Supplementary material for
Stromal cell–derived factor-1 is upregulated by dipeptidyl peptidase-4 inhibition and has protective roles in progressive diabetic nephropathy
Satoru Takashima, MD1*
Hiroki Fujita, MD, PhD1*
Hiromi Fujishima, MS1
Tatsunori Shimizu, MD1
Takehiro Sato, MD1
Tsukasa Morii, MD, PhD1
Katsushi Tsukiyama, MD, PhD2
Takuma Narita, MD, PhD1
Takamune Takahashi, MD, PhD3
Daniel J. Drucker, MD4,5
Yutaka Seino, MD, PhD6
Yuichiro Yamada, MD, PhD1
1 Division of Endocrinology, Metabolism and Geriatric Medicine, Akita University Graduate School of Medicine, Akita, Japan
2 Division of Metabolism and Clinical Nutrition Science, Akita University Graduate School of Medicine, Akita, Japan
3 Division of Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, TN, USA
4 Department of Medicine, University of Toronto, Toronto, Ontario, Canada
5 The Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada
6 Kansai Electric Power Medical Research Institute, Osaka, Japan
* These authors contributed equally to this work.
Correspondence: Hiroki Fujita, Division of Endocrinology, Metabolism and Geriatric Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. Tel.: +81 18 884 6769; fax: +81 18 884 6449. E-mail: hirofuji@gipc.akita-u.ac.jp
Running headline: SDF-1, DPP-4 inhibition, and Diabetic Nephropathy
MATERIALS AND METHODS
Measurement of blood, urine and physiological parameters
Blood glucose was measured after a 6-hour daytime fast using Glutestmint (Sanwa Chemistry, Nagoya, Aichi, Japan). Blood urea nitrogen (BUN), plasma creatinine, plasma total cholesterol, plasma triglycerides, urinary sodium, and urinary potassium were measured by an autoanalyzer (Fuji Dry-Chem 800 and 5500, Fuji Film, Tokyo, Japan). Urinary albumin excretion was determined on morning spot urine as described previously.1 Urinary excretion of sodium and potassium was expressed as a ratio to urinary creatinine. Systolic blood pressure was measured using a non-invasive tail cuff and pulse transducer system (BP-98A, Softron, Tokyo, Japan). GFR was determined by a single-bolus FITC-inulin injection and clearance method as described previously.2
Histologic analysis and immunohistochemistry
The kidneys were perfused via left ventricle with PBS followed by 4% paraformaldehyde in PBS, removed, and fixed in 4% paraformaldehyde in PBS for overnight at 4°C. Two m-thick paraffin sections were stained with PAS and Masson trichrome, and used for immunohistochemistry. The degree of glomerular mesangial expansion was assessed using a semi-quantitative score as described previously.3 Glomerular podocyte was stained by WT1 immunohistochemistry, and twenty cortical glomeruli were evaluated in each mouse. The podocyte number was calculated using the Weibel-Gomez method as reported previously.4, 5 The immunohistochemistry for MDA, SDF-1, TSP-1, and fibronectin was performed using rabbit anti-MDA polyclonal antibody (1:100, Alpha Diagnostic, San Antonio, TX), mouse anti-SDF-1 monoclonal antibody (1:100, R & D Systems), mouse anti-TSP-1 monoclonal antibody (1:100; Invitrogen, Camarillo, CA), and mouse anti-fibronectin monoclonal antibody (1:100; Thermo Scientific, Fremont, CA). The glomerular superoxide levels are assessed by dihydroethidium (DHE) histochemistry as described previously.6 The NO production in the glomeruli was evaluated by the fluorescent intensity of the DAF-2DA reaction as reported previously.7, 8 The fluorescent images were observed using confocal laser microscopy (LSM510; Carl Zeiss, Jena, Germany). The fluorescence intensity in twenty glomeruli in each mouse was semiquantified using Adobe Photoshop (version CS5; Adobe systems, San Jose, CA).
REFERENCES
1. Qi Z, Fujita H, Jin J, et al. Characterization of susceptibility of inbred mouse strains to diabetic nephropathy. Diabetes 2005; 54: 2628-2637.
2. Qi Z, Whitt I, Mehta A, et al. Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. Am J Physiol Renal Physiol 2004; 286: F590-596.
3. Fujita H, Fujishima H, Chida S, et al. Reduction of renal superoxide dismutase in progressive diabetic nephropathy. J Am Soc Nephrol 2009; 20: 1303-1313.
4. Tanabe K, Lanaspa MA, Kitagawa W, et al. Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse. Am J Physiol Renal Physiol 2012; 302: F1151-1160.
5. Nicholas SB, Basgen JM, Sinha S. Using stereologic techniques for podocyte counting in the mouse: shifting the paradigm. Am J Nephrol 2011; 33 Suppl 1: 1-7.
6. Fujita H, Morii T, Fujishima H, et al. The protective roles of GLP-1R signaling in diabetic nephropathy: possible mechanism and therapeutic potential. Kidney Int 2014; 85: 579-589.
7. Fujita H, Fujishima H, Takahashi K, et al. SOD1, but not SOD3, deficiency accelerates diabetic renal injury in C57BL/6-Ins2(Akita) diabetic mice. Metabolism 2012; 61: 1714-1724.
8. Satoh M, Fujimoto S, Haruna Y, et al. NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy. Am J Physiol Renal Physiol 2005; 288: F1144-1152.
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