Data supporting the regulation of FOXC2 in podocyte dysfunction

This data article shows the expression levels of specific podocyte injury markers and podocyte slit diaphragm protein nephrin in obese and lean Zucker rat glomeruli. It also contains information on the effect of the overexpression of transcription factor FOXC2 on the ratio of F- and G-actin and the expression level of ZO-1 in differentiated human podocytes. The article also shows data on the effect of treatments of differentiated podocytes with various factors associated with obesity and diabetes on the expression level of FOXC2. The detailed interpretation of these data and other aspects of podocyte injury mediated by upregulation of FOXC2 can be found in “Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility [1].


Value of the data
The obese Zucker rats show a trend towards upregulation of podocyte injury markers in glomeruli.
The obese Zucker rats with the highest level of proteinuria express least nephrin in glomeruli.
Overexpression of FOXC2 in differentiated human podocytes in vitro does not change the F-actin/Gactin ratio, or the expression level of the tight junction protein ZO-1.
Several obesity and diabetes-associated factors were found not to upregulate FOXC2 in differentiated human podocytes.

Data
Quantitative Western blotting reveals that podocyte injury markers active beta-catenin, desmin and fibronectin show a trend of upregulation in the glomeruli of 40 weeks old obese Zucker rats compared to lean controls ( Fig. 1A and B). Nephrin, the key protein of the interpodocyte slit diaphragm, shows a trend of downregulation in the glomeruli of obese rats ( Fig. 1C and D), with the most albuminuric rats expressing least nephrin (Fig. 1E). Exposure of differentiated human podocytes to factors associated with obesity, insulin resistance and type 2 diabetes did not increase the expression of FOXC2 as observed by quantitative RT-PCR for tumor necrosis factor-α (TNF-α) and transforming growth factor β (TGF-β) ( Fig. 2A and B), and by quantitative Western blotting for angiotensin II and a combination of glucose and palmitate ( Fig. 2C-F). The data also show that overexpression of FOXC2 in differentiated human podocytes by lentiviral transduction does not change the ratio of filamentous (F) actin and globular (G) actin ( Fig. 3A and B) or the expression level of the tight junction protein ZO-1 ( Fig. 4A and B).

Animal model and preparation of glomerular lysates
Obese (fa/fa) and lean (fa/ þ) Zucker rats (Crl:ZUC-Leprfa) were obtained from Charles River Laboratories (Sulzfeld, Germany). Blood glucose values were measured from tail vein samples using OneTouch Ultra glucometer (Lifescan, Milpitas, CA). The urinary albumin to creatinine ratio was determined from spot urine samples. Albumin was measured with rat albumin ELISA kit (CellTrend, Luckenwalde, Germany) and creatinine using CREA plus enzymatic assay (Roche, Basel, Switzerland) and Roche clinical chemistry analyzer ( Table 1). The experiments were approved by the National Animal Experiment Board. Glomerular fractions were isolated from 40 weeks old rat kidney cortices (E) Comparison of urine albumin/creatinine ratio to nephrin/actin ratio (C) in Zucker lean and obese rats reveals that the obese rats with highest albuminuria ( 415 mg/mg) express less nephrin in the glomeruli than the obese rats with low albuminuria ( o15 mg/mg). Data are mean 7 SD (n ¼3 for lean, n ¼2 for obese with low alb/cre, n ¼4 for obese with high alb/cre). * r 0.05.

(Invitrogen) and
IRDye 800 (LI-COR) anti-mouse, anti-rabbit, anti-guinea pig or anti-sheep IgGs. The signal was detected using an Odyssey Infrared Imager (LI-COR) and subsequently quantified using Odyssey software.
Treatment of cultured human podocytes with obesity-and diabetes-associated factors Conditionally immortalized human podocytes (AB8/13) were cultured as described in Datta et al. [1]. Differentiated podocytes were serum starved for 12 h in medium supplemented with 1% fetal bovine serum (FBS) and independently treated with 10 ng/ml TNF-α (R&D Systems) for 2-24 h, 4 ng/ ml TGF-β (R&D Systems) for 3 or 6 days, 1 μM angiotensin II (Sigma-Aldrich, St. Louis, MO, USA) for 24 h, or 20 mM glucose (Sigma-Aldrich) together with 200 μM palmitate (Sigma-Aldrich) for the last 7 days of the differentiation period. Sodium palmitate was conjugated with fatty acid-free low endotoxin BSA (Sigma-Aldrich) as described earlier [4]. Solvent only was used as a control for all treatments. Cells were either lysed and immunoblotted as described above or used for preparation of total RNA and quantitative RT-PCR as described below.

Total RNA preparation and quantitative RT-PCR
Total RNA was isolated, treated with DNase I and reverse transcribed into complementary DNA (cDNA). The quantitative PCR was performed using TaqMan gene expression assays (Applied Biosystems, Foster City, CA) for hFOXC2 (Assay ID: Hs00270951_s1) and hGAPDH (glyceraldehyde 3phosphate dehydrogen-ase; Assay ID: Hs99999905_m1) as in [5]. Measurements were performed in triplicate using an iCyclerIQ s (BIO-RAD, Hercules, CA, USA). The expression levels of FOXC2 mRNA were normalized to GAPDH using the comparative Ct method (DDCt).

Quantification of G-actin and F-actin
FOXC2 was lentivirally overexpressed in differentiated human podocytes as described in Datta et al. [1]. The G-actin and F-actin quantification in FOXC2 and control vector-transduced podocytes was performed as described in the G-actin/F-actin in vivo Assay Kit (Cytoskeleton Inc., Denver, CO, USA). Briefly, cells were lysed in a detergent-based lysis buffer that stabilizes and maintains the Gand F-forms of cellular actin. This was followed by a 100,000g centrifugation at 37°C for 1 h that pellets the F-actin and leaves the G-actin in the supernatant. Samples of supernatant and pellet were separated by SDS-PAGE and actin was quantified by Western blotting using the rabbit polyclonal antiactin antibody provided in the kit.

Statistical analysis
Results are presented as mean 7SD. Statistical analysis was performed using Student's t test (Microsoft Excel, Redmond, WA). Table 1 Weight, blood glucose and urinary albumin to creatinine values of 40 weeks old lean and obese Zucker rats. The weights and blood glucose levels were not significantly different between the obese and lean rats, but the obese rats had significantly higher urine albumin to creatinine ratios.