Comparison between characteristics of immunoglobulin M nephropathy and other glomerular diseases.

Background: Immunoglobulin M (IgM) nephropathy (IgMN) is characterized by the IgM deposition in the kidney's mesangium. We assessed the impact of electron-dense deposits (EDDs) on IgMN and compared it to other kidney diseases. Methods: We enrolled 63 adult patients with IgMN who underwent renal biopsy from May 2003 to June 2017. We compared clinicopathological features of IgMN based on EDD presence; compared characteristics to 91 minimal change disease (MCD), 103 focal segmental glomerulosclerosis (FSGS), and 469 immunoglobulin A nephropathy (IgAN) patients. Renal events were defined as a >50% decrease in estimated glomerular filtration rate (eGFR), eGFR of <15 mL/min/1.73 m2, or end-stage renal disease development. Results: IgMN patients with EDDs had increased mesangial cellularity, matrix accumulation, prominent immunofluorescent staining, and more diffuse podocyte effacement than those without EDD. Clinical characteristics and renal outcomes did not differ significantly based on EDD presence. During 79.5 ± 58.8 months of follow-up, renal events developed in 46.2% and 46.0% of IgMN cases with and without EDD. IgMN (46.0%) and FSGS cases (40.8%) had similar frequencies of renal events and higher frequency than MCD (18.7%) or IgAN cases (26.4%). IgMN cases had more severe manifestations than MCD and IgAN; higher blood pressure, lower proteinuria, and eGFR levels at biopsy than MCD cases; higher blood pressure, proteinuria, frequency of acute kidney injury, and lower eGFR levels. Conclusion: Clinical characteristics of IgMN did not differ based on EDD presence. Therefore, IgMN should be defined based on IF findings. IgMN shared clinical characteristics with FSGS but had more severe than MCD and IgAN.

Efficacy of Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles in Ameliorating Cisplatin Nephrotoxicity, as Modeled Using Three-Dimensional, Gravity-Driven, Two-Layer Tubule-on-a-Chip (3D-MOTIVE Chip).

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) are known to have a therapeutic effect on nephrotoxicity. As animal models require significant time and resources to evaluate drug effects, there is a need for a new experimental technique that can accurately predict drug effects in humans. We evaluated the therapeutic effect of MSC-derived EVs in cisplatin nephrotoxicity using a three-dimensional, gravity-driven, two-layer tubule-on-a-chip (3D-MOTIVE chip). In the 3D-MOTIVE chip, 10 µM cisplatin decreased the number of attached cells compared to the vehicle. Conversely, annexin V and reactive oxygen species (ROS) were increased. Cell viability was increased 2.8-fold and 2.5-fold after treatment with EVs at 4 and 8 µg/mL, respectively, compared to the cisplatin-induced nephrotoxicity group. Cell attachment was increased 2.25-fold by treatment with 4 µg/mL EVs and 2.02-fold by 8 µg/mL EVs. Annexin V and ROS levels were decreased compared to those in the cisplatin-induced nephrotoxicity group. There were no significant differences in annexin V and ROS levels according to EV concentration. In sum, we created a cisplatin-induced nephrotoxicity model on a 3D-MOTIVE chip and found that MSC-derived EVs could restore cell viability. Thus, MSC-derived EVs may have the potential to ameliorate cisplatin-induced nephrotoxicity.

Outcomes of minimal change disease without nephrotic range proteinuria.

Minimal change disease (MCD) is characterized by edema and nephrotic range proteinuria (NS). However, the fate of MCD without nephrotic proteinuria requires elucidation. We retrospectively reviewed 79 adults diagnosed with primary MCD at their initial renal biopsy at a tertiary hospital between May 2003 and June 2017. Clinicopathologic features were compared between patients with and without NS. The frequency of flaring to nephrotic proteinuria and renal outcomes were assessed during follow-up. There were 20 and 59 patients in the Non-NS and NS groups, respectively. The Non-NS group had a lower frequency of acute kidney injury (AKI) during the follow-up period [5.0% vs. 59.3%, p <0.001]. The response rate to steroid treatment was 100% in the Non-NS group and 92.3% in the NS group (p = 1.000). Except for one patient, the Non-NS group was treated with steroids when their proteinuria increased to a nephrotic level. There were no differences in the frequency of the first relapse or the number of relapses among patients with initial remission from nephrotic range proteinuria. At the final visit, the complete remission rate was 73.4%. The estimated glomerular filtration rate during follow-up was significantly better in the NS group than the Non-NS group, given the higher rates of AKI at renal biopsy. The rates of renal events, end-stage renal disease, and mortality did not differ between the groups. Adult MCD patients with nephrotic and non-nephrotic range proteinuria showed similar outcomes. Accordingly, this population must be carefully managed, regardless of the amount of proteinuria at renal biopsy.

SIRT-1 regulates TGF-ß-induced dermal fibroblast migration via modulation of Cyr61 expression.

SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-ß further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-ß or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-ß or RSV. TGF-ß, RSV, or SIRT1 overexpression enhanced ß-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/ß-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

PPARγ agonist rosiglitazone inhibits migration and invasion by downregulating Cyr61 in rheumatoid arthritis fibroblast-like synoviocytes.

AIM: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have anti-inflammatory properties that reduce inflammatory cytokine production in rheumatoid arthritis (RA). Cysteine-rich angiogenic inducer 61 (Cyr61) is associated with diseases related to chronic inflammation. The aim of this study was to investigate the mechanisms underlying the effects of PPARγ agonists on tumor necrosis factor (TNF)-α-induced fibroblast-like synoviocyte (FLS) invasion and migration, as well as Cyr61 production, in RA-FLS. METHODS: FLS were cultured with TNF-α and Cyr61 in the presence or absence of PPARγ agonists. Matrix metalloproteinase and Cyr61 expression levels in RA-FLS and culture supernatants were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The migration and invasion phenotypes of RA-FLS were determined by wound healing and Boyden chamber assays. RESULTS: Cyr61 protein was expressed in RA-FLS, and its intracellular expression and secretion levels were increased by TNF-α. Moreover, Cyr61 directly promoted RA-FLS migration and invasion. Rosiglitazone (RSG) significantly decreased TNF-α-induced Cyr61 expression. RSG decreased TNF-α-induced nuclear factor (NF)-κB activation and inhibitor of κBα degradation. Furthermore, RSG inhibited TNF-α-induced RA-FLS migration and invasion and decreased Cyr61 treatment-induced RA-FLS invasion. Finally, blocking Cyr61 significantly attenuated TNF-α-induced migration. CONCLUSIONS: Our results demonstrate for the first time that PPARγ agonists may have beneficial effects on the migration and invasion of RA-FLS via the downregulation of Cyr61. Therefore, PPARγ agonists could be potential treatment targets for RA.

Agmatine modulates melanogenesis via MITF signaling pathway.

Agmatine contained in soybean is also found in Manaca, an anti-aging plant, inhabited in Amazon and induces vasodilation by the promotion of NO synthesis in blood vessel. However, the research of agmatine on melanin synthesis related to hair greying is lacking. The aim of this study was to investigate the melanogenic effect of agmatine via regulation of MITF signaling pathway in B16F1 cells. It was determined whether agmatine regulates melanin synthesis at cellular level in addition to the effect of agmatine on mushroom tyrosinase in vitro in the presence of different concentrations of agmatine. Furthermore, the effect of agmatine on the protein expressions of tyrosinase, TRP-1, TRP-2, BMP-4, BMP-6, C-KIT, p-p38, MITF and C-FOS were examined by western blot analysis. In addition, immunofluorescence staining was carried out to visualize the location of MITF expression in cell. Agmatine at 256µM or more increased melanin synthesis as well as tyrosinase activity. Moreover, whereas agmatine increased the expression levels of TRP-1, BMP-6, p-p38 and MITF, it reduced the expression level of BMP-4. It was also found that agmatine enhanced the expression level of MITF in nucleus. These results suggest that agmatine could induce melanin synthesis though the regulation of MITF transcription factor via BMP-6/p38 signaling pathway.

Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes.

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress (ERS), and triggers the unfolded protein response (UPR) that consequently reduces accumulation of unfolded proteins by increasing the quantity of ER chaperones. Calumenin, a Ca(2+)-binding protein with multiple EF hand motifs, which is located in the ER/SR, is highly expressed during the early developmental stage of the heart, similar to other ER-resident chaperones. The aim of this study was to investigate the functional role of calumenin during ERS in the heart. Like other chaperones (e.g., GRP94 and GRP78), calumenin expression was highly upregulated during ERS induced by 10 µg/ml tunicamycin, but attenuated in the presence of 500 µM PBA, the chemical chaperone in neonatal rat ventricular cardiomyocytes (NRVCs). Upon 7.5-fold overexpression of calumenin using a recombinant adenovirus system, the expression levels of ERS markers (GRP78, p-PERK, and p-elF2α) and ER-initiated apoptosis markers (CHOP and p-JNK) were reduced, whereas the survival protein BCL-2 was upregulated during ERS compared to the control. Evaluation of cell viability by TUNEL assay showed that apoptosis was also significantly reduced by calumenin overexpression in ERS-induced cells. Taken together, our results suggest that calumenin plays an essential role in the alleviation of ERS in neonatal rat cardiomyocytes.

CD26/DPP4 levels in peripheral blood and T cells in patients with type 2 diabetes mellitus.

CONTEXT: Dipeptidyl peptidase 4 (CD26/DPP4) is expressed on blood T cells and also circulates in a soluble form (sCD26/DPP4). OBJECTIVE: We aimed to evaluate blood T cell and circulating CD26/DPP4 and its association with metabolic parameters in patients with type 2 diabetes mellitus (T2DM). DESIGNS: We measured CD26/DPP4 expression (percentage of CD26(+) cells using flow cytometry) on CD4(+) and CD8(+) T cells, serum CD26/DPP4 level and activity, and various metabolic parameters in T2DM patients not on DPP4 inhibitor therapy (n = 148). Nondiabetic subjects (n = 50) were included as a control group. RESULTS: Compared with the healthy controls, CD26/DPP4 expression on CD4(+) T cells and CD8(+) T cells was higher in T2DM patients. Serum CD26/DPP4 levels and enzymatic activities were also higher in patients with T2DM than in the control group only when metformin and/or thiazolidinedione-treated T2DM patients were excluded; metformin and/or thiazolidinedione-treated T2DM patients had lower values compared with other T2DM patients. Various parameters in T2DM patients were related to CD26/DPP4 expression on the T cells (hemoglobin A1c), serum sCD26/DPP4 (hemoglobin A1c and insulin resistance assessed by updated homeostasis model assessment), and serum CD26/DPP4 activity (insulin resistance assessed by updated homeostasis model assessment, γ-glutamyl transferase, and alanine aminotransferase) by multivariate analyses. After active glucose control for 12 weeks in drug-naive T2DM patients (n = 50), CD26/DPP4 expression on blood T cells was significantly decreased. CONCLUSIONS: Our results suggest that the CD26/DPP4 level on blood T cells was associated with glucose control status in patients with T2DM.

AAV-mediated knock-down of HRC exacerbates transverse aorta constriction-induced heart failure.

BACKGROUND: Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca(2+) cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca(2+) homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca(2+) cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively. METHODOLOGY/PRINCIPAL FINDINGS: AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca(2+) cycling and increased activities of RyR2 and SERCA2 without change in SR Ca(2+) load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay. CONCLUSIONS/SIGNIFICANCE: Increased Ca(2+) leak and cytosolic Ca(2+) concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca(2+) cycling.

Melanocortins induce interleukin 6 gene expression and secretion through melanocortin receptors 2 and 5 in 3T3-L1 adipocytes.

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. alpha-Melanocyte-stimulating hormone (alpha-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and IkappaB kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the alpha-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, alpha-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that alpha-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.

E2F1 represses beta-catenin transcription and is antagonized by both pRB and CDK8.

The E2F1 transcription factor can promote proliferation or apoptosis when activated, and is a key downstream target of the retinoblastoma tumour suppressor protein (pRB). Here we show that E2F1 is a potent and specific inhibitor of beta-catenin/T-cell factor (TCF)-dependent transcription, and that this function contributes to E2F1-induced apoptosis. E2F1 deregulation suppresses beta-catenin activity in an adenomatous polyposis coli (APC)/glycogen synthase kinase-3 (GSK3)-independent manner, reducing the expression of key beta-catenin targets including c-MYC. This interaction explains why colorectal tumours, which depend on beta-catenin transcription for their abnormal proliferation, keep RB1 intact. Remarkably, E2F1 activity is also repressed by cyclin-dependent kinase-8 (CDK8), a colorectal oncoprotein. Elevated levels of CDK8 protect beta-catenin/TCF-dependent transcription from inhibition by E2F1. Thus, by retaining RB1 and amplifying CDK8, colorectal tumour cells select conditions that collectively suppress E2F1 and enhance the activity of beta-catenin.

Transcriptional regulation of the Drosophila caudal homeobox gene by bHLH-PAS proteins.

Caudal-related homeobox transcription factors are involved in the definition of the anteroposterior axis and intestinal development. Recent reports indicate that dysregulation of CDX1 and CDX2, the human homologues of Drosophila caudal, are associated with several types of cancer. Very little is known, however, about the regulatory mechanisms that direct the caudal-related homeobox gene expression. In this study, we have identified the binding sites for bHLH-PAS proteins, referred to as CNS midline element (CME), in the 5'-flanking region of the Drosophila caudal gene. Analyses using transgenic flies carrying a caudal-lacZ fusion gene bearing a wild-type or mutant CME indicate that the CME sites are required for caudal gene expression in vivo. We also determined that the caudal promoter activity can be regulated by Trachealess (Trh)/Tango (Tgo) bHLH-PAS proteins, via the CME sites. Our results suggest that the Drosophila caudal gene is a target of the Trh/Tgo bHLH-PAS proteins.

G1 cyclin-dependent kinases are insufficient to reverse dE2F2-mediated repression.

Here we show that the cell cycle defects of dE2F1-depleted cells depend on the cooperative effects of dE2F2 and DACAPO (DAP), an inhibitor of Cyclin E/cyclin-dependent kinase 2 (CycE/cdk2). The different properties of cells lacking dE2F1/dE2F2 and dE2F1/DAP lead to the surprising observation that dE2F2-mediated repression differs from retinoblastoma family protein 1 (RBF1) inhibition of dE2F1, and is resistant to both CycE/cdk2 and Cyclin D/cyclin-dependent kinase 4 (CycD/cdk4). This resistance occurs even though dE2F2/RBF1 complexes are disrupted by CycE/cdk2, and may explain why dE2F2 is so potent in the absence of de2f1. The implication of these results is that cells containing dE2F2 require dE2F1 to either prevent, or reverse, dE2F-mediated repression.

The caudal homeodomain protein activates Drosophila E2F gene expression.

The Drosophila caudal homeobox gene is required for definition of the anteroposterior axis and for gut development, and CDX1 and CDX2, human homologs, play crucial roles in the regulation of cell proliferation and differentiation in the intestine. Most studies have indicated tumor suppressor functions of Cdx2, with inhibition of proliferation, while the effects of Cdx1 are more controversial. The influence of Drosophila Caudal on cell proliferation is unknown. In this study, we found three potential Caudal binding sequences in the 5'-flanking region of the Drosophila E2F (DE2F) gene and showed by transient transfection assays that they are involved in Caudal transactivation of the dE2F gene promoter. Analyses with transgenic flies carrying an E2F-lacZ fusion gene, with and without mutation in the Caudal binding site, indicated that the Caudal binding sites are required for expression of dE2F in living flies. Caudal-induced E2F expression was also confirmed with a GAL4-UAS system in living flies. In addition, ectopic expression of Caudal with heat-shock promotion induced melanotic tumors in larvae. These results suggest that Caudal is involved in regulation of proliferation through transactivation of the E2F gene in Drosophila.

Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity.

Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.