Blood samples were collected at the end of the metabolic cage study. fatty acids . Accordingly, we investigated the effects on DN of the lack of eNOS and a high fat (HF) diet that is adjusted to diets consumed in Western societies. We here show that these two factors additively increase TF expression in diabetic kidneys, and that, Nipradilol when combined, they dramatically exacerbate DN. Administration of anti-TF antibody corrected the increase in the expression of inflammatory genes in the kidney of diabetic mice by lack of eNOS and HF, indicating that TF contributes to the severity of DN in diabetic eNOS-/- mice fed HF. Methods Animals Animal experiments were conducted in accordance with the guideline of IACUC at UNC at Chapel Hill. Male eNOS -/- mice () and their WT littermates, backcrossed at least Rabbit Polyclonal to PPP4R1L 10 times to C57BL/6J, were used in this study. Diabetes was induced in 46-month-old male mice by intraperitoneal injection of streptozotocin (STZ, 40 mg/kg, Sigma) for 5 consecutive days after 4-hour fasting as previously described . Animals were maintained without insulin treatment for 6 months. Mice having plasma glucose concentration equal to or greater than 300 mg/dl throughout the study after STZ injection were defined as diabetic and included in the study. Both diabetic and non-diabetic control mice (injected with buffer only) were randomly divided and fed either normal chow (NC, 14% calories from fat) or a HF diet (42% calories from fat, TD88137, Harlan Teklad). At 3 and 6 months after inducing diabetes, the individual mice were placed in metabolic cages. Body weight, food and water intake, and urine volume were measured at 24 and 48 hrs. Blood samples were collected at the end of the metabolic cage study. Mice were then sacrificed for further analysis. For testing short-term effect of diabetes, 34 month-old male Nipradilol eNOS-/- and WT mice were injected with STZ or buffer only, fed with NC or HF, and analyzed 5 weeks later. To test whether TF exacerbates DN a single dose at 100 g/mouse of an anti-mouse TF neutralizing antibody AF3178 (R&D) was administered intraperitoneally 5 weeks after STZ injection. Mice were sacrificed 4 days later for further analysis. [Our data show that 100 g/mouse of anti-TF neutralizing antibody inhibits 70 %70 % of the TF activity in the kidney without bleeding complication.] BP and glomerular filtration rate (GFR) measurements BP was measured by the computerized tail-cuff method for 6 days . All mice were trained 10 cycles of measurements on the BP apparatus before 30 measurements were made each day. GFR was estimated by measuring the plasma and urinary creatinine using LC-MS/MS . Biochemical measurements Urinary albumin was determined using Albuwell-M kits (Exocell Inc.). Urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) was measured with an ELISA kit (The Japan Institute for The Control of Aging). Plasma CML was measured with an ELISA kit (CycLex Co.). Plasma thrombin-antithrombin (TAT) complexes were measured with the AssayMax TAT complexes ELISA kit (Assaypro Co.). For PT and aPTT determination, blood was collected in glass tubes containing 3.8% trisodium citrate (1 vol citrate plus 9 vol blood), and PT and aPTT were measured using a thromboplastin reagent and aPTT reagent (Biomerieux Inc.), respectively [20, 21]. Kidney cortex (10 mg) was homogenized in 1ml PBS for measuring CML content  and TF-dependent procoagulant activity . Quantitative RT-PCR The kidney tissue was snap frozen in liquid nitrogen, and the RNA was extracted using Trizol (Life Technologies). Gene expression was quantified with TaqMan real-time quantitative RT-PCR (Applied Biosystems) with -actin as a reference gene . The primers and probes used are listed in the Online Appendix Table 1. Kidney morphometry and immunohistochemistry Cross paraffin sections of kidneys containing papilla (4 m thick) were stained with Periodic Acid-Schiff (PAS) and with Masson’s trichrome, and scanned using a NIKON Microphot-FCA 216567. Glomerulosclerosis was defined as synechiae formation with global obliteration of the capillary loops . Nipradilol The number of sclerosed glomeruli was expressed as a percentage of the total number of glomeruli. The mesangial matrix score was defined as the ratio of the mesangial matrix area divided by the glomerular tuft area [24, 25], and was measured using the Image J program. All of the glomeruli (90 to 110) in each section were measured. Tubulointerstitial fibrosis was scored by using 1015 fields of the cortex at a magnification of 4 with a scale of 0 to 4: 0, no fibrosis;.