ICAM-1-linked long noncoding RNA is associated with progression of IgA nephropathy and fibrotic changes in proximal tubular cells
Patients and samples
We conducted a retrospective cohort study of 413 patients with renal biopsy-confirmed IgAN at the First Affiliated Hospital of Zhengzhou University between May 2013 and March 2018. All of these patients had additional biopsy specimens for this research. We excluded 26 patients with missing baseline clinical data, 29 with missing follow-up data, and 21 with missing treatment regimens. All recruited patients were followed for at least 3 years. Patients with secondary IgAN, including IgA vasculitis, hepatitis B virus-associated glomerulonephritis, liver cirrhosis, systemic lupus erythematosus, were excluded. Therefore, the final cohort included 337 patients with primary IgAN. Kidney biopsy specimens were collected from patients included with clinical kidney biopsy and were immediately frozen at -80°C until measurement. Additionally, normal kidney tissue from nephrectomy specimens from 89 age- and sex-matched patients with renal cell carcinoma served as biopsy controls (Supplementary Table S3).
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, China (Approval No: 2019-KY-361). Informed consent was obtained from all recruited participants.
Clinical and histological manifestations
Clinical data, including gender, age, MAP, serum creatine, and 24-hour urinary protein excretion, were collected at the time of renal biopsy (defined as baseline). Patients’ treatment regimens, including use of steroids or other immunosuppressants, were recorded during follow-up. eGFR was calculated using the CKD-epidemiology Collaboration formula29. All kidney biopsy sections were reviewed by two independent pathologists blinded to the clinical data of the patients. Pathological lesions were assessed according to the Oxford classification (MEST-C score)30. Briefly, it includes 4 parameters as follows: mesangial hypercellularity (M0/M1), endocapillary hypercellularity (E0/E1), segmental sclerosis (S0/S1), interstitial fibrosis/tubular atrophy (T0/T1/T2) and cellular/fibrocellular growths (C0/C1/C2).
The ESRD event was defined as an eGFR 2 or need renal replacement therapy. The composite event of disease progression was defined as a permanent reduction ≥40% in eGFR from baseline or ESRD, whichever came first. Both of these reported results were confirmed by a second assessment at least 4 weeks later.
Cell culture and transfection
HK-2 cell is an immortalized human proximal tubule cell line obtained from Wanleibio (Shenyang, China) and cultured in DMEM (Lonza, Basel, Switzerland) supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were incubated at 37°C and 5% CO2 in a humidified atmosphere. When cell confluence reached approximately 60%, the growth medium was replaced with serum-free medium for another 12 h incubation. After that, the cells were incubated with 10 ng/ml of TGF-β1 for 24 h or 48 h. Plasmids containing the ICR shRNA were engineered to knock down ICR expression and purchased from Wanleibio. HK-2 cells were seeded in a 6-well plate 24 h before transfection. Cells were transfected with the sh-ICR plasmid or its negative control using Lipofectamine 3000 transfection reagent according to the manufacturer’s instructions. QPCR was used to assess the transfection efficiency of the sh-ICR plasmid 24 h after transfection. These transfected cells were then stimulated with TGF-β1 for another 48 h incubation. Cell morphology images were obtained with a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany).
Fluorescence in situ hybridization
In situ hybridization by fluorescence was carried out with an anti-ICR oligonucleotide probe labeled with Cy3. Cells were fixed in 4% paraformaldehyde, dehydrated with ethanol, then incubated with an ICR probe in the dark overnight at 37°C. The next day, cell nuclei were counterstained with DAPI. A confocal microscope (Carl Zeiss, Oberkochen, Germany) was used to capture the images.
Western blot analysis
Cellular proteins were obtained by lysing cells in lysis buffer containing 1% Triton X-100, 150 mM NaCl and 50 mM Tris.HCl. Proteins extracted from cells were separated by SDS-PAGE and analyzed by Western blot as previously described31. The following antibodies purchased from Wanleibio were used: AKT antibody (1:500, WL0003b), p-AKT antibody (1:500, WLP001a), mTOR antibody (1:500, WL02477), p-mTOR antibody (1:500 , WL03694), collagen I antibody (1:500, WL0088), α-SMA antibody (1:500, WL02510) and β-actin antibody as loading control (1:1000, WL01372). Horseradish peroxidase-conjugated sheep anti-rabbit immunoglobulins (WLA023) were used for detection with the ECL substrate. ImageJ 1.47 software was used to quantify the signal.
Total RNA extraction and qPCR analysis
Amounts of ICR in kidney tissues of patients were measured using qPCR by KangChen Bio-tech (Shanghai, China)32. Briefly, total RNA was purified from kidney tissue using TRIzol® Reagent (Thermo Fisher Scientific, Waltham, USA). Then 20 μl of reaction including 1.5 μg of total RNA and 1 μl (0.5 μg/μl) of Oligo(dT)18 The primer was used for reverse transcription. QPCR was performed on the QuantStudio™ 5 real-time PCR system (Applied Biosystems, Foster, USA). For qPCR analysis in HK-2 cells, total RNA was purified from the cells by TRIpure (BioTeke, Beijing, China). Complementary DNA was produced by RNA and qPCR was performed with SYBR Green. ICR expression in kidney tissue and cells was normalized to 18S rRNA and β-actin, respectively. Relative changes in ICR were calculated using the 2−ΔΔCt method, where ΔCt = CtRIC − CT18S/β-actinand ΔΔCt = ΔCt experimental− ACt control. The primers purchased from KangChen Bio-tech were as follows: ICR: forward: 5′-CGTCAGCCGGCATAGAACA-3′, reverse: 5′-TCGGTGAGGCACCCCTGTAA-3′; 18S: sense: 5′-CAGCCACCCGAGATTGAGCA-3′, antisense: 5′-TAGTAGCGACGGGCGGTGTG-3′; β-actin: sense: 5′-GGCACCCAGCACAATGAA-3′, antisense: 5′-TAGAAGCATTTGCGGTGG-3′.
Quantitative data were expressed as mean ± standard deviation or median (IQR) and compared using t-test, Mann-Whitney U-test or Kruskal-Wallis test, as appropriate. Categorical data were expressed as frequencies and percentages and compared by the chi-square test. The patients were divided into 3 groups according to renal ICR level tertiles (group 1, 3.04). We used Kaplan-Meier analysis to produce cumulative renal survival curves and used the log-rank test to analyze differences between curves. Cox’s unadjusted multivariate proportional hazards models were applied to assess the effect of renal ICR on the risk of disease progression in IgAN. In the models, the renal ICR was analyzed either as a 3-level categorical variable defined by the tertiles of the renal ICR level, or as a continuous variable. Results were expressed as HR and 95% CI. P– trend values in the Cox proportional hazards models were calculated by entering the median value of each tertile of the renal ICR level as a continuous variable. We used interaction terms to test the interaction between renal ICR level and proteinuria level on disease progression in IgAN. In addition, C-statistic, NRI and IDI were calculated to assess the additional prognostic value of renal ICR for 5-year renal survival outcome after biopsy beyond traditional risk factors.33, 34. Statistical analysis was performed using SPSS software (version 19.0; IBM Corp., Armonk, USA) and R version 4.0.2. A P– value