisms involved in ageing and age-related diseases. However, there is still a lack of awareness of modelling approaches in many areas of biomedical research. We previously developed a stochastic computer model to examine some of the key pathways involved in the aggregation of amyloid-beta (Aβ) and the micro-tubular binding protein tau. Here we show how we extended this model to include the main processes involved in passive and active immunisation against Aβ and then demonstrate the effects of this intervention on soluble Aβ, plaques, phosphorylated tau and tangles. The model predicts that immunisation leads to clearance of plaques but only results in small reductions in levels of soluble Aβ, phosphorylated tau and tangles. The behaviour of this model is supported by neuropathological observations in Alzheimer patients immunised against Aβ. Since, soluble Aβ, phosphorylated tau and tangles more closely correlate with cognitive decline than plaques, our model suggests that immunotherapy against Aβ may not be effective unless it is performed very early in the disease process or combined with other therapies.

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1-7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

Glioblastoma is one of the deadliest forms of cancer, in part because of its highly invasive nature. The tumor suppressor PTEN is frequently mutated in glioblastoma and is known to contribute to the invasive phenotype. However the downstream events that promote invasion are not fully understood. PTEN loss leads to activation of the atypical protein kinase C, PKCι. We have previously shown that PKCι is required for glioblastoma cell invasion, primarily by enhancing cell motility. Here we have used time-lapse videomicroscopy to more precisely define the role of PKCι in glioblastoma.

Recent evidence suggests that glycogen synthase kinase-3beta (GSK3beta) is implicated in both sporadic and familial forms of Alzheimer's disease. The transcription factor, p53 also plays a role and has been linked to an increase in tau hyperphosphorylation although the effect is indirect. There is also evidence that GSK3beta and p53 interact and that the activity of both proteins is increased as a result of this interaction. Under normal cellular conditions, p53 is kept at low levels by Mdm2 but when cells are stressed, p53 is stabilised and may then interact with GSK3beta. We propose that this interaction has an important contribution to cellular outcomes and to test this hypothesis we developed a stochastic simulation model.

The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood.

Expanded polyglutamine (polyQ) proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. PolyQ tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyQ into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of polyQ, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyQ protein inclusion, body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Renal ubiquitin C-terminal hydrolase L1 (UCHL1) is upregulated in a subset of human glomerulopathies, including focal segmental glomerulosclerosis (FSGS), where it may serve to promote ubiquitin pools for degradation of cytotoxic proteins. In the present study, we tested whether UCHL1 is expressed in podocytes of a mouse model of ACTN4-associated FSGS. Podocyte UCHL1 protein was detected in glomeruli of K256E-ACTN4(pod+)/UCHL1+/+ mice. UCHL1+/- mice were intercrossed with K256E-ACTN4(pod+) mice and monitored for features of glomerular disease. 10-week-old K256E-ACTN4(pod+)/UCHL1-/- mice exhibited significantly ameliorated albuminuria, glomerulosclerosis, tubular pathology and blood pressure. Interestingly, while UCHL1 deletion diminished both tubular and glomerular apoptosis, WT1-positive nuclei were unchanged. Finally, UCHL1 levels correlated positively with poly-ubiquitinated proteins but negatively with K256E-α-actinin-4 levels, implying reduced K256E-α-actinin-4 proteolysis in the absence of UCHL1. Our data suggest that UCHL1 upregulation in ACTN4-associated FSGS fuels the proteasome and that UCHL1 deletion may impair proteolysis and thereby preserve K256E/wt-α-actinin-4 heterodimers, maintaining podocyte cytoskeletal integrity and protecting the glomerular filtration barrier.

Despite its high prevalence and economic burden, the aetiology of human hypertension remains incompletely understood. Here we identify the transcription factor GATA5, as a new regulator of blood pressure (BP). GATA5 is expressed in microvascular endothelial cells and its genetic inactivation in mice (Gata5-null) leads to vascular endothelial dysfunction and hypertension. Endothelial-specific inactivation of Gata5 mimics the hypertensive phenotype of the Gata5-null mice, suggestive of an important role for GATA5 in endothelial homeostasis. Transcriptomic analysis of human microvascular endothelial cells with GATA5 knockdown reveals that GATA5 affects several genes and pathways critical for proper endothelial function, such as PKA and nitric oxide pathways. Consistent with a role in human hypertension, we report genetic association of variants at the GATA5 locus with hypertension traits in two large independent cohorts. Our results unveil an unsuspected link between GATA5 and a prominent human condition, and provide a new animal model for hypertension.

In yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed.

Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Akt phosphorylation. Exposure of hypoxic endothelial cells to conditioned medium from ECFCs pretreated with anti-miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti-miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury.

The C-type lectin-related protein, Clr-f, encoded by Clec2h in the mouse NK gene complex (NKC), is a member of a family of immune regulatory lectins that guide immune responses at distinct tissues of the body. Clr-f is highly expressed in the kidney; however, its activity in this organ is unknown. To assess the requirement for Clr-f in kidney health and function, we generated a Clr-f-deficient mouse (Clr-f-/-) by targeted deletions in the Clec2h gene. Mice lacking Clr-f exhibited glomerular and tubular lesions, immunoglobulin and C3 complement protein renal deposits, and significant abdominal and ectopic lipid accumulation. Whole kidney transcriptional profile analysis of Clr-f-/- mice at 7, 13, and 24 weeks of age revealed a dynamic dysregulation in lipid metabolic processes, stress responses, and inflammatory mediators. Examination of the immune contribution to the pathologies of Clr-f-/- mouse kidneys identified elevated IL-12 and IFNγ in cells of the tubulointerstitium, and an infiltrating population of neutrophils and T and B lymphocytes. The presence of these insults in a Rag1-/-Clr-f-/- background reveals that Clr-f-/- mice are susceptible to a T and B lymphocyte-independent renal pathogenesis. Our data reveal a role for Clr-f in the maintenance of kidney immune and metabolic homeostasis.

The SIRT1 protein deacetylase is reported to have a remarkably wide spectrum of biological functions affecting such varied processes as aging, cancer, metabolism, neurodegeneration and immunity. However, the SIRT1 literature is also full of contradictions. To help establish the role(s) of SIRT1 in these and other biological processes, we set out to create a mouse in which the SIRT1 activity could be toggled between on and off states by fusing the estrogen receptor ligand-binding domain (ER) to the C terminus of the SIRT1 protein. We found that the catalytic activity of the SIRT1-ER fusion protein increased 4-5 fold in cells treated with its ligand, 4-hydroxy-tamoxifen (4OHT). The 4OHT-induced activation of SIRT1-ER was due in large part to a 2 to 4-fold increase in abundance of the SIRT1-ER protein in cells in culture and in tissues in vivo. This increase is reversible and is a consequence of 4OHT-induced stabilization of the SIRT1-ER protein. Since changes in SIRT1 level or activity of 2-4 fold are frequently reported to be sufficient to affect its biological functions, this mouse should be helpful in establishing the causal relationships between SIRT1 and the diseases and processes it affects.

Endothelial colony forming cell (ECFC)-derived exosomes protect mice against ischemic kidney injury, via transfer of microRNA-(miR)-486-5p. Mechanisms mediating exosome recruitment to tissues are unclear. We hypothesized that ECFC exosomes target ischemic kidneys, involving interaction between exosomal CXC chemokine receptor type 4 (CXCR4) and stromal cell-derived factor (SDF)-1α. Ischemia-reperfusion was induced in mice by bilateral renal vascular clamp, with intravenous infusion of exosomes at reperfusion. Optical imaging determined exosome biodistribution, and miR-486-5p was measured by real-time PCR. Human umbilical vein endothelial cells (HUVECs) were cultured to study the CXCR4/SDF-1α interaction. Targeting of administered exosomes to ischemic kidneys was detected 30 min and 4 hrs after reperfusion. Exosomes increased miR-486-5p levels only in kidneys, within proximal tubules, glomeruli, and endothelial cells. Uptake of fluorescently-labeled exosomes into HUVECs, and exosomal transfer of miR-486-5p were enhanced by hypoxia, effects blocked by neutralizing antibody to SDF-1α or by the CXCR4 inhibitor plerixafor. Infusion of ECFC exosomes prevented ischemic kidney injury in vivo, an effect that was not observed when exosomes were pre-incubated with plerixafor. These data indicate that ECFC exosomes selectively target the kidneys after ischemic injury, with rapid cellular transfer of miR486-5p. Targeting of exosomes may involve interaction of CXCR4 with endothelial cell SDF-1α.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

Canagliflozin (CANA) is a sodium-glucose cotransporter 2 (SGLT2) inhibitor with blood glucose lowering effects. CANA also promotes kidney protection in patients with cardiovascular diseases and type 2 diabetes (T2D), as well as in normoglycemic patients with hypertension or heart failure. Clinical studies, although conduct in both sexes, do not report sex-dependent differences in T2DM treated with CANA. However, the impact of CANA in type 1 diabetes, as well in sex-dependent outcomes in such cohort needs further understanding. To analyze the effects of CANA in mice with combined hypertension and type 1 diabetes, diabetes was induced by STZ injection (5 days, 50mg/kg/day) in both male and female 8 weeks old genetic hypertensive mice (Lin), whereas the control (Lin) received 0.1M sodium citrate injections. 8 weeks after STZ. Mice were fed either regular or CANA-infused diet for 4 weeks. 8 weeks after STZ, hyperglycemia was present in both male and female mice. CANA reversed BG increase mice fed regular diet. Male LinSTZ mice had elevated water intake, urine output, urinary albumin to creatinine ratio (ACR), kidney lesion score, and creatinine clearance compared to the Lin control group. Kidney injury was improved in male LinSTZ + CANA group in male mice. Water intake and urine output were not statistically significantly different in female LinSTZ compared to female LinSTZ+ CANA. Moreover, CANA did not improve kidney injury in female mice, showing no effect in creatinine clearance, lesion score and fibrosis when compared to LinSTZ fed regular diet. Here we show that Canagliflozin might exert different kidney protection effects in male compared to female mice with hypertension and type 1 diabetes. Sex-dimorphisms were previously found in the pathophysiology of diabetes induced by STZ. Therefore, we highlight the importance of in-depth investigation on sex-dependent effects of CANA, taking in consideration the unique characteristics of disease progression for each sex.