Supplemental Methods

Bioinformatic analysis, IHG-1 cDNA assembly and plasmid constructs

Database searching and alignments were performed at the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool Algorithm (BLAST).44 SSH analysis16-18 yielded a 198bp cDNA fragment which we have called IHG-1 (Genbank accession no. AF110136). A sequence that encoded a complete putative open-reading frame of 894bp was generated by EST walking, as previously described,45 this cDNA was generated from human mesangial cells by RT-PCR, sequenced and found to be identical to UniGene cluster HS353090. A mammalian cell expression vector encoding a carboxy-terminus V5 tagged IHG-1 protein was generated in pcDNA6 V5-His (Invitrogen). Vectors encoding FLAG-tagged Smad3, and the promoter-reporter 3TP-lux were kindly provided by Dr Rik Derynck, University of California, San Francisco, and Dr Joan Massague, Sloan Kettering Institute, New York, respectively.

Northern blot analysis and real-time (Taqman) PCR

Northern blots were performed as described previously.17 Multi-tissue northern used was FirstChoice Human Blot 1 (Ambion). Total RNA from tissue and cultured cells was isolated using TRIzol (Invitrogen) and cDNA-generated by reverse transcription using random hexamers.. Transcript levels were determined by quantitative real-time Taqman PCR using a PerkinElmer 7700 analyzer, as described previously.46 Probe and primer sequences for IHG-1 (within the open reading frame) were sense primer: AAAGATGGCAGTGACCCGG and antisense primer: CATCCCCGATGATATCGCA and competitor probe: CAGGACAAAGCCAGTGCCCTTGCA. All other primer/probes used were predeveloped Taqman reagents (Perkin-Elmer).

Quantitative PCR analysis of cDNA from human DN kidney biopsies

Renal biopsies were micro-dissected to yield the tubule and interstitium rich fraction. cDNA, which was generated from total RNA isolated from the microdissected renal biopsies from DN patients (n=13), normal pre-transplantation kidney (isolated under conditions of cold ischemia) (n=4) and normal histologically verified non-affected regions of tumor nephrectomies (n=4) and is held at the European Renal cDNA renal bank (ECRB).47 IHG-1 transcript levels were measured by Taqman real-time PCR and are expressed relative to GAPDH levels. These human kidney biopsy segments not required for diagnostic evaluation were obtained from patients after informed consent and with the approval of their local ethical committees.44

Assessment of the pattern of IHG-1 expression in human diabetic nephropathy by insitu hybridization (ISH)

Diagnostic kidney biopsy specimens were studied after obtaining patient consent and local ethical approval. Biopsies from patients with diabetic nephropathy and controls (unaffected kidney from tumor-nephrectomies) were studied. ISH was performed as described previously.48 Biotin-labeled human IHG-1 probes (GIBCO BRL) were used. The specificity of the reaction was confirmed by demonstrating the disappearance of hybridization signal with addition of RNAse (Sigma) and by the use of a sense probe. Southwestern analysis was performed as described previously.49 Briefly, dewaxed sections (4-m) were incubated with 5 mmol/L levamisole (Sigma) to quench endogenous alkaline phosphatase and fixed with 0.2% p-formaldehyde. Following digestion with 0.5% pepsin A (433 U/mg; Sigma) in 1 N HCl for 30 minutes at 37°C they were incubated with 0.1 mg/mL DNAse I for 30 minutes at 30°C. Labeled probe (100 pmol/L) was added to the sections overnight at 37°C in a humidified box. Following washing and blocking, sections were incubated with an antidigoxigenin antibody conjugated to alkaline phosphatase (1:250, Roche) overnight at 4°C. Alkaline phosphatase was visualized with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP; Dako). Sections were dehydrated in ethanol series and mounted in Canadian balsam (Polysciences Inc.).

Unilateral Ureteric obstruction (UUO)

The UUO model used was carried out under license and with the approval of the UCD animal research ethical committee.UUO was performed as described previously.50 Following visual confirmation of obstructive nephropathy, cold (4ºC) isotonic saline was perfused via the abdominal aorta to clear the kidneys of blood. The kidneys were then excised, weighed, snap frozen in liquid nitrogen, and stored at -80ºC prior to RNA extraction and cDNA synthesis. In some cases one-half of the kidney was immersed in 10% buffered formalin for 24 hours prior to inclusion in paraffin for subsequent Gomori’s Trichrome staining, carried out according to the manufacturers protocol (Polysciences Europe GmBH). Contralateral non-obstructed kidneys (NL) were used as no-fibrosis internal controls.

Cell culture, transfections and reporter gene assay

Mv 1 Lu, HEK 293T and HeLa cells were obtained from ATCC. Cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 2 mM glutamine, and 1% penicillin-streptomycin. Human proximal tubular cells (HK-2) (ATCC) were maintained in DMEM-F12containing 10% fetal bovine serum, 2 mM glutamine, and 1% penicillin-streptomycin. Stably transfected cell lines were generated using HK-2 cells following transfection with Fugene6 (Invitrogen) as described by the manufacturer with plasmid pIRESpuro3 and pIRESpuro3-IHG-1-V5 (Clontech). Stable transfectants were selected for and maintained with 5ng/ml puromycin (Sigma). Transient transfection of cells with plasmids was performed using the Nucleofector kit (Amaxa) or Fugene 6 reagent (Roche) according to the manufacturer’s instructions; 24h later they were washed twice in PBS and cultured for a further 24h in serum free medium. The cells were stimulated with TGF-1 as detailed in the figure legends. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega).

Recombinant lentivirus production and cell transduction

HEK 293T cells were transfected with (pCMVΔR8.9), (pMD.2G) and LLCIEP or IHG-1-V5-LLCIEP using a calcium phosphate transfection kit (Invitrogen) exactly as directed by manufacturer. Forty-eight hours after transfection, the virus enriched supernatant was removed and filtered through a 0.45μm low protein binding syringe filter (Millipore), snap frozen and stored at -80oC. HK-2 cells were infected with a 1 in 20 dilution of thawed virus in medium. Optimal transduction of HK-2 cells was determined with serial dilutions of virus followed by western blot analysis.

siRNA inhibition of IHG-1 production

Optimization of transfection of HK-2 cells was carried out as described previously.51 Transfection of siRNAs targeted against IHG-1 (Dharmacon, SMARTpool reagent) was achieved using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 40 nM of each siRNA was incubated with lipofectamine 2000 for 20 min at room temperature and transfected into HK-2 cell in suspension at 37°C for 20 min. Cells were then plated onto 24 well plates and maintained in the presence of transfection mix for a further 2 h prior to changing the medium.

SDS-PAGE and western blotting

Cells were washed twice in ice-cold PBS, scraped and pelleted. Whole cell lysates were prepared in RIPA buffer.46 Protein concentration was determined using the Bradford assay. Western blotting was performed as previously reported.46 Antibodies were obtained from the following suppliers;Smad3 (Zymed Laboratories), Fibronectin (BD Biosciences), V5 (Invitrogen), -actin (Sigma) and CTGF (Santa Cruz Biotechnology). Anti phospho-Smad3 antibody was a gift from Dr Ed Leof, Mayo Clinic College of Medicine, Rochester. A rabbit IHG-1 antibody to the peptide SKFEYVRDFEADDTC was generated by Sigma-Genosys Ltd (UK). Densitometry was performed using UN-SCAN-IT.

Statistical analysis

Results are expressed as mean ± SEM and were analyzedfor significance by unpaired t-test. Statistical significance was set at p ≤0.05.