**2.2 Analytical procedures**

#### **Renal Function Assessment and Blood Pressure Measurement**

Serum creatinine (Scr) and Blood urea nitrogen (BUN) were measured using a Beckman Cx4 analyser (Fullerton, CA, USA), respectively.

24h Urinary protein concentrations were determined by the Bradford method, adapted to a microtiter plate assay. Coomassie reagent (USB, Cleveland, OH) was added to the diluted urine samples. After 10 minutes, the absorbance at 595-nm wavelength was read on ELX800 microplate reader (Bio-Tek Instruments, VT). The protein concentrations were calculated by reference to bovine serum albumin (Sigma) standards.

Systolic blood pressure was recorded by tail plethysmography using the BP2000 blood pressure analysis system (Visitech Systems, Inc., Apex, NC) in conscious rats at baseline and every 2 weeks throughout the experimental time course.

Molecular Mechanisms of Nephro-Protective

expressed as the percentage of mean±SEM.

**4. Characterization of monoclonal anti-TGF–β antibody** 

terminated with 1.3 M H2SO4. The absorption at 492 nm was measured.

GTCAGT AACTGGAC-5'.

relevant figures.

**4.1 Statistical analysis** 

Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure 179

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

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

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

The reactivity of the produced monoclonal antibodies with Urine TGF–β was screened by enzyme-linked immunosorbent assay (ELISA) using kit produced by Section living creature technique limited company of Hangzhou, China (NO,13409007) The sample solution (40 µl) was incubated with the monoclonal anti- TGF–β antibody (40 µl) at room temperature for 1 h in an TGF–β–transferrin attached microplate. After washing with phosphate-buffered saline (PBS) containing 0.05% Tween 20, 0.1 ml of peroxidase-labelled goat F(ab')2 fragment to mouse IgG(Fc) was added into the microplate, followed by incubation at room temperature for 1 h. After washing with PBS containing 0.05% Tween 20, 0.2 ml of ophenylenediamine hydrochloride (1 mg/ml) containing 0.0124% H2O2 was added to the microplate, and then incubated at room temperature for 30 min. The reaction was

Data obtained from this study are expressed as the means ± SEM. Statistical analyses were performed using GraphPad Prism 3.0 (GraphPad Software, Inc., San Diego, CA). Differences in blood pressure, serum creatinine, blood urea nitrogen, 24h urine protein and Urine TGF– β at different time points (weeks 0 to 8) within the groups, and differences of Ang II and

#### **2.3 Immunohistochemical analysis**

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

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 preincubated with NF-κb.

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 component from 70 to 123°.
