Antibodies and reagents:
The TNF-α (NBP1-19532), TNFR1 (NBP1-97453), CD11b (NB110-89474), MCP1 (NBP2-22-115), Podocin (NBP2-75624) and Nephrin (NBP1-77303) were purchased from Novus Biologicals (Minneapolis, Minnesota). The NF-kb (ab32536), pIKB-α (ab133462), IKB-α (ab32518), Lamin-B1(ab16048) were purchased from Abcam (Cambridge, MA); F4/80 from Biolegend (San Diego, CA); Phospho IKKa/b (ser176/180) (#2697), CD14 (D7A2T) (#56082) and β-actin (#4970) were from Cell Signaling Technology (Danvers, MA). CD68 Antibody (sc-17832) Santa Cruz Biotechnology (Dallas, TX). Mouse/Rabbit PolyDetector DAB HRP Brown Immunochemistry detection system (#BSB020, Santa Barbara, CA). Phalloidin fluorescein isothiocyanate labeled (#P5282), glutaraldehyde solution (G5882) and Giemsa stain (#26149-01) were obtained from Sigma Aldrich (St. Louis, MO, USA). Precision Plus Protein Dual Color Standards (Bio-Rad, Hercules, CA) and ProLongTM Diamond Antifade Mountant (#P36961) were purchased from Molecular Probes Life Technologies. DyLight 488 and DyLight 564, and Cy5-conjugated secondary antibody were purchased from Vector Laboratories (Burlingame, CA). Primers used in this study procured from Integrated DNA Technologies (Coralville, IA). JAK2 inhibitor (AG490) were purchased from Tocris Bioscience (Pune, India). Human TNF-α Recombinant Protein (#PHC3015) TNF-α ELISA kit (#KHC3011) and MCP1 ELISA kit (#BMS281INST) were purchased from ThermoFisher Scientific. Paraformaldehyde (#P6148-500G), bovine serum albumin (BSA, A3294-50G), 4, 6-diamidino-2-phenylindole (DAPI, #P36971) were obtained from Sigma Aldrich (St. Louis, MO, USA).
Podocyte culture and treatment:
In this study, conditionally immortalized human podocytes (a gift from Prof. Moin Saleem, University of Bristol) cells were cultured essentially as described earlier21. Briefly, after 14 days of differentiation at 37°C, podocytes were treated with or without GH and GH + AG490. Unless otherwise mentioned, all the experimental conditions for podocyte cells were given for 1 h. The cell lysate was prepared for RNA isolation, immunoblotting, and enzyme-linked immunosorbent assay. For immunofluorescence, cells were cultured on coverslips, followed by treatment as mentioned above, subsequent fixation with paraformaldehyde (4%), and blocking with PBS containing normal BSA (5%) before incubation with primary antibodies. The next day, the samples were incubated with Alexa Fluor-conjugated secondary antibodies, and DAPI for nuclear stain, for 1 h at room temperature. Images were acquired using a laser scanning microscope (ZEISS, Germany). For F-actin staining in podocytes cells essentially as described earlier 22. Podocytes were incubated with Phalloidin fluorescein isothiocyanate labeled (#P5282) for 40 min at room temperature. Next counterstaining by DAPI, mounting and images were acquired using a Leica trinocular microscope or Apotome Axio Imager Z2 (Zeiss). We analyzed images using LASX Industry Software and ImageJ (NIH).
RNA extraction, library construction, sequencing and analysis:
Total RNA was isolated from with or without GH treated HPC cells using Trizol (Invitro-gen#15596026) according to manufacturer instructions. RNA was further purified using a commercially available kit (RNeasy Mini Kit, QIAGEN). 20 μg of purified RNA from each sample was treated with 10 Units of DNase1 (Ambion TURBO DNase, Life Technologies) and further purified by using G Sure cell culture RNA isolation kit. From each RNA sample, Ribosomal RNA was removed using Ribo-Zero kit (NEB#E6310L), and further mRNAs were enriched using Oligo (dT) beads. Illumina paired-end library was prepared as per the NEBNext® Ultra™ RNA Library Prep Kit (NEB#E7530S). The paired-end libraries were prepared and sequencing performed using Illu-mina HiSeq-2500 sequencing platform. The resultant raw reads were compressed to fastq.gz format and deposited in GEO repository (GSE140308). The raw reads quality check report was generated and trimmed the reads as per the fastQC report. Trimmed reads were aligned to the human hg38 reference genome (GRCh38) using TopHat v2.1.0. The mismatch parameter along with all other parameters were set to default.
Gene expression quantification was performed using ‘Cufflinks’ tool and derived Fragments per Kilobase exon per million reads sequenced (FPKM) values. Differentially expressed genes were derived using 'Cuffdiff' tool a log2 Fold change value of ≥ 1 is taken as a cutoff to define upregulated and a value of ≤−1 to define downregulated genes with p-value threshold 0.001 to maintain statistical significance. KEGG pathway analysis was performed for differentially expressed genes using KEGG pathways and functional annotations performed using g:profiler tool. Expressed genes Venn diagram was generated using Ugent web tool (http://bioinformatics.psb.ugent.be/webtools/Venn/). Heatmap and volcano plots were generated using R programming. Gene Set Enrichment Analysis (GSEA) was performed using GSEA v4.1.0 software (Broad Institute).
Quantitative Real time PCR:
The total RNA was isolated from human podocytes and mouse podocytes using a TRIzol reagent. Next, 1 μg of total RNA was reverse transcribed using the cDNA synthesis kit (#6110 A, PrimeScript 1st strand cDNA Synthesis kit, TaKaRa). The qRT-PCR analysis was performed by the QuantStudio 3 system (Applied Biosystems) with SYBR Green (Kappa Biosystem) Master Mix as mentioned in the following protocol: initial denaturation at 95 °C for 3 min, followed by 35 cycles of three steps each at 95 °C for the 30 s, 60 °C for 30 s, and 72 °C for 30 s. The expression levels of β-actin did not change under the different experimental conditions and were used as an internal control to normalize each gene’s mRNA expression. The primers and siRNA sequence used are shown in Supplementary Table.1.
THP1 cell culture and treatment:
THP-1 cells were cultured as described earlier 23. Cells were maintained at 37°C in an atmosphere containing 5% CO2. For exposure, the THP-1 monocytes were seeded in 12 well Corning™ Costar™ Flat Bottom Cell Culture Plates (Fisher Scientific Hampton, New Hampshire, USA) to a density of 5x105 cells/mL in a volume of 1 mL and incubated for 24 h. To assess the impact on THP1 differentiation into macrophage, the cells were exposed to 50% of conditioned media (CM; prepared by adding spent media with fresh RPMI) from with or without GH treatment, GH+AG490 and from GH neutralizing CM and incubated for another 72 h to initiate differentiation into macrophage-like cells. The effect of CM from with or without GH and GH+AG490-CM to podocyte on THP1 differentiation into macrophages Giemsa staining was performed as recommended by the manufacturer protocol and image captured using EVOS M5000 cell imaging system (Invitrogen) light microscope. TNF-α neutralization in CM was performed as recommended by the manufacturer’s protocol (#7321).
Animal and tissues:
Eight-week-old Swiss Webster male mice weighing 30±3 g were used. Mice were allocated randomly (not blinded) into to three groups: (1) control group, (2) GH-treated group and (3) GH + AG490. Mice received a single i.p. injection of hGH (1.5 mg/kg) or an equal saline volume per day for 6 weeks. We used a predefined value of n = 6 mice/group. The inhibitor group mice were received AG490 (1 mg/kg of b.w.) per day in addition to GH. If animal dies prematurely the data excluded from the analysis. After 6 weeks of the experimental period, the mice were placed in individual metabolic cages for collecting 24 h urine to estimate albumin (#COD11573) and creatinine (#COD11502) levels, as recommended by the manufacturer’s protocol (Biosystems, Barcelona, Spain). Disease establishment was confirmed by estimating the UACR. An aliquot of urine from mice was subjected to SDS-PAGE gel, and silver staining was performed to compare the urinary protein profile for all three groups. Further, we have also measured the GFR in these mice, as described previously22. Mouse podocytes were isolated from the kidney of mice, as described in earlier protocol24. Briefly, glomeruli were prepared by filtration of the kidney’s cortex with mesh sieves, whose holes were 100, 76, and 54 μm in diameter. The tissues left on the mesh sieve with 54 μm holes were collected and prepared for the qRT-PCR and immunoblotting. For histological analysis, the kidney cortex was fixed with 4% paraformaldehyde before embedding in paraffin. Paraffin-embedded tissues were sliced longitudinally into 3–4 μm thick sections, subjected to staining with H&E and PAS staining. We obtained TEM images for glomerular sections from all the experimental mice groups described earlier25. The experiments with animals were approved by IAEC of University of Hyderabad, Hyderabad, India.
Flow cytometry analysis:
In order to confirm that CM from GH treated podocytes induces Cd11b differentiation marker from THP-1 cells to macrophages by flow cytometry. THP1 cells (2 x 106 cells/ml) are treated with or without CM from GH and GH+AG490 for 72 h. Cell surface staining were performed by direct immunofluorescent assay with fluorescence-conjugated CD11b antibody and corresponding isotype control antibodies for 30 min at room temperature in the dark. Next cells were centrifuge (500xg maximum) at 4OC for 5 min and resuspended the pellet in 200 μl of PBS for flow cytometric analysis using S3e Cell Sorter flow cytometer (Bio-Rad). Data were analyzed using FCS Express 7 program.
Western blot analysis:
Cytoplasmic extract for immunoblotting was prepared as described previously 22. Briefly, human podocytes and isolated mouse primary podocytes were lysed by RIPA buffer (#9806, Cell Signaling Technology) containing protease inhibitor mixture (#05056489001, Sigma Aldrich) and phosphatase inhibitor tablets (Roche), centrifuged and collected as supernatants. Nuclear protein extract for immunoblotting was prepared as described earlier 21. Briefly, cell pellet was resuspended in nuclear extracting buffer and vortexed for 20 s. Incubate the cell lysate for 25 min on ice and vortex every 10 min for 10 s. Next, cell lysate was centrifuged for 12 min at 19,400xg at 4°C, and the supernatant (nuclear) was collected. The protein concentrations of cell and mouse podocyte lysates were determined using a bicinchonic acid reagent (ThermoFischer Scientific), using BSA as a standard. An equal amount of protein from corresponding samples was subjected (25-30 μg) to SDS-PAGE and immunoblotting, and bands were visualized using a ChemiDocTM XRS System (Bio-Rad, Hercules, CA).
Enzyme-linked immunosorbent assay:
The CM from with or without treated podocytes were collected for the detection of related indexes. The expressions of TNF-α and MCP-1 in CM were detected by ELISA. The operation was carried out according to the kit instructions (ThermoFischer Scientific) with the double antibody sandwich ELISA. Briefly, the only media, CM from podocyte with or without GH-Ab and GH-Ab+AG490 treatment for 1 h were collected and applied to TNF-α and MCP1 ELISA plates. Next washing and enzyme-labeled antibodies were added to combine with the antigen to form the enzyme-labeled antibody-antigen-solid antibody complex. Finally, substrate was added for color development, and the depth of color was measured at 450nm using a fluorescent microplate reader (Multiskan Microplate reader, ThermoFisher Scientific).
The invasion of THP-1 cells was estimated using a modified method as described earlier 26. Briefly, THP1 cells were spun down at 1000 rpm and resuspended in 1 ml of 0.1% BSA-RPMI. After adjusting the cell density to 1x105 cells/ml, 2.5 ml of only media (CTL), CM from podocytes treated with or without GH-Ab or GH-Ab+AG490 were add to each 24 well plate. Next, the transwell insert was placed into the each 24 well plate (6.5 mm Transwell with 5-μm pore size, CLS3421-48EA; Corning, NY) with 10,000 cells in 100 μl incubated for 4 h at 37°C in an atmosphere containing 5% CO2. The insets were removed and migrated cell in the bottom well were transferred to 17x120 mm tube (#352096) and cell counting was performed using a FACScan (Becton-Dickinson, San Jose, CA).
Human kidney specimens:
Immunohistochemistry of human kidney biopsies were not performed for the purpose of this study, but collected from archived kidney biopsies without patient identifiers from Guntur Medical College, AP, India. Informed consent was obtained from the patients that these kidney biopsies obtained for patient care could be used for research purpose. The selected cases were with biopsy proven DN and proteinuria. This study was approved by the Institutional Review Board of Guntur Medical College and Government General Hospital, Guntur, Andhra Pradesh, India (application number GMC/IEC/120/2018). Our study protocols were abide by the Declaration of Helsinki principles.