Role of Superoxide Dismutase in Murine Diabetic Nephropathy

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, MD^1*

Hiroki Fujita, MD, PhD^1*

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, MD^4,5

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.: [phone]; fax: [phone]. E-mail: [e-mail]
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

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