Role of the CCL20/CCR6 axis in tubular epithelial cell injury: Kidney-specific translational insights from acute kidney injury to chronic kidney disease.

This study investigated the role of the axis involving chemokine receptor 6 (CCR6) and its ligand chemokine (C-C motif) ligand 20 (CCL20) in acute kidney disease (AKD) using an ischemia-reperfusion injury (IRI) model. The model was established by clamping the unilateral renal artery pedicle of C57BL/6 mice for 30 min, followed by evaluation of CCL20/CCR6 expression at 4 weeks post-IRI. In vitro studies were conducted to examine the effects of hypoxia and H2 O2 -induced oxidative stress on CCL20/CCR6 expression in kidney tissues of patients with AKD and chronic kidney disease (CKD). Tubular epithelial cell apoptosis was more severe in C57BL/6 mice than in CCL20 antibody-treated mice, and CCR6, NGAL mRNA, and IL-8 levels were higher under hypoxic conditions. CCL20 blockade ameliorated apoptotic damage in a dose-dependent manner under hypoxia and reactive oxygen species injury. CCR6 expression in IRI mice indicated that the disease severity was similar to that in patients with the AKD phenotype. Morphometry of CCL20/CCR6 expression revealed a higher likelihood of CCR6+ cell presence in CKD stage 3 patients than in stage 1-2 patients. Kidney tissues of patients with CKD frequently contained CCL20+ cells, which were positively correlated with interstitial inflammation. CCL20/CCR6 levels were increased in fibrotic kidneys at 4 and 8 weeks after 5/6 nephrectomy. These findings suggest that modulating the CCL20/CCR6 pathway is a potential therapeutic strategy for managing the progression of AKD to CKD.

Antibiotic-induced intestinal microbiota depletion can attenuate the acute kidney injury to chronic kidney disease transition via NADPH oxidase 2 and trimethylamine-N-oxide inhibition.

Intestinal microbiota and their metabolites affect systemic inflammation and kidney disease outcomes. Here, we investigated the key metabolites associated with the acute kidney injury (AKI)-to chronic kidney disease (CKD) transition and the effect of antibiotic-induced microbiota depletion (AIMD) on this transition. In 61 patients with AKI, 59 plasma metabolites were assessed to determine the risk of AKI-to-CKD transition. An AKI-to-CKD transition murine model was established four weeks after unilateral ischemia-reperfusion injury (IRI) to determine the effects of AIMD on the gut microbiome, metabolites, and pathological responses related to CKD transition. Human proximal tubular epithelial cells were challenged with CKD transition-related metabolites, and inhibitory effects of NADPH oxidase 2 (NOX2) signals were tested. Based on clinical metabolomics, plasma trimethylamine N-oxide (TMAO) was associated with a significantly increased risk for AKI-to-CKD transition [adjusted odds ratio 4.389 (95% confidence interval 1.106-17.416)]. In vivo, AIMD inhibited a unilateral IRI-induced increase in TMAO, along with a decrease in apoptosis, inflammation, and fibrosis. The expression of NOX2 and oxidative stress decreased after AIMD. In vitro, TMAO induced fibrosis with NOX2 activation and oxidative stress. NOX2 inhibition successfully attenuated apoptosis, inflammation, and fibrosis with suppression of G2/M arrest. NOX2 inhibition (in vivo) showed improvement in pathological changes with a decrease in oxidative stress without changes in TMAO levels. Thus, TMAO is a key metabolite associated with the AKI-to-CKD transition, and NOX2 activation was identified as a key regulator of TMAO-related AKI-to-CKD transition both in vivo and in vitro.

Inflammatory chemokine (C-C motif) ligand 8 inhibition ameliorates peritoneal fibrosis.

Peritoneal fibrosis (PF) is an irreversible complication of peritoneal dialysis (PD) that leads to loss of peritoneal membrane function. We investigated PD effluent and serum levels and the tissue expression of chemokine (C-C motif) ligand 8 (CCL8) in patients with PD. Additionally, we investigated their association with PF in a mouse model. Eighty-two end-stage renal disease (ESRD) patients with PD were examined. CCL8 levels were measured via enzyme-linked immunosorbent assays in PD effluents and serum and analyzed with peritoneal transport parameters. Human peritoneal mesothelial cells (hPMCs) were obtained from the PD effluents of 20 patients. Primary cultured hPMCs were treated with recombinant (r) transforming growth factor (TGF)-ß, and CCL8 expression was assessed via western blotting. As the duration of PD increased, the concentration of CCL8 in PD effluents significantly increased. Correlations between peritoneal transport parameters and dialysate CCL8 levels were observed. Western blotting analysis showed that CCL8 was upregulated via rTGF-ß treatment, accompanied by increases in markers of inflammation, fibrosis, senescence, and apoptosis in hPMCs after induction of fibrosis with rTGF-ß. Anti-CCL8 monoclonal antibody (mAb) treatment suppressed the rTGF-ß-induced increase in all analyzed markers. Immunohistochemical analysis revealed that CCL8 along with fibrosis- and inflammation-related markers were significantly increased in the PF mouse model. Functional blockade of CCL8 using a CCR8 inhibitor (R243) abrogated peritoneal inflammation and fibrosis in vivo. In conclusion, high CCL8 levels in PD effluents may be associated with an increased risk of PD failure, and the CCL8 pathway is associated with PF. CCL8 blockade can ameliorate peritoneal inflammation and fibrosis.

Macrophage transcription factor TonEBP promotes systemic lupus erythematosus and kidney injury via damage-induced signaling pathways.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients - in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN.

Phonozen-mediated photodynamic therapy comparing two wavelengths in a mouse model of peritoneal carcinomatosis.

BACKGROUND: This study assessed the therapeutic efficacy of intraperitoneal photodynamic therapy (PDT) using photosensitizer activation at two different wavelengths, 405 and 664 nm, in a mouse model of peritoneal carcinomatosis. METHODS: The dark and light cytotoxicity of chlorin e6-polyvinylpyrrolidone (Phonozen) were measured in vitro under 402 ± 14 and 670 ± 18 nm LED activation in bioluminescent human gastric cancer cells, MKN45-luc. Cell viability was measured at 6 h after irradiation using the PrestoBlue assay. Corresponding in vivo studies were performed in athymic nude mice by intraperitoneal injection of 1 × 106 MKN45-luc cells. PDT was performed 10 d after tumor induction and comprised intraperitoneal injection of Phonozen followed by light irradiation at 3 h, delivered by a diffusing-tip optical fiber placed in the peritoneal cavity and coupled to a 405 or 664 nm diode laser to deliver a total energy of 50 J (20 mice per cohort). Whole-body bioluminescence imaging was used to track the tumor burden after PDT out to 130 days, and 5 mice in each cohort were sacrificed at 4 h post treatment to measure the acute tumor necrosis. RESULTS: Photosensitizer dose-dependent photocytotoxicity was higher in vitro at 405 than 664 nm. In vivo, PDT reduced the tumor growth rate at both wavelengths, with no statistically significant difference. There was substantial necrosis, and median survival was significantly prolonged at both wavelengths compared with controls (46 and 46 vs. 34 days). CONCLUSIONS: Phonozen-mediated PDT results in significant cytotoxicity in vitro as well as tumor necrosis and prolonged survival in vivo following intraperitoneal light irradiation. Blue light was more photocytotoxic than red in vitro and had marginally higher efficacy in vivo.

Multisample Mass Spectrometry-Based Approach for Discovering Injury Markers in Chronic Kidney Disease.

Urinary proteomics studies have primarily focused on identifying markers of chronic kidney disease (CKD) progression. Here, we aimed to determine urinary markers of CKD renal parenchymal injury through proteomics analysis in animal kidney tissues and cells and in the urine of patients with CKD. Label-free quantitative proteomics analysis based on liquid chromatography-tandem mass spectrometry was performed on urine samples obtained from 6 normal controls and 9, 11, and 10 patients with CKD stages 1, 3, and 5, respectively, and on kidney tissue samples from a rat CKD model by 5/6 nephrectomy. Tandem mass tag-based quantitative proteomics analysis was performed for glomerular endothelial cells (GECs) and proximal tubular epithelial cells (PTECs) before and after inducing 24-h hypoxia injury. Upon hierarchical clustering, out of 858 differentially expressed proteins (DEPs) in the urine of CKD patients, the levels of 416 decreased and 403 increased sequentially according to the disease stage, respectively. Among 2965 DEPs across 5/6 nephrectomized and sham-operated rat kidney tissues, 86 DEPs showed same expression patterns in the urine and kidney tissue. After cross-validation with two external animal proteome data sets, 38 DEPs were organized; only ten DEPs, including serotransferrin, gelsolin, poly ADP-ribose polymerase 1, neuroblast differentiation-associated protein AHNAK, microtubule-associated protein 4, galectin-1, protein S, thymosin beta-4, myristoylated alanine-rich C-kinase substrate, and vimentin, were finalized by screening human GECs and PTECs data. Among these ten potential candidates for universal CKD marker, validation analyses for protein S and galectin-1 were conducted. Galectin-1 was observed to have a significant inverse correlation with renal function as well as higher expression in glomerulus with chronic injury than protein S. This constitutes the first multisample proteomics study for identifying key renal-expressed proteins associated with CKD progression. The discovered proteins represent potential markers of chronic renal cell and tissue damage and candidate contributors to CKD pathophysiology.

RNA-seq profiling of tubulointerstitial tissue reveals a potential therapeutic role of dual anti-phosphatase 1 in glomerulonephritis.

Transcriptome profiling of tubulointerstitial tissue in glomerulonephritis may reveal a potential tubulointerstitial injury-related biomarker. We profiled manually microdissected tubulointerstitial tissue from biopsy cores of 65 glomerulonephritis cases, including 43 patients with IgA nephropathy, 3 with diabetes mellitus nephropathy, 3 with focal segmental glomerulosclerosis, 3 with lupus nephritis, 4 with membranous nephropathy and 9 with minimal change disease, and additional 22 nephrectomy controls by RNA sequencing. A potential biomarker was selected based on the false discovery rate, and experiments were performed in TNF-α-stimulated primary cultured human tubular epithelial cells (hTECs). We identified 3037 genes with low expression and 2852 genes with high expression in the disease samples compared to the controls. Dual-specificity phosphatase 1 (DUSP1) exhibited universal low expression in various diseases (log2 fold change, -3.87), with the lowest false discovery rate (7.03E-132). In further experimental validation study, DUSP1 overexpression ameliorated inflammatory markers related to MAP kinase pathways in hTECs, while pharmacologic inhibition of DUSP1 increased these markers. The combination of DUSP1 overexpression with low-concentration corticosteroid treatment resulted in more potent suppression of inflammation than high-concentration corticosteroid treatment alone. The profiled transcriptomes provide insights into the pathophysiology of tubulointerstitial injury in kidney diseases and may reveal a potential therapeutic biomarker.

Blockade of STAT3 signaling alleviates the progression of acute kidney injury to chronic kidney disease through antiapoptosis.

Signal transducer and activator of transcription 3 (STAT3) is a pivotal mediator of IL-6-type cytokine signaling. However, the roles of its full-length and truncated isoforms in acute kidney injury (AKI) and its transition to chronic kidney disease (CKD) remain elusive. Herein, the role of STAT3 isoforms in the AKI-to-CKD transition was characterized using an ischemia-reperfusion injury (IRI) mouse model. The STAT3 inhibitor Stattic was administered to C57BL/6 mice 3 h before IRI. Intrarenal cytokine expression was quantified using real-time PCR and FACS. The effect of Stattic on human tubular epithelial cells cultured under hypoxic conditions was also evaluated. Phosphorylated (p)STAT3 isoforms were detected by Western blot analysis. Stattic treatment attenuated IRI-induced tubular damage and inflammatory cytokine/chemokine expression while decreasing macrophage infiltration and fibrosis in mouse unilateral IRI and unilateral ureteral obstruction models. Similarly, in vitro STAT3 inhibition downregulated fibrosis and apoptosis in 72-h hypoxia-induced human tubular epithelial cells and reduced pSTAT3α-mediated inflammation. Moreover, pSTAT3 expression was increased in human acute tubular necrosis and CKD tissues. STAT3 activation is associated with IRI progression, and STAT3α may be a significant contributor. Hence, STAT3 may affect the AKI-to-CKD transition, suggesting a novel strategy for AKI management with STAT3 inhibitors.NEW & NOTEWORTHY We found that IRI increased expression of STAT3 in murine kidneys, along with inflammation markers. Through the investigation of the role of STAT3 in the AKI-to-CKD transition mechanism using mouse unilateral IRI and unilateral ureteral obstruction models and 24- or 72-h hypoxic induction of primary cultured human tubular epithelial cells, we found that STAT3 could affect the AKI-to-CKD transition. We also observed different degrees of expression in STAT3 isoforms in these processes.

Clinical Significances of Anti-Collagen Type I and Type III Antibodies in Antibody-Mediated Rejection.

It is important to determine the clinical significance of non-human leukocyte antigen (HLA) antibodies and their association with antibody-mediated rejection (ABMR) of kidney allografts. We collected post-transplant sera from 68 ABMR patients, 67 T-cell mediated rejection (TCMR) patients, and 83 control subjects without rejection, and determined the titers of 39 non-HLA antibodies including antibodies for angiotensin II receptor type I and MICA. We compared all these non-HLA antibody titers among the study groups. Then, we investigated their association with the risk of death-censored graft failure in ABMR cases. Among the antibodies evaluated, anti-collagen type I (p = 0.001) and type III (p < 0.001) antibody titers were significantly higher in ABMR cases than in both TCMR cases and no-rejection controls. Both anti-collagen type I [per 1 standard deviation (SD), adjusted odds ratio (OR), 11.72 (2.73-76.30)] and type III [per 1 SD, adjusted OR, 6.22 (1.91-31.75)] antibodies were significantly associated with the presence of ABMR. Among ABMR cases, a higher level of anti-collagen type I [per 1 SD, adjusted hazard ratio (HR), 1.90 (1.32-2.75)] or type III per 1 SD, [adjusted HR, 1.57 (1.15-2.16)] antibody was associated with a higher risk of death-censored graft failure. In conclusion, post-transplant anti-collagen type I and type III antibodies may be novel non-HLA antibodies related to ABMR of kidney allografts.

Role of the IL-33/ST2 pathway in renal allograft rejection.

The interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) pathway modulates immune response and inflammation, associated with allograft dysfunction and rejection. We hypothesized that IL-33/ST2 is a marker of renal allograft rejection and IL-33/ST2 expression may differ according to rejection type. IL-33/ST2 expression was measured in sera and kidney tissues from recipients with acute antibody-mediated rejection (AAMR), acute cell-mediated rejection (ACMR), chronic antibody-mediated rejection (CAMR), and healthy controls. The soluble ST2 and IL-33/ST2 expression levels were higher in participants with all three rejection types than in controls. Although the expression levels in recipients with AAMR and ACMR were significantly higher than those with CAMR, there was no significant difference between the expression levels in AAMR and ACMR. Although IL-33, IL-8, and fibronectin expression were significantly increased after the addition of the recipients' serum in primary cultured human renal proximal tubular epithelial cells, the levels decreased after treatment with an anti-ST2 antibody. Furthermore, the anti-ST2 antibody specifically suppressed the upregulation of the mixed lymphocyte reaction. Boyden chamber assays demonstrated that anti-ST2 antibody abrogated chemotaxis induced by recombinant IL-33. Thus, IL-33 and ST2 are potent mediators of rejection. Treatment with an anti-ST2 antibody ameliorates rejection and could be a potential therapeutic strategy for renal allograft rejection.

ADABase: A Multimodal Dataset for Cognitive Load Estimation.

Driver monitoring systems play an important role in lower to mid-level autonomous vehicles. Our work focuses on the detection of cognitive load as a component of driver-state estimation to improve traffic safety. By inducing single and dual-task workloads of increasing intensity on 51 subjects, while continuously measuring signals from multiple modalities, based on physiological measurements such as ECG, EDA, EMG, PPG, respiration rate, skin temperature and eye tracker data, as well as behavioral measurements such as action units extracted from facial videos, performance metrics like reaction time and subjective feedback using questionnaires, we create ADABase (Autonomous Driving Cognitive Load Assessment Database) As a reference method to induce cognitive load onto subjects, we use the well-established n-back test, in addition to our novel simulator-based k-drive test, motivated by real-world semi-autonomously vehicles. We extract expert features of all measurements and find significant changes in multiple modalities. Ultimately we train and evaluate machine learning algorithms using single and multimodal inputs to distinguish cognitive load levels. We carefully evaluate model behavior and study feature importance. In summary, we introduce a novel cognitive load test, create a cognitive load database, validate changes using statistical tests, introduce novel classification and regression tasks for machine learning and train and evaluate machine learning models.

Comprehensive metabolomic profiling in early IgA nephropathy patients reveals urine glycine as a prognostic biomarker.

Identification of a urinary metabolite biomarker with diagnostic or prognostic significance for early immunoglobulin A nephropathy (IgAN) is needed. We performed nuclear magnetic resonance-based metabolomic profiling and identified 26 metabolites in urine samples. We collected urine samples from 201, 77, 47, 36 and 136 patients with IgAN, patients with membranous nephropathy, patients with minimal change disease, patients with lupus nephritis and healthy controls, respectively. We determined whether a metabolite level is associated with the prognosis of IgAN through Cox regression and continuous net reclassification improvement (cNRI). Finally, in vitro experiments with human kidney tubular epithelial cells (hTECs) were performed for experimental validation. As the results, the urinary glycine level was higher in the IgAN group than the control groups. A higher urinary glycine level was associated with lower risk of eGFR 30% decline in IgAN patients. The addition of glycine to a predictive model including clinicopathologic information significantly improved the predictive power for the prognosis of IgAN [cNRI 0.72 (0.28-0.82)]. In hTECs, the addition of glycine ameliorated inflammatory signals induced by tumour necrosis factor-α. Our study demonstrates that urinary glycine may have diagnostic and prognostic value for IgAN and indicates that urinary glycine is a protective biomarker for IgAN.

NK1.1- natural killer T cells upregulate interleukin-17 expression in experimental lupus nephritis.

Interleukin (IL)-17-secreting invariant natural killer T (NKT) cells are involved in several inflammatory diseases. However, their role in lupus nephritis (LN) has not been fully characterized. Samples from patients with LN or glomerulonephritis and healthy controls were obtained, and elevated IL-17+ NKT cell numbers and IL-17 expression were observed in blood cells and kidneys, respectively, in patients with LN. Comparison of a mouse model of experimental autoimmune LN with the parental strain (NKT-deficient B6.CD1d-/- mice) revealed improved proteinuria, disease severity, and histopathology and decreased levels of chemokine (C-X-C motif) ligand 16 and T cell receptor-α variable 14 expression. Spleens and kidneys of B6.CD1d-/- mice also showed downregulation of inflammatory markers and IL-17. In coculture with renal mesangial and NKT cells, inflammatory markers and IL-17 were upregulated following α-galactosylceramide treatment and downregulated after treatment with IL-17-blocking antibodies. This was most prominent with killer cell lectin-like receptor subfamily B member 1 C (NK1.1)- NKT cells. Thus, IL-17 is upregulated in LN. Activation of NKT cells regulates IL-17-related immune responses systemically and in the kidneys, primarily via NK1.1- NKT cells. IL-17-secreting NK1.1- NKT cells could serve as diagnostic and therapeutic targets for LN.NEW & NOTEWORTHY This study makes a significant contribution to the literature because our results indicate that IL-17 is upregulated in lupus nephritis and that natural killer T (NKT) cells are involved in its pathogenesis. Activation of NKT cells regulates IL-17-related immune responses, both systemically and in the kidney, and this mainly involves NK1.1- NKT cells. Furthermore, IL-17-secreting NK1.1- NKT cells could serve as a diagnostic and therapeutic target for lupus nephritis.

Fumarate modulates phospholipase A2 receptor autoimmunity-induced podocyte injury in membranous nephropathy.

Downstream mechanisms that lead to podocyte injury following phospholipase A2 receptor (PLA2R) autoimmunity remain elusive. To help define this we compared urinary metabolomic profiles of patients with PLA2R-associated membranous nephropathy (MN) at the time of kidney biopsy with those of patients with minimal change disease (MCD) and to healthy individuals. Among the metabolites differentially expressed in patients with PLA2R-associated MN compared to healthy individuals, fumarate was the only significant differentially expressed metabolite in PLA2R-associated MN compared to MCD [fold-difference vs. healthy controls and vs. MCD: 1.76 and 1.60, respectively]. High urinary fumarate levels could predict the composite outcome of PLA2R-associated MN. Fumarate hydratase, which hydrolyzes fumarate, colocalized with podocalyxin, and its expression was lower in glomerular sections from patients with PLA2R-associated MN than in those from healthy individuals, patients with non-PLA2R-associated MN or MCD. Podocytes stimulated with IgG purified from serum with a high anti-PLA2R titer (MN-IgG) decreased expression of fumarate hydratase and increased fumarate levels. These changes were coupled to alterations in the expression of molecules involved in the phenotypic profile of podocytes (WT1, ZO-1, Snail, and fibronectin), an increase in albumin flux across the podocyte layer and the production of reactive oxygen species in podocytes. However, overexpression of fumarate hydratase ameliorated these alterations. Furthermore, knockdown of fumarate hydratase exhibited synergistic effects with MN-IgG treatment. Thus, fumarate may promote changes in the phenotypic profiles of podocytes after the development of PLA2R autoimmunity. These findings suggest that fumarate could serve as a potential target for the treatment of PLA2R-associated MN.

cMet agonistic antibody prevents acute kidney injury to chronic kidney disease transition by suppressing Smurf1 and activating Smad7.

We aimed to investigate the role of cMet agonistic antibody (cMet Ab) in preventing kidney fibrosis during acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Additionally, we explored the effect of cMet Ab on TGF-ß1/Smad pathway during the pathogenesis of kidney fibrosis. A unilateral ischemia-reperfusion injury (UIRI) mouse model was established to induce AKI-to-CKD transition. Furthermore, we incubated human proximal tubular epithelial cells (hPTECs) under hypoxic conditions as in vitro model of kidney fibrosis. We analyzed the soluble plasma cMet level in patients with AKI requiring dialysis. Patients who did not recover kidney function and progressed to CKD presented a higher increase in the cMet level. The kidneys of mice treated with cMet Ab showed fewer contractions and weighed more than the controls. The mice in the cMet Ab-treated group showed reduced fibrosis and significantly decreased expression of fibronectin and α-smooth muscle actin. cMet Ab treatment decreased inflammatory markers (MCP-1, TNF-α, and IL-1ß) expression, reduced Smurf1 and Smad2/3 level, and increased Smad7 expressions. cMet Ab treatment increased cMet expression and reduced the hypoxia-induced increase in collagen-1 and ICAM-1 expression, thereby reducing apoptosis in the in vitro cell model. After cMet Ab treatment, hypoxia-induced expression of Smurf1, Smad2/3, and TGF-ß1 was reduced, and suppressed Smad7 was activated. Down-regulation of Smurf1 resulted in suppression of hypoxia-induced fibronectin expression, whereas treatment with cMet Ab showed synergistic effects. cMet Ab can successfully prevent fibrosis response in UIRI models of kidney fibrosis by decreasing inflammatory response and inhibiting the TGF-ß1/Smad pathway.

Krüppel-like factor 15 is a key suppressor of podocyte fibrosis under rotational force-driven pressure.

Krüppel-like factor 15 (KLF15) is a well-known transcription factor associated with podocyte injury and fibrosis. Recently, hypertensive nephropathy was discovered to be closely related to podocyte injury and fibrosis. However, methods to stimulate hypertension in vitro are lacking. Here, we constructed an in vitro model mimicking hypertension using a rotational force device to identify the role of KLF15 in fibrosis due to mechanically induced hypertensive injury. First, we found that KLF15 expression was decreased in patients with hypertensive nephropathy. Then, an in vitro study of hypertension due to rotational force was conducted, and an increase in fibrosis markers and decrease in KLF15 levels were determined after application of 4 mmHg pressure in primary cultured human podocytes. KLF15 and tight junction protein levels increased with retinoic acid treatment. siRNA-mediated inhibition of KLF15 exacerbated pressure-induced fibrosis injury, and KLF15 expression after treatment with angiotensin II was similar to that observed after treatment with the blood pressure modeling device. Furthermore, the reduced KLF15 levels after mechanical pressure application were restored after the administration of an antihypertensive drug. KLF15 expression was also low in vivo. We confirmed the protective role of KLF15 in fibrosis using a mechanically induced in vitro model of hypertensive injury.

Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury.

Chemokine receptor 5 (CCR5) is a pivotal regulator of macrophage trafficking in the kidneys in response to an inflammatory cascade. We investigated the role of CCR5 in experimental ischaemic-reperfusion injury (IRI) pathogenesis. To establish IRI, we clamped the bilateral renal artery pedicle for 30 min and then reperfused the kidney. We performed adoptive transfer of lipopolysaccharide (LPS)-treated RAW 264.7 macrophages following macrophage depletion in mice. B6.CCR5-/- mice showed less severe IRI based on tubular epithelial cell apoptosis than did wild-type mice. CXCR3 expression in CD11b+ cells and inducible nitric oxide synthase levels were more attenuated in B6.CCR5-/- mice. B6.CCR5-/- mice showed increased arginase-1 and CD206 expression. Macrophage-depleted wild-type mice showed more injury than B6.CCR5-/- mice after M1 macrophage transfer. Adoptive transfer of LPS-treated RAW 264.7 macrophages reversed the protection against IRI in wild-type, but not B6.CCR5-/- mice. Upon knocking out CCR5 in macrophages, migration of bone marrow-derived macrophages from wild-type mice towards primary tubular epithelial cells with recombinant CCR5 increased. Phospho-CCR5 expression in renal tissues of patients with acute tubular necrosis was increased, showing a positive correlation with tubular inflammation. In conclusion, CCR5 deficiency favours M2 macrophage activation, and blocking CCR5 might aid in treating acute kidney injury.

Urinary cMet as a prognostic marker in immunoglobulin A nephropathy.

The prediction of prognosis in patients with immunoglobulin A nephropathy (IgAN) is challenging. We investigated the correlation between urinary cMet (ucMet) levels and clinical parameters and examined the effects of cMet agonistic antibody (cMet Ab) in an in vitro IgAN model. Patients diagnosed with IgAN (n = 194) were divided into three groups representing undetectable (Group 1), below-median (Group 2) and above-median (Group 3) levels of ucMet/creatinine (ucMet/Cr). Stained kidney biopsy samples were graded according to cMet intensity. Primary-cultured human mesangial cells were stimulated with recombinant tumour necrosis factor (TNF)-α and treated with cMet Ab. Our results showed that ucMet/Cr levels positively correlated with proteinuria (P < .001). During the follow-up, patients in Group 3 showed a significantly lower probability of complete remission (CR; uPCr < 300 mg/g) than those in groups 1 and 2, after adjusting for blood pressure, estimated glomerular filtration rate, and proteinuria, which influence clinical prognosis (HR 0.60, P = .038); moreover, ucMet/Cr levels were also associated with glomerular cMet expression. After TNF-α treatment, the proliferation of mesangial cells and increased interleukin-8 and intercellular adhesion molecule-1 expression were markedly reduced by cMet Ab in vitro. In conclusion, ucMet/Cr levels significantly correlated with proteinuria, glomerular cMet expression, and the probability of CR. Further, cMet Ab treatment alleviated the inflammation and proliferation of mesangial cells. Hence, ucMet could serve as a clinically significant marker for treating IgAN.

cMet agonistic antibody attenuates apoptosis in ischaemia-reperfusion-induced kidney injury.

Acute kidney injury (AKI) is a very common complication with high morbidity and mortality rates and no fundamental treatment. In this study, we investigated whether the hepatocyte growth factor (HGF)/cMet pathway is associated with the development of AKI and how the administration of a cMet agonistic antibody (Ab) affects an AKI model. In the analysis using human blood samples, cMet and HGF levels were found to be significantly increased in the AKI group, regardless of underlying renal function. The administration of a cMet agonistic Ab improved the functional and histological changes after bilateral ischaemia-reperfusion injury. TUNEL-positive cells and Bax/Bcl-2 ratio were also reduced by cMet agonistic Ab treatment. In addition, cMet agonistic Ab treatment significantly increased the levels of PI3K, Akt and mTOR. Furthermore, after 24 hours of hypoxia induction in human proximal tubular epithelial cells, treatment with the cMet agonistic Ab also showed dose-dependent antiapoptotic effects similar to those of the recombinant HGF treatment. Even when the HGF axis was blocked with a HGF-blocking Ab, the cMet agonistic Ab showed an independent dose-dependent antiapoptotic effect. In conclusion, cMet expression is associated with the occurrence of AKI. cMet agonistic Ab treatment attenuates the severity of AKI through the PI3K/Akt/mTOR pathway and improves apoptosis. cMet agonistic Ab may have important significance for the treatment of AKI.

RNA-Seq profiling of microdissected glomeruli identifies potential biomarkers for human IgA nephropathy.

Few studies have examined gene expression changes occurring in the glomeruli of IgA nephropathy (IgAN) using a sensitive transcriptomic profiling method such as RNA sequencing (RNA-Seq). We collected glomeruli from biopsy specimens from patients with IgAN with relatively preserved kidney function (estimated glomerular filtration rate ≥ 60 mL·min-1·1.73 m-2 and urine protein-to-creatinine ratio < 3 g/g) and from normal kidney cortexes by hand microdissection and performed RNA-Seq. Differentially expressed genes were identified, and gene ontology term annotation and pathway analysis were performed. Immunohistochemical labeling and primary mesangial cell cultures were performed to confirm the findings of RNA-Seq analysis. Fourteen patients with IgAN and ten controls were included in this study. Glomerulus-specific genes were highly abundant. Principal component analysis showed clear separation between the IgAN and control groups. There were 2,497 differentially expressed genes, of which 1,380 were upregulated and 1,117 were downregulated (false discovery rate < 0.01). The enriched gene ontology terms included motility/migration, protein/vesicle transport, and immune system, and kinase binding was the molecular function overrepresented in IgAN. B cell signaling, chemokine signal transduction, and Fcγ receptor-mediated phagocytosis were the canonical pathways overrepresented. In vitro experiments confirmed that spleen tyrosine kinase (SYK), reported as upregulated in the IgAN transcriptome, was also upregulated in glomeruli from an independent set of patients with IgAN and that treatment with patient-derived IgA1 increased the expression of SYK in mesangial cells. In conclusion, transcriptomic profiling of the IgAN glomerulus provides insights in the intraglomerular pathophysiology of IgAN before it reaches profound kidney dysfunction. SYK may have a pathogenetic role in IgAN.