bluuebunny/crossref_metadata_2025_split huggingface dataset:
481-P: KCNMA1 Transcriptional Regulation by Nrf2 in Diabetic Vessels
Authors: LEE HON-CHI , SUN XIAOJING , WANG XIAO-LI , LU TONG 
Published: June 2019

KCNMA1 encodes the large conductance Ca2+-activated K+ channel pore-forming subunits (BK-α), a major ionic mechanism that regulates vascular smooth muscle physiology and is impaired in high fat diet-induced (HFD) diabetic mice. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor that regulates a wide variety of cellular proteins by binding to the antioxidant response elements (AREs). We found that the promoter region of KCNMA1 contains multiple AREs, but whether Nrf2 regulates KCNMA1 expression is unknown. In this study, we hypothesize that Nrf2 is a transcriptional regulator of KCNMA1 and the reduced Nrf2 activity in diabetes contributes to BK channelopathy. Using videomicroscopy, patch clamp recordings, molecular and cellular approaches, we found that BK channel activity and BK channel-mediated vasodilation were diminished in the coronary arteries of mice after 4 months on HFD. This was accompanied by significant reductions in BK-α and Nrf2 protein expression. In cultured coronary artery smooth muscle cells (CASMCs), adenoviral transduction with Nrf2 (50 MOI) resulted in an increase of BK-α mRNA by 10-fold and BK-α protein expression by 2-fold, while a 70% knockdown of Nrf2 by shRNA produced a 30% downregulation of BK-α protein levels. Luciferase reporter assays showed robust luciferase signals in cells 48 h after co-transfection with Nrf2 vectors and reporter vectors containing a segment of the KCNMA1 promoter region with a predicted ARE-Nrf2 binding site, but not those with empty vectors or KCNMA1 reporter vectors with the ARE-Nrf2 binding site deleted. Most importantly, treatment with dimethyl fumarate (a Nrf2 activator) increased BK-α mRNA and protein expression in cultured CASMCs, and rescued BK channel function and BK channel-mediated vasodilation in coronary arteries of HFD mice. Our results indicate that Nrf2 transcriptionally regulates KCNMA1 expression in CASMCs and has a therapeutic potential in the treatment of BK channelopathy and vasculopathy in diabetes.

Disclosure
H. Lee: None. X. Sun: None. X. Wang: None. T. Lu: None.

Funding
American Diabetes Association (1-18-IBS-210 to H-C.L.); National Institutes of Health (R01HL74180)
490-P: Administration of AAV-Containing Glyoxalase-I Attenuated Nephropathy in Rats with Chronic Diabetes
Authors: BIDASEE KESHORE , ALOMAR FADHEL AHMED 
Published: June 2019

Supra-physiologic flux/production of methylglyoxal (MG) has emerged as an underlying cause of nephropathy in chronic type 1 diabetes mellitus (T1DM). This increased MG arises in part from a reduction in expression of its detoxifying enzyme, glyoxalase-I (Glo-I). Adeno-associated virus (AAV)-mediated gene transfer has materialized as a promising strategy to increase expression of deficit proteins in disease states. Here we compared the efficacies of AAV2/9 containing Glo-I driven by two promoters, endothelin-1 (AAV2/9-Endo-Glo-I) and cytomegalovirus (AAV2/9-CMV-GloI) to attenuate nephropathy in T1DM rat. After eight weeks of T1DM, urine production increased 4-fold, urine norepinephrine increased 2.5-fold, serum creatinine increased 4-fold, creatinine clearance decreased 4-fold, BUN increased 5-fold, and proteinuria increased 8-fold. Renal Glo-I protein was 50% lower, MG was 2.5-fold higher, and extravasation of albumin from peritubular and glomerular capillaries and fibrosis were prominent. TNF-α and NF-κB (p65) phosphorylation were increased 3-4 folds. A single bolus injection of AAV/2/9 Glo-I and AAV2/9-Glo-I (1.7×1012 viron particles/kg) ten days after T1DM onset blunted increases in water consumption and urine assayed eight weeks later. AAV2/9-Glo-I also attenuated increases in norepinephrine, creatinine, BUN, proteinuria, albumin extravasation from renal microvessels, fibrosis, MG, TNF-α, NF-κB, activation and increased creatinine clearance rate. The magnitudes of the effects were higher in AAV2/9-Endo-Glo-I injected rats in part to higher expression of Glo-I in tubules and reduced expression of the MG-synthesizing enzyme vascular adhesion protein-1 within glomeruli. These new data demonstrate that intravenous administration of AAV2/9-containing Glo-I after the onset of T1DM attenuated renal dysfunction in rats via multiple mechanisms involving reductions in sympatho-excitation, inflammation, microvascular leakage and fibrosis.

Disclosure
K. Bidasee: None. F.A. Alomar: None.

Funding
National Institutes of Health (P30GM103335); University of Nebraska Medical Center (GTRP 1053); UNeMed
494-P: Whole-Kidney 3D Imaging and Image Analysis Enables Detailed Quantification of Diabetic Nephropathy Features in the Uninephrectomized db/db Mouse Model
Authors: FINK LISBETH N. , SEMBACH FREDERIKKE E. , ROOSTALU URMAS , JOHANSEN THEA , SKYTTE JACOB LERCKE , SECHER THOMAS , FOSGERAU KELD , VRANG NIELS , JELSING JACOB , HECKSHER-SØRENSEN JACOB , PEDERSEN TANJA X. 
Published: June 2019

Diabetic nephropathy (DN) is a serious complication of diabetes and the leading cause of end-stage renal disease. To reproducibly test novel therapies, there is a need for precise quantification of DN pathology in rodent models. We compared the accelerated development of DN in male and female uninephrectomized db/db mice using advanced imaging techniques. Unilateral nephrectomy (UNx) was performed in 7-8 weeks old male and female db/db mice. Sham-operated db/+ mice served as controls. Kidneys were preserved for histology and stereology. In a separate study, UNx was performed in 18 weeks old male db/db mice. These mice and control db/+ mice were injected with lectin-594 prior to termination to visualize glomerular morphology by 3D light sheet microscopy. All mice were terminated at 24 weeks of age. Male and female db/db UNx mice showed comparable increases in albuminuria. Total glomerular volume was markedly higher in male and female db/db UNx mice compared to db/+ controls (mean males; 4.42 vs. 2.71 mm3; p&lt;0.001; females; 5.04 vs. 2.69 mm3; p&lt;0.001). Tubulointerstitial collagen III immuno-staining was significantly increased only in female db/db UNx mice compared to db/+ mice. In contrast, total glomerular collagen IV levels were increased in male db/db UNx mice compared to db/+ controls (mean 1.30 vs. 0.80 mm3; p&lt;0.01), and female db/db UNx mice displayed increased glomerular collagen IV compared to male db/db UNx mice (mean 1.80 vs. 1.30 mm3; p&lt;0.05). 3D imaging at 24 weeks following UNx at 18 weeks of age confirmed expansion in glomerular volume (median db/+ 96.000 vs. db/db 145.000 µm3). Moreover, a marked increase was seen in cortical lectin-594 staining, indicating glomerular leakage in db/db UNx mice.
In conclusion, male and female db/db UNx mice displayed comparable features of DN and both constitute valid models for DN. The use of whole-kidney 3D imaging provides a novel avenue for assessment of individual glomerular volume and permeability.

Disclosure
L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. F.E. Sembach: Consultant; Self; Novo Nordisk A/S. Employee; Self; Gubra. U. Roostalu: Employee; Self; Gubra. T. Johansen: Employee; Self; Gubra. J.L. Skytte: Employee; Self; Gubra. T. Secher: Employee; Self; Gubra. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Gubra. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. K. Fosgerau: Stock/Shareholder; Self; Gubra. N. Vrang: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. J. Jelsing: Stock/Shareholder; Self; Gubra. J. Hecksher-Sørensen: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. T.X. Pedersen: Employee; Self; Gubra.
497-P: Fyn-Dependent Tyrosine Phosphorylation of Tgm2 Modulates p53-Tgm2-p62 Complex in Renal Proximal Tubular Epithelial Cells
Authors: YAMADA EIJIRO , OKADA SHUICHI , BASTIE CLAIRE C. , YAMADA MASANOBU 
Published: June 2019

Diabetic kidney disease (DKD) is one of the major diabetic complications, the leading cause of the end stage renal disease (ESRD). Recently autophagy, which is also implicated in diabetes and insulin resistance, was shown to regulate DKD. Previously, we reported that Fyn functions as a muscle mass regulator by suppressing autophagy but it is still not known whether Fyn regulates autophagy in kidney and functions as a pathogenesis of DKD. Recently, we demonstrated that over-expressing of Fyn in HK2 cells as an in vitro cell model of renal proximal tubular epithelial cells inhibits autophagy. SiRNA mediating knockdown of Fyn increased autophagy demonstrating Fyn has important role for autophagy in HK2 cells. Phospho-proteomic analysis revealed that Fyn phosphorylates Transglutaminase 2 (Tgm2), a known autophagic inhibitor through Beclin1, on Y369 and Y617. More recently, we demonstrated that Fyn dependent phosphorylation of Tgm2 could regulate autophagy in HK2 cells.
While it has been reported that Tgm2 could cross-link Beclin1 to inhibits its activity, no cross-linking of Beclin1 was seen in Tgm2 expressing HK2 cells, indicating there could be another mechanism of Fyn-Tgm2 regulating autophagy in HK2 cells. Recently, Tgm2 was shown to make a complex with p53 and p62, known autophagy regulator/substrate to degrade p53 in autophagosome in cancer cells. Interestingly, we found that p53 expression was decreased in Tgm2 knock-downed HK2 cells and increased in Tgm2 expressing HK2 cells. More importantly co-immunoprecipitation assay showed that Tgm2-Y369,617F which could not be phosphorylated by Fyn bind to p53 more than WT and dissociated from p62. Taken together, these data suggest that Fyn phosphorylates Tgm2 on Y369, 617 to modulate p53-Tgm2-p62 complex in the renal tubulus cells leading to a pathogenesis of DKD.

Disclosure
E. Yamada: None. S. Okada: None. C.C. Bastie: None. M. Yamada: None.
50-OR: Identifying Risk of Blindness in Patients with Diabetic Retinopathy
Authors: NGUYEN QUAN DONG , HALL REBECCA , KELLY SCOTT P. , LUM FLORA , STOILOV IVAYLO , ABBASS IBRAHIM M. , TO TU MY , ABOLIAN ANNA , GARMO VINCENT 
Published: June 2019

Purpose: Using real-world data from the American Academy of Ophthalmology (Academy) IRIS® Registry (Intelligent Research in Sight), this retrospective study sought to identify risk factors for developing sustained blindness (SB) in eyes with newly diagnosed diabetic retinopathy (DR).
Methods: IRIS Registry data were reviewed to identify eyes newly diagnosed with DR between Jan 1, 2013 and Dec 31, 2017 (index date). Included eyes had ≥ 2 visual acuity (VA) readings of 20/40 or better before or ≤ 3 months post index; eyes with evidence of prior DR, retinal disease or retinal treatment were excluded. Patients were followed until the development of SB (defined as ≥ 2 VA readings of 20/200 or worse ≥ 3 months apart), or most recent VA reading. Kaplan-Meier analyses estimated time to SB by DR severity; multivariable Cox proportional hazards models assessed the impact of baseline characteristics and ocular conditions during follow-up on the risk of developing SB.
Results: A total of 53,535 eyes were analyzed, 678 of which developed SB during follow-up. At index, 49.4% of eyes were diagnosed with mild non-proliferative DR (NPDR), 9.1% moderate NPDR, 1.5% severe NPDR, 10.5% proliferative DR (PDR), and 29.5% unspecified DR. Relative to eyes with mild NPDR at index, eyes diagnosed with severe NPDR and PDR were 3.6 and 4.0 times more likely to develop SB within 2 years, respectively. Factors associated with increased risk of developing SB included black (hazard ratio (HR), 1.24; P &lt; 0.0001) and Asian race (1.27; P &lt; 0.011), female sex (HR 1.08; P = 0.01), and enrollment in Medicare or Medicaid compared with private plans (HR 0.70-0.91; P &lt; 0.05), in addition to diabetic macular edema and retinal vein occlusion during follow-up (HR 1.96-4.91; P &lt; 0.0001).
Conclusions: Our model identified that race, sex, and other characteristics can predict likelihood of developing SB in patients with DR. Close monitoring or early intervention should be considered for patients who are black or Asian race, female, or have developed other ocular conditions.

Disclosure
Q. Nguyen: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Genentech, Inc., Regeneron Pharmaceuticals. R. Hall: Employee; Self; American Academy of Ophthalmology. S.P. Kelly: None. F. Lum: None. I. Stoilov: Employee; Self; Genentech, Inc. I.M. Abbass: Employee; Self; Genentech, Inc. T. To: Employee; Self; Genentech, Inc. Stock/Shareholder; Self; Genentech, Inc. A. Abolian: Employee; Self; Genentech, Inc. V. Garmo: Employee; Self; Genentech, Inc.
500-P: The Performance of Four Different Exosome Isolation Methods for Urinary Exosomal microRNA Profiling in Patients with Diabetic Kidney Disease
Authors: LEE KIYOUNG , LEE DAEHO , PARK IE BYUNG 
Published: June 2019

Both exosome and microRNAs (miRNAs) in biological fluids can be good biomarkers for pathologic conditions. Thus, urinary exosome miRNAs would be reasonable non-invasive tools for biomarker development for diabetic kidney disease. To be eligible for exosome miRNA biomarker development, the performance of exosome isolation methods for miRNA profiling need to be tested. In the present study, using urine samples from patients with type 2 diabetes, we compared four different exosome isolation methods for their performance in urine exosome isolation and detectable urinary exosome miRNA profiles. Four methods compared in the present study were ultracentrifugation (UC), size exclusion chromatography using qEV column (qEV), ExoQuick-TC (ExoQuick), and Amicon tube 100K (Amicon). Isolated exosomes were subjected to next generation sequencing (NGS) to profile miRNAs. The counts, RNA concentrations, and protein marker expressions of isolated urine exosome were variable between the extraction methods, with the highest exosome count with the qEV method. The number of miRNAs detected (read count ≥1) in 100 mL urine each were 314, 174, 91, and 130 from UC, qEV, ExoQuicK, and Amiconmethods, respectivley. And, 57 miRNAs were detected with all 4 methods concordantly. There were correlations in the levels of exosomal miRNAs between samples from UC, qEV, and ExoQuick methods, whereas exosome miRNA profiles from Amicon sample showed only a weak correlation with those from other methods. Our results suggest that exosome isolation by using UC or qEV method may be a better option among different isolation methods to profile urinary exosome miRNAs in diabetic kidney disease. However, ultrafiltration method using Amicon tube produced a different profile of miRNAs with other methods. Further studies are needed to characterize both exosome and their genetic materials from different extraction sources.

Disclosure
K. Lee: None. D. Lee: None. I. Park: None.
505-P: ROCK2 Regulates TGF-Beta-Induced Expression of CTGF and Profibrotic Genes via NF-kappa B and Cytoskeleton Dynamics in the Mesangial Cells
Authors: NAGAI YOSUKE , KAWANAMI DAIJI , MATOBA KEIICHIRO , TAKEDA YUSUKE , UTSUNOMIYA KAZUNORI 
Published: June 2019

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic kidney disease. Accumulating evidence shows profibrotic mediators, including connective tissue growth factor (CTGF), are key regulators of glomerulosclerosis under diabetic conditions. However, the interactions of Rho-kinase and these profibrotic mediators in the development of renal dysfunction remain unclear. Glomeruli isolated from type 2 diabetic db/db mice demonstrated increased gene expression of transforming growth factor β (TGF-β) and its downstream profibrotic mediators. Chemical inhibition of Rho-kinase suppressed the expression of profibrotic mediators in both isolated glomeruli and cultured mesangial cells. An investigation of mechanisms underlying this observation revealed activated Rho-kinase functions through the phosphorylation of JNK and Erk, and the nuclear translocation of nuclear factor-kappa B (NF-κB). Immunostaining studies demonstrated that a Rho-kinase inhibitor and Cytochalasin D, an inhibitor of actin polymerization, attenuated TGF-β-mediated nuclear translocation of NF-κB, indicating that Rho-kinase-mediated actin reorganization regulates NF-κB activation. Knockdown by small interfering RNA against two Rho-kinase isoforms, ROCK1 and ROCK2, showed that ROCK2 but not ROCK1 controls this fibrotic machinery. SLx-2119, a chemical ROCK2 inhibitor, also modulated the expression of TGF-β-induced profibrotic mediators via a similar mechanism by downregulating ROCK2 activity. A further in vivo study showed that ROCK2 activity in the renal cortex of db/db mice was elevated compared to control db/m mice. These observations indicate that ROCK2 is a key player in the development of diabetic renal injury. Glomerular ROCK2 may be a potential therapeutic target for the treatment of diabetic kidney disease.

Disclosure
Y. Nagai: None. D. Kawanami: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. K. Matoba: Research Support; Self; Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Sanofi, Shionogi & Co., Ltd., Takeda Pharmaceutical Company Limited. Y. Takeda: None. K. Utsunomiya: None.
512-P: Sexual Differences in Progression of Diabetic Nephropathy in over 26 Type 1 Diabetic Mice
Authors: WANG WANNING , CAI LU 
Published: June 2019

As a transgenic type 1 diabetic model, OVE26 mice develop diabetic nephropathy (DN) with the feature close to advanced DN in human, which makes it a valuable rodent model to study DN. Nevertheless, sexual differences in renal lesions, especially dynamic progression with different ages, remain undefined in OVE26 mice. To explore these issues, male and female OVE26 and wild type nondiabetic FVB mice at 4-, 12-, 24 - and 36-weeks old were used (N=6). Urine albumin-to-creatinine ratio (UACR) was detected to evaluate renal function. Renal histopathology and immunochemical and Western blotting protein expression of inflammation, tubular epithelial-myofibroblast transdifferentiation and fibrosis markers were examined. In FVB mice, blood glucose, triglyceride, UACR and podocytes numbers were constant during aging and comparable in male and female. Age-dependent slightly increase of glomerular mesangial matrix, inflammatory cells infiltration and renal fibrosis were observed in FVB mice. Compared with the age- and sex- matched FVB mice, male OVE26 mice developed hyperglycemia, mesangial matrix accumulation and increased trend of triglyceride at 4-week-old and albuminuria, glomerular enlargement, and upregulations of TGF-β1, α-SMA at 12 weeks old. Significant loss of podocytes, tubular dilation, collagen accumulation, increased trend of macrophage and T lymphocytes infiltrations, and increased protein levels of TNF-α, ICAM and fibronectin in kidney were observed in 24-week old male OVE26 mice. Compared with age-matched male OVE26 mice, female OVE26 mice had significant hypertriglyceridemia, worse renal function and kidney lesions at older age, including higher UACR and sustained loss of podocytes at age of 24 weeks; more inflammatory cells infiltration and renal fibrosis at age of 36 weeks. Our findings suggest the sexual difference in the progression of DN in OVE26 mice to provide evidence for researchers to properly select the age and sex of OVE26 mice in the future type 1 DN study.

Disclosure
W. Wang: None. L. Cai: None.
514-P: Group 2 Innate Lymphoid Cells Promote Renal Fibrosis through the Production of Th2 Cytokines in Diabetic Kidney Disease
Authors: QIN LU DAN , LIU CUIPING , ZHOU LUPING , GAO CHENLIN , ZHANG TING , GUO MAN , HUANG WEI , JIANG ZONGZHE , DING JING YA , XU YONG 
Published: June 2019

Aim: To explore the role of Group 2 innate lymphoid cells (ILC2s) in the pathogenesis of renal fibrosis in diabetic kidney disease (DKD).
Methods: The proportion of ILC2s and ILC2s-producted Th2 cytokines (IL-4, IL-5, IL-13) in the peripheral blood of normal control subjects (NC) or patients with type 2 diabetes (DM), early diabetic kidney disease (DKD1), or late diabetic kidney disease (DKD2) were analyzed using flow cytometry and ELISA. Renal tubular epithelial cells (HK-2) were stimulated with IL-4 or IL-13 in the presence or absence of high glucose and the expression and release of transforming growth factor-β1 (TGF-β1) and fibronectin (FN) was analyzed using qPCR and ELISA.
Results: The proportion of ILC2s and the level of IL-4, IL-5, and IL-13 were significantly increased with the severity of DKD (P&lt;0.05). The expression and release of TGF-β1 and FN were significantly upregulated by IL-4 or IL-13 in HK-2 cells (P&lt;0.05). Compared with high glucose stimulation alone, treatment with IL-4 or IL-13 combined with high glucose significantly stimulated HK-2 cells to increase expression of FN and TGF-β1 (P&lt;0.05).
Conclusions: ILC2s may promote the development and progression of diabetic kidney disease by secreting Th2 cytokines (IL-4 and IL-13) and promoting renal fibrosis.

Disclosure
C. Liu: None. L. Zhou: None. C. Gao: None. T. Zhang: None. M. Guo: None. W. Huang: None. Z. Jiang: None. J.Y. Ding: None. Y. Xu: None.
530-P: Early Insulin Initiation in T2DM Is Associated with Prevention of eGFR Decline and Lower Insulin Dose Requirements
Authors: KESAVADEV JOTHYDEV , WARRIER ROHIT , RAMACHANDRAN LAKSHMY , SHANKAR ARUN , DAVID ASHWIN , AJAI NEETHU A. , SANAL GEETHU , THAMPIRAJ KAVITHA , KRISHNAN GOPIKA , JOTHYDEV SUNITHA 
Published: June 2019

Early initiation of insulin therapy often remains contentious due to various patient and physician-related barriers. Insulin is delayed by several years despite high A1c and is the foremost reason for costly future complications. In addition, the problem of early aggressive use of insulin depleting drugs causes β-cell fatigue leading to "brittle diabetes" and higher insulin dose requirements. In this observational cohort study, a comparison has been made between early insulin initiators (EII) (within 7-8 years of diabetes onset) and late insulin initiators (LII) (&gt;8 years of onset), in terms of progression of CKD (eGFR decline), if any. A total of 200 subjects in each arm were matched at baseline for T2DM duration (16.78±4.32 year), BP, BMI, HbA1c and presence of co-morbid illnesses. Both groups were closely followed-up through our Diabetes Tele Management System based health care model that have previously shown to be successful in safely achieving glycemic targets compared to conventional models of care. A paired t test was done for analyzing the eGFR decline within each arm. EII arm required a significantly lower total daily dose of insulin (TDD) (average of 9 units/day) to maintain glycemic control throughout than the LII arm (average of 30 units/day). The decline in eGFR was significant in the LII arm, whereas in the EII arm, eGFR was maintained throughout. Change in A1c was also significant.

Disclosure
J. Kesavadev: None. R. Warrier: None. L. Ramachandran: None. A. Shankar: None. A. David: None. N.A. Ajai: None. G. Sanal: None. K. Thampiraj: None. G. Krishnan: None. S. Jothydev: None.