Kim 1

Total RNA was extracted from kidney tissue with Trizol^® (Invitrogen). cDNA was synthesized using oligo d(T)₁₆ (Applied Biosystems) primers and the SuperScript III Reverse Transcriptase kit (Invitrogen 18080-044). Taqman Real-Time PCR was employed using the Roche Lightcycler 480 (Roche Applied Science) for the following genes: TLR2, TLR4, NLRP3, ASC, TNFα, IL6, IL18, IL1β, IL4, IL10, IFNγ, CXCL2, CCL2, CXCL10, iNOS, KIM1, MMP2, MMP9, TGFβ1, HDAC1-11, and GAPDH (Applied Biosystems). Results were normalized to GAPDH expression.

Total RNA was prepared from isolated kidney by using ultra-pure TRIzol reagent (GIBCO-BRL, Grand Island, NY, USA). The RNA (1 μg) was retro-transcripted using a specific kit (ROCHE, Monza, Italy). KIM-1 and GAPDH, specific TaqMan probes, were from Applied Biosystems (Applied Biosystems Inc., Foster City, CA, USA). Each sample was run in triplicate. The 2^–ΔΔCt method was used to quantify the relative expression and the comparative threshold cycle method was used to quantify fold increase (2^–ΔΔCt) compared to controls.

Total RNA was extracted from 10–20 mg frozen kidney tissue and isolated using the RNeasy mini kit (Qiagen, Venlo, the Netherlands) as previously described [10 (link), 22 (link)]. The RNA concentration and purity were determined using NanoDrop 1000 (NanoDrop Technologies, Wilmington, DE, USA). A total of 1 μg RNA was transcribed into complementary DNA using an iScript cDNA synthesis kit (Bio-Rad, Veenendaal, the Netherlands) using oligo-dT priming. mRNA abundance was measured using a CFX384 Touch real-time PCR detection system (Bio-Rad, Veenendaal, the Netherlands). The following primers were used for quantitative polymerase chain reaction: Tie2, NGAL, KIM-1, ICAM-1, VCAM-1, E-selectin, P-selectin, RhoA and Rac-1 (Applied Biosystems, Foster City, California, USA). ΔΔcT values were calculated and mRNA expression levels were normalized to Arbp abundance (Applied Biosystems, Foster City, California, USA).

The isolation of total RNA from the renal cortex or brown adipose tissue (BAT), cDNA synthesis and quantitative real-time PCR were performed as previously described [16 (link)]. TaqMan probes for type 3 collagen (Col3) (Product ID: Rn01437681), Cd68 (Rn01495634), interleukin-6 (Il6) (Rn01410330), C-C motif chemokine ligand 2 (Ccl2) (Rn00580555), Toll-like receptor 4 (Tlr4) (Rn00569848), kidney injury molecule-1 (Kim-1) (RN00597703) and uncoupling protein1 (Ucp1) (Rn00562126) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) [16 (link)]. The analytical data were normalized to the levels of 18 s (Rn03928990) mRNA expression as an internal control.

Kidney tissue sections stored in RNAlater were homogenized, and RNA was extracted using the ISOLATE II RNA Mini Kit as per manufacturer’s instructions (Bioline/Meridian Life Science, TN, USA). One microgram of RNA was reverse transcribed using the SensiFAST cDNA synthesis kit (Bioline/Meridian Life Science). cDNA amplification was performed in triplicate in volumes of 10 μL, consisting of cDNA, SensiFAST Probe No-ROX, and the relevant gene-specific primer/Taqman™ probes (HPRT1 – MM00446968_m1; IL-6 – MM00446190_m1; TNF-α – MM00443258_m1; IL-1β – MM00434228_m1; CCL2 – MM00441242_m1; CXCL2 – MM00436450_m1; KIM-1 – MM00506686_m1) (Thermo Fisher Scientific, MA, USA). Real-time polymerase chain reaction (RT-PCR) was performed on a Bio-Rad CFX384 machine – 95 °C for 10 mins, 95 °C for 30 sec (40 cycles), and 60 °C for 45 sec (40 cycles). The ∆∆Ct method was used to calculate expression fold changes normalized to HPRT1, with the 0.9% NaCl group utilized as the control.

Western blotting was performed using antibodies against CD38 (1:1000), β-actin (1:1000), Acetylated (Lys-413)-IDH2 (1:1000), IDH2 (1:1000), Acetylated (Lys-68)-SOD2 (1:1000), SOD2 (1:1000), Sirt3 (1:1000) and COX-IV (1:1000) as previously described [9 (link)]. Total RNA was isolated from the renal cortex, and cDNA synthesis and quantitative real-time PCR were performed. TaqMan probes for collagen III, CD38, Kim-1, IL-6 and TNF-α were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The data were normalized to the level of 18S mRNA, which was used as an internal control, as previously described [9 (link)].