Genome-Wide DNA Methylation Signatures Predict the Early Asymptomatic Doxorubicin-Induced Cardiotoxicity in Breast Cancer.

Chemotherapy with doxorubicin (DOX) may cause unpredictable cardiotoxicity. This study aimed to determine whether the methylation signature of peripheral blood mononuclear cells (PBMCs) prior to and after the first cycle of DOX-based chemotherapy could predict the risk of cardiotoxicity in breast cancer patients. Cardiotoxicity was defined as a decrease in left ventricular ejection fraction (LVEF) by >10%. DNA methylation of PBMCs from 9 patients with abnormal LVEF and 10 patients with normal LVEF were examined using Infinium HumanMethylation450 BeadChip. We have identified 14,883 differentially methylated CpGs at baseline and 18,718 CpGs after the first cycle of chemotherapy, which significantly correlated with LVEF status. Significant differentially methylated regions (DMRs) were found in the promoter and the gene body of SLFN12, IRF6 and RNF39 in patients with abnormal LVEF. The pathway analysis found enrichment for regulation of transcription, mRNA splicing, pathways in cancer and ErbB2/4 signaling. The preliminary results from this study showed that the DNA methylation profile of PBMCs may predict the risk of DOX-induced cardiotoxicity prior to chemotherapy. Further studies with larger cohorts of patients are needed to confirm these findings.

Levofloxacin-Induced Acute Hyperpigmentation Changes in a Chronic Kidney Disease Patient.

Medication-induced cutaneous hyperpigmentation has variable clinical presentations and is dependent on the specific drug involved. Most commonly, an attentive patient observes such changes early in the course; when missed by the patient, such changes are usually noted by an observant clinician. Clinical diagnosis can be challenging if the patient is on multiple medications because other causes must be excluded. This condition occurs via multiple mechanisms. Frequently, the pigmentary change is reversible with discontinuation of the drug. Causative medications include nonsteroidal; anti-inflammatory agents, antimalarials, antibiotics, psychotropics, amiodarone, and chemotherapeutic agents. The; antimicrobials responsible for hyperpigmentation are antimalarials, tetracyclines, tigecycline, dapsone, rifampicin, and antiretrovirals such as zidovudine. Sunlight exposure can worsen the pigmentation seen with some of the above antimicrobials (e.g., dapsone). Here, we describe an older adult white woman presenting with acute cutaneous; hyperpigmentation of the bilateral lower extremities while on levofloxacin therapy. Hyperpigmentation resolved after cessation of the agent. Our case highlights this unique acute presentation after only a few days of oral levofloxacin.

A Uromodulin Mutation Drives Autoimmunity and Kidney Mononuclear Phagocyte Endoplasmic Reticulum Stress.

We identified a family with a UMOD gene mutation (C106F) resulting in glomerular inflammation and complement deposition. To determine if the observed phenotype is due to immune system activation by mutant uromodulin, a mouse strain with a homologous cysteine to phenylalanine mutation (C105F) in the UMOD gene was generated using CRISPR-Cas9 gene editing and the effect of this mutation on mononuclear phagocytic cells was examined. Mutant mice developed high levels of intracellular and secreted aggregated uromodulin, resulting in anti-uromodulin antibodies and circulating uromodulin containing immune complexes with glomerular deposition and kidney fibrosis with aging. F4/80+ and CD11c+ kidney cells phagocytize uromodulin. Differential gene expression analysis by RNA sequencing of F4/80+ phagocytic cells revealed activation of the activating transcription factor 5 (ATF5)-mediated stress response pathway in mutant mice. Phagocytosis of mutant uromodulin by cultured dendritic cells resulted in activation of the endoplasmic reticulum stress response pathway and markers of cell inactivation, an effect not seen with wild-type protein. Mutant mice demonstrate a twofold increase in T-regulatory cells, consistent with induction of immune tolerance, resulting in decreased inflammatory response and improved tissue repair following ischemia-reperfusion injury. The C105F mutation results in autoantibodies against aggregated misfolded protein with immune complex formation and kidney fibrosis. Aggregated uromodulin may induce dendritic cell tolerance following phagocytosis through an unfolded protein/endoplasmic reticulum stress response pathway, resulting in decreased inflammation following tissue injury.

Depletion of senescent-like neuronal cells alleviates cisplatin-induced peripheral neuropathy in mice.

Chemotherapy-induced peripheral neuropathy is among the most common dose-limiting adverse effects of cancer treatment, leading to dose reduction and discontinuation of life-saving chemotherapy and a permanently impaired quality of life for patients. Currently, no effective treatment or prevention is available. Senescence induced during cancer treatment has been shown to promote the adverse effects. Here, we show that cisplatin induces senescent-like neuronal cells in primary culture and in mouse dorsal root ganglia (DRG), as determined by the characteristic senescence markers including senescence-associated beta-galactosidase, accumulation of cytosolic p16INK4A and HMGB1, as well as increased expression of p16Ink4a, p21, and MMP-9. The accumulation of senescent-like neuronal cells in DRG is associated with cisplatin-induced peripheral neuropathy (CIPN) in mice. To determine if depletion of senescent-like neuronal cells may effectively mitigate CIPN, we used a pharmacological 'senolytic' agent, ABT263, which inhibits the anti-apoptotic proteins BCL-2 and BCL-xL and selectively kills senescent cells. Our results demonstrated that clearance of DRG senescent neuronal cells reverses CIPN, suggesting that senescent-like neurons play a role in CIPN pathogenesis. This finding was further validated using transgenic p16-3MR mice, which permit ganciclovir (GCV) to selectively kill senescent cells expressing herpes simplex virus 1 thymidine kinase (HSV-TK). We showed that CIPN was alleviated upon GCV administration to p16-3MR mice. Together, the results suggest that clearance of senescent DRG neuronal cells following platinum-based cancer treatment might be an effective therapy for the debilitating side effect of CIPN.

Id1 expression in kidney endothelial cells protects against diabetes-induced microvascular injury.

The inhibitor of differentiation (Id) transcription regulators, which are induced in response to oxidative stress, promote cell proliferation and inhibit senescence. Inhibitor of differentiation 1 (Id1) expression is limited to endothelial cells (EC) in the normal mouse kidney and is required for a normal response to injury. Endothelial dysfunction leads to the development of diabetic nephropathy, and so, we hypothesized that endothelial Id1 may help protect against hyperglycemia-induced microvascular injury and nephropathy. Here, we tested this hypothesis by using streptozotocin to induce diabetes in Id1 knockout (KO) mice and WT B6;129 littermates and examining the mice at 3 months. Expression of Id1 was observed to be increased 15-fold in WT kidney EC, and Id1 KO mice exhibited increased mesangial and myofibroblast proliferation, matrix deposition, and albuminuria compared with WT mice. Electron microscopy demonstrated peritubular capillary EC injury and lumen narrowing, and fluorescence microangiography showed a 45% reduction in capillary perfusion area with no reduction in CD31-stained areas in Id1 KO mice. Microarray analysis of EC isolated from WT and KO control and diabetic mice demonstrated activation of senescence pathways in KO cells. Kidneys from KO diabetic mice showed increased histological expression of senescence markers. In addition, premature senescence in cultured KO EC was also seen in response to oxidative stress. In conclusion, endothelial Id1 upregulation with hyperglycemia protects against microvascular injury and senescence and subsequent nephropathy.

Young blood for old kidneys? More questions than answers so far.

The aging kidney has increased susceptibility to injury and decreased regenerative capacity. It is unknown if the effects of aging on acute kidney injury are reversible. In this issue, Liu et al., using a mouse heterochronic parabiosis ischemia-reperfusion model, demonstrate that a shared circulation between a young and older mouse results in decreased acute tubular injury. This study is an important first step using an interesting model, but many questions remain about how young blood reduces acute kidney injury in an older mouse.

Microparticles from kidney-derived mesenchymal stem cells act as carriers of proangiogenic signals and contribute to recovery from acute kidney injury.

We recently demonstrated the use of in vitro expanded kidney-derived mesenchymal stem cells (KMSC) protected peritubular capillary endothelial cells in acute renal ischemia-reperfusion injury. Herein, we isolated and characterized microparticles (MPs) from KMSC. We investigated their in vitro biologic effects on human endothelial cells and in vivo renoprotective effects in acute ischemia-reperfusion renal injury. MPs were isolated from the supernatants of KMSC cultured in anoxic conditions in serum-deprived media for 24 hours. KMSC-derived MPs demonstrated the presence of several adhesion molecules normally expressed on KMSC membranes, such as CD29, CD44, CD73, α4, 5, and 6 integrins. Quantitative real time PCR confirmed the presence of 3 splicing variants of VEGF-A (120, 164, 188), bFGF and IGF-1 in isolated MPs. MPs labeled with PKH26 red fluorescence dye were incorporated by cultured human umbilical vein endothelial cells (HUVEC) via surface molecules such as CD44, CD29, and α4, 5, and 6 integrins. MP dose dependently improved in vitro HUVEC proliferation and promoted endothelial tube formation on growth factor reduced Matrigel. Moreover, apoptosis of human microvascular endothelial cell was inhibited by MPs. Administration of KMSC-derived MPs into mice with acute renal ischemia was followed by selective engraftment in ischemic kidneys and significant improvement in renal function. This was achieved by improving proliferation, of peritubular capillary endothelial cell and amelioration of peritubular microvascular rarefaction. Our results support the hypothesis that KMSC-derived MPs may act as a source of proangiogenic signals and confer renoprotective effects in ischemic kidneys.

INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury.

The molecular mechanisms that lead to tubular atrophy, capillary loss, and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. The INK4a/ARF locus encodes overlapping genes for two proteins, a cyclin kinase inhibitor, p16(INK4a), and a p53 stabilizer, p19(ARF), from independent promoters. To determine if decreased INK4a gene expression results in improved kidney regeneration, INK4a knockout (KO) and wild-type (WT) mice were subjected to ischemia-reperfusion injury (IRI). p16(INK4a) and p19(ARF) levels were increased markedly in WT mice at 1-28 days after injury. Kidneys were examined to determine the localization and levels of p16(INK4a), apoptosis, cell proliferation, and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis, increased cell proliferation, and lower creatinine levels after injury. KO mice had significantly higher capillary density compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating factor (G-CSF) increased after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1(+)/Cd11b(+) myeloid cells by flow cytometry. KO mice treated with a Gr-1-depleting antibody displayed reduced vascular endothelial growth factor mRNA, plasma G-CSF, and capillary density, and an increase in serum creatinine and medullary myofibroblasts, compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16(INK4a) and p19(ARF) following IRI has a protective effect on the kidney through improved epithelial and microvascular repair, in part by enhancing the mobilization of myeloid cells into the kidney.

INK4a knockout mice exhibit increased fibrosis under normal conditions and in response to unilateral ureteral obstruction.

The INK4a proteins p16(INK4a) and p19(ARF) regulate cell cycle arrest and senescence. However, the role of these proteins in controlling these processes in the normal kidney and following injury is unknown. We performed unilateral ureteral obstruction (UUO) to induce fibrosis in 2- to 3-mo-old wild-type (WT) C57/B6 and INK4a knockout mice. By quantitative RT-PCR, p16(INK4a) levels were increased sixfold in WT mice 7 days after UUO and p19(ARF) remained undetectable. Kidney sections were examined to determine levels and localization of p16(INK4a), apoptosis, fibrosis, and senescent cells. INK4a knockout mice displayed mesangial cell proliferation, increased matrix deposition, and myofibroblast differentiation under normal conditions. Following UUO, INK4a knockout mice displayed 10-fold increased tubular and interstitial cell proliferation, 75% decreased collecting duct apoptosis, 2-fold greater collagen and fibronectin deposition, and no cell senescence by senescence-associated ß-galactosidase staining compared with WT mice. Both INK4a knockout mesangial cells and kidney lysates from knockout mice following injury showed elevated levels of IL-6 by ELISA compared with WT samples. INK4a knockout epithelial cell cultures displayed increased mesenchymal cell markers when exposed to transforming growth factor-ß. These results confirm that p16(INK4a) controls cell proliferation and matrix production and mitigates fibrosis following injury and suggest that the mechanism involves a role in limiting inflammation and cell proliferation.

Mesenchymal stem cells, used as bait, disclose tissue binding sites: a tool in the search for the niche?

We developed an ex vivo approach characterizing renal mesenchymal stem cell (MSC) adhesion to kidney sections. Specificity of MSC adhesion was confirmed by demonstrating a) 3T3 cells displayed 10-fold lower adhesion, and b) MSC adhesion was CXCR4/stromal-derived factor-1 (SDF-1)-dependent. MSC adhesion was asymmetrical, with postischemic sections exhibiting more than twofold higher adhesion than controls, and showed preference to perivascular areas. Pretreating kidney sections with cyclic arginine-glycine-aspartic acid peptide resulted in increased MSC adhesion (by displacing resident cells), whereas blockade of CXCR4 with AMD3100 and inhibition of alpha4beta1(VLA4) integrin or vascular cellular adhesion molecule-1, reduced adhesion. The difference between adhered cells under cyclic arginine-glycine-aspartic acid peptide-treated and control conditions reflected prior occupancy of binding sites with endogenous cells. The AMD3100-inhibitable fraction of adhesion reflected CXCR4-dependent adhesion, whereas maximal adhesion was interpreted as kidney MSC-lodging capacity. MSC obtained from mice overexpressing caveolin-1 exhibited more robust adhesion than those obtained from knockout animals, consistent with CXCR4 dimerization in caveolae. These data demonstrate a) CXCR4/SDF-1-dependent adhesion increases in ischemia; b) CXCR4/SDF-1 activation is dependent on MSC surface caveolin-1; and c) occupancy of MSC binding sites is decreased, while d) capacity of MSC binding sites is expanded in postischemic kidneys. In conclusion, we developed a cell-bait strategy to unmask renal stem cell binding sites, which may potentially shed light on the MSC niche(s) and its characteristics.

Regulation of the bHLH transcription factor E2A in epithelial cells by interaction with the Na/K-ATPase beta1 subunit.

The bHLH transcription factor E2A controls proliferation and differentiation in many cell types including kidney epithelial cells. To identify regulatory binding partners of E2A in the kidney, a yeast two-hybrid assay with a human adult kidney cDNA library was performed. Results demonstrated E2A interactions with other HLH proteins including Id1-3 and Pod1 and the Na/K-ATPase beta1 subunit. The specificity of beta1 subunit binding was confirmed by co-immunoprecipitation of E2A and beta1 subunit deletion constructs in HEK cells demonstrating E2A binding to the cytoplasmic tail of the beta1 subunit. Immunofluorescence and Western analysis of HEK cells co-transfected with GFP-beta1 subunit and E2A demonstrated E2A membrane binding and increased beta1 subunit membrane localization. Increased beta1 subunit expression resulted in decreased nuclear E2A expression and protein half-life and reduced E2A induced gene expression. These results suggest that E2A and Na/K-ATPase beta1 subunit expression in epithelial cells are regulated by interactions between these proteins.

Kidney-derived mesenchymal stem cells contribute to vasculogenesis, angiogenesis and endothelial repair.

We isolated a clonal cell line (4E) from kidneys of mice expressing green fluorescent protein controlled by the endothelial-specific Tie2 promoter. When grown in a three-dimensional matrigel matrix they formed a fluorescent capillary network. In vivo angiogenesis assays using growth factor-depleted matrigel implanted plugs promoted a moderate angiogenesis of host endothelial cells. Using vascular endothelial growth factor (VEGF)-A and fibroblast growth factor-2 in the plugs containing 4E-cells resulted in a robust vasculogenesis. Transplantation of 4E cells into mice with acute renal ischemia showed selective engraftment in the ischemic kidney which promoted tubular regeneration by increasing epithelial proliferation and inhibiting apoptosis. This resulted in an accelerated functional recovery 3 days after ischemia. These mice showed a 5-fold increase in tissue VEGF expression compared to controls, but no difference in plasma VEGF level corresponding with better preservation of peritubular capillaries, perhaps due to a local paracrine effect following systemic 4E infusion. One month after ischemia, 9% of engrafted 4E cells expressed green fluorescent protein in the peritubular region while half of them expressed alpha-smooth muscle actin. Our study shows that kidney mesenchymal stem cells are capable of differentiation toward endothelial and smooth muscle cell lineages in vitro and in vivo, support new blood vessel formation in favorable conditions and promote functional recovery of an ischemic kidney.

Glial cell line-derived neurotrophic growth factor increases motility and survival of cultured mesenchymal stem cells and ameliorates acute kidney injury.

Glial cell line-derived neurotrophic growth factor (GDNF), a member of the transforming growth factor family, is necessary for renal organogenesis and exhibits changes in expression in models of renal disease. Nestin is an intermediate filament protein originally believed to be a marker of neuroepithelial stem cells and recently proposed as a marker of mesenchymal stem cells (MSC). Having demonstrated the participation of nestin-expressing cells in renoprotection during acute renal ischemia, we hypothesized that growth factors and transcription factors similar to those operating in the nervous system should be also operant in the kidney and may be induced after noxious stimuli, such as an ischemic episode. Using cultured kidney-derived MSC, which abundantly express nestin, we confirmed expression of GDNF by these cells and demonstrated the GDNF-induced expression of GDNF. The cellular expression of nestin paralleled that of GDNF: serum starvation decreased the expression, whereas application of GDNF resulted in a dose-dependent increase in nestin expression. Immunohistochemical and Western blot analyses of kidneys obtained from control and postischemic mice showed that expression of GDNF was much enhanced in the renal cortex, a pattern similar to the previously reported expression of nestin. Based on the observed GDNF-induced GDNF expression, we next explored the effect of supplemental GDNF administered early after ischemia on renal function postischemia. GDNF-treated mice were protected against acute ischemia. To address potential mechanisms of the observed renoprotection, in vitro studies showed that GDNF accelerated MSC migration in a wound-healing assay. Hypoxia did not accelerate, but rather slightly reduced, the motility of MSC and reduced the expression of GDNF in MSC by approximately twofold. Furthermore, GDNF was cytoprotective against oxidative stress-induced apoptotic death of MSC. Collectively, these data establish 1) an autoregulatory circuit of GDNF-induced GDNF expression in renal MSC; 2) induction of GDNF expression in postischemic kidneys; 3) the ability of exogenous GDNF to ameliorate ischemic renal injury; and 4) a possible contribution of GDNF-induced motility and improved survival of MSC to renoprotection.

Pod1 induces myofibroblast differentiation in mesenchymal progenitor cells from mouse kidney.

The class II basic helix-loop-helix (bHLH) transcription factor Pod1 is expressed in mesenchymal cells including smooth muscle progenitors during development and in interstitial cells in adult organs. To determine the role of Pod1 in mesenchymal cell smooth muscle and myofibroblast differentiation, we examined a kidney progenitor cell line (4E) that endogenously expresses Pod1 and its class I bHLH partner E2A. In vitro-translated Pod1 co-immunoprecipitated E2A and increased E2A binding to a calponin promoter E-box sequence as determined by an electrophoresis mobility shift assay (EMSA). Overexpression of Pod1 and E2A resulted in increased smooth muscle and myofibroblast gene expression including calponin, SM22alpha, alphaSMA, fibronectin, and connective tissue growth factor (CTGF) compared with overexpression of E2A alone. Suppression of Pod1 by siRNA resulted in increased cell proliferation and reduced expression of alphaSMA, fibronectin, and CTGF, and myofibroblast secreted proteins including pro-fibrotic cytokines and inhibitors of matrix metalloproteinases. Examination of the signaling pathways for myofibroblast differentiation including Rho/Rho kinase and p38 MAPK showed that inhibition of actin polymerization by Rho kinase inhibitors decreased nuclear Pod1 levels while inhibition of p38 MAPK decreased Pod1 expression. These results indicate that Pod1 increases myofibroblast differentiation in combination with E2A and promotes a myofibroblast phenotype in mesenchymal progenitor cells.

Mesenchymal cells from adult kidney support angiogenesis and differentiate into multiple interstitial cell types including erythropoietin-producing fibroblasts.

Mesenchymal cells have been isolated from embryos and multiple adult organs where they may differentiate into various connective tissue cell types and provide paracrine support for surrounding cells. With the use of a technique for culturing multipotent mesenchymal cells from adult tissues, a fibroblast-like cell clone (4E) was isolated from adult mouse kidney. 4E cells were able to differentiate along multiple mesodermal lineages including cell types located in the renal interstitium such as fibroblasts and pericytes. Coculture of 4E cells with ureteric bud and epithelial cell lines and analysis of resulting changes in gene expression revealed that these cells support angiogenesis and tubulogenesis and expressed genes characteristic of embryonic renal stromal cells. Following subcapsular injection after unilateral ischemia-reperfusion in adult mice, 4E cells migrated to a peritubular interstitial location and expressed interstitial cell markers, whereas cells injected in control kidneys remained stationary. Incubation in hypoxic or anoxic conditions resulted in erythropoietin expression in a small subset of ecto-5'-nucleotidase-positive cells and resulted in increased vascular endothelial growth factor expression in the same cell population. Our findings suggest that the adult kidney may contain interstitial mesenchymal cell progenitors with embryonic stromal cell characteristics that are able to provide paracrine support for surrounding vessels and tubular epithelial cells and differentiate into erythropoietin producing fibroblasts.

Therapeutic use of stem and endothelial progenitor cells in acute renal injury: ça ira.

Acute renal failure (ARF) seriously worsens prognosis of hospitalized patients. The dysfunction and apoptosis/necrosis of tubular epithelial cells is of key importance for the pathophysiological consequences of the syndrome. ARF also affects the structure and function of the renal endothelium because these cells undergo an early swelling with narrowing of the vascular lumen, resulting in prolonged renal hypoperfusion. The dysfunctional renal epithelium and endothelium have remarkable capacity to recover. With the growing knowledge in the field of stem cell research, these regenerative mechanisms become more and more elucidated. Recent data suggest that bone marrow derived mesenchymal stem cells can ameliorate ARF through both paracrine effects and repair of injured microvasculature by providing endothelial progenitor cells. Evidence for resident adult renal stem cells is also now emerging.

Identification of the gamma-aminobutyric acid receptor beta(2) and beta(3) subunits in rat, rabbit, and human kidneys.

The properties and functions of gamma-aminobutyric acid (GABA(A)) receptors in the mammalian central nervous system are well studied. However, the presence and significance of GABA(A) receptors in nonneural tissue is less clear. The goal of this study was to examine the expression and localization of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney. Reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and cerebellum and rabbit S(2) segments were amplified by use of PCR and GABA(A) beta(2) and beta(3) subunit-specific primers. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(2) subunit were identical in nucleotide composition. The rabbit kidney and rabbit neuronal GABA(A) receptor beta(2) subunit were 99% identical in nucleotide composition. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(3) subunits were 93% and 95% identical in nucleotide and amino acid composition, and rabbit kidney cortex and rabbit neuronal GABA(A) receptor beta(3) subunits were 95% and 98% identical in nucleotide and amino acid composition, respectively. PCR screening of a human kidney cDNA library and sequencing revealed that the human kidney cortex and neuronal beta(3) subunits were identical in nucleotide composition. Immunoblot analysis of rat kidney cortex and brain identified immunoreactive proteins in the 55 to 57 kD region, corresponding to the GABA(A) receptor beta(2) and beta(3) subunits. Immunohistochemistry revealed cytosolic and basolateral staining of the proximal convoluted and straight tubule. These results provide compelling evidence for the expression of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney of multiple species and the localization of the beta(2)/beta(3) subunits to the renal proximal tubule.