**3. Real-time quantitative Polymerase Chain Reaction (PCR) for TNF**-**α, Ang II and AT1R**

To investigate the expression of TNF–α mRNA, Ang II and AT1R real-time PCR (BC living creature technique company, Shanghai, China) was performed with the Opticon real-time PCR machine (FX scientific research Inc. Shanghai, China). Briefly, total RNA was extracted from renal tissues. All of the RNA samples were treated with the RNase-free DNase I (GIBCO BRC Inc, Shanghai, China) before the RT-PCR. Real-time quantitative one-step RT-PCR assay was performed to quantify mRNA using real-time PCR machine (FX scientific research Inc. Shanghai, China). The primers used for real-time RT-PCR were as follows: TNF-a: forward 5'-CTCATTCCCGCTCGTGG-3' reverse 3'-CGTTTGGTGGTTCGTCTCC- 5';

Immunostaining of NF-κb (Sigma) in renal tissue sections was performed using the streptavidin–biotinylated peroxidase complex (SABC) method. The tissue specimens were divided into thin sections (4-µm thick) that were then deparaffinized. The sections were washed three times with distilled water for 5 min. The sections were treated with Protease K (Try box produced by BSD living creature technique company of Wuhan) in distilled water at 37°C for 15 min, and washed three times with PBS for 10 min. Endogenous peroxidase activity was blocked by incubating the sections with 0.3% H2O2 in methanol for 20 min at room temperature. The sections were washed three times with PBS for 5 min. The sections were incubated with 10% rabbit serum at 37°C for 60 min to reduce the non-specific background staining, and washed three times with PBS for 5 min. Then, the sections were incubated with a monoclonal anti- NF-κb antibody (7 µg/ml) dissolved in PBS containing 3% BSA and 0.1% NaN3 at 4°C overnight, and washed three times with PBS for 10 min; followed by incubation with a biotinylated rabbit antibody against mouse IgG+IgA+IgM (10 µg/ml) at 37°C for 40 min. The sections were washed three times with PBS for 5 min, and then incubated with peroxidase-labelled streptavidin at 37°C for 30 min. After washing three times with PBS for 10 min, the reaction was completed by the addition of diaminobenzidine–H2O2 solution for 15 min, and washed three times with distilled water

The primary anti- NF-κb antibody (1 : 100) was incubated with NF-κb (10 mg/ml) at 4°C overnight. After centrifuging the mixture at 10,000xg for 30 min, the supernatant was used as negative control for the primary antibody solution followed by the usual SABC method. There was no positive staining in the renal cortex when the primary antibody was pre-

The immunostaining of NF-κb was quantified using an image analyser IMS (FUDAN university of medical science portrait examination center) by evaluating the positively stained area of the sections under the same light intensity for microscopy. The intensity of colour component for red, green or blue was graded from 0 to 256°. Areas which showed intense brown color were extracted from the microscopic fields (number of fields for each tissue sample, six fields; magnification on the display: x300) under the following conditions; red component ranging from 104 to 158°, green component from 81 to 129°, and blue

**3. Real-time quantitative Polymerase Chain Reaction (PCR) for TNF**-**α, Ang II** 

To investigate the expression of TNF–α mRNA, Ang II and AT1R real-time PCR (BC living creature technique company, Shanghai, China) was performed with the Opticon real-time PCR machine (FX scientific research Inc. Shanghai, China). Briefly, total RNA was extracted from renal tissues. All of the RNA samples were treated with the RNase-free DNase I (GIBCO BRC Inc, Shanghai, China) before the RT-PCR. Real-time quantitative one-step RT-PCR assay was performed to quantify mRNA using real-time PCR machine (FX scientific research Inc. Shanghai, China). The primers used for real-time RT-PCR were as follows: TNF-a: forward 5'-CTCATTCCCGCTCGTGG-3' reverse 3'-CGTTTGGTGGTTCGTCTCC- 5';

for 5 min, then the slides were counter-stained with methylgreen.

**2.3 Immunohistochemical analysis** 

incubated with NF-κb.

component from 70 to 123°.

**and AT1R** 

AT1R: forward 5'-CTTGTTCCCTTTCCTTATC -3'reverse 3'-ACTCCACCTCACTGTCCA - 5'. Ang II : forward 5'- ACCTG CATGA GTGTT GATAGG-3' reverse 3'-ACTTCA ATATC GTCAGT AACTGGAC-5'.

Total RNA of osteoblasts was isolated by using TRIzol reagent (Invitrogen) and reverse transcription was performed follow manufacturer's manual(BioTNT, Shanghai, China). Quantitative real-time PCR, enabling the quantification of relative gene expression, was performed using SYBR green DNA binding fluorescent dye. 10 μL of QuantiTect TM SYBR Green PCR Master Mix, 4 μL of QuantiTect TM SYBR Green primer assay (osteocalcin, b-actin; all provided by BioTNT), 5 μL of RNase free water and 1 μL of cDNA (1 ng/μL) were used for one reaction. Quantitative real-time PCR was performed in triplicates with the following cycler program: 95°C 10 min, denaturation step: 95°C 15 s, annealing step: 60°C 15 s, elongation step: 72°C 30 s; dissociation: 95°C 15 s, 60°C 1min, 95°C 15 s, 40 cycles were performed in total. B-actin was taken as an endogenous standard and relative gene expression was determined using the ΔΔCt method. Gene expression was compared by setting control cultures to 1 (reference value) as indicated in the relevant figures.

Quantitative analyses of TNF, α, Ang II and AT1R expression were performed using a quantitative image analysis system (FR-2000,FR Science and technology Inc, Shanghai China). Because the pattern of expression of TNFα, Ang II and AT1R are diffuse in nature, the percentage of positive staining in the renal tissue was quantified under a ×20 power field of microscope. Briefly, up to 10 random areas of kidney with the early stage (media:intima ≥1) and advanced stage (media:intima <1) were chosen from each tissue section and examined. The examined area was outlined, the positive staining patterns were identified, and the percent positive area in the examined area was then measured. Data were expressed as the percentage of mean±SEM.
