Long noncoding (Lnc)RNA np_5318 has been proved to be involved in renal injury, while its functionality in renal ischemia-reperfusion (I/R) injury is unknown. Therefore, the present study aimed to investigate the role of lncRNA np_5318 in the development of renal I/R injury. Renal I/R injury model and I/R cell model were established
LncRNA np_5318 may participate in the development of renal I/R injury through TGF-β/Smad signaling pathway.
Acute renal failure (ARF) of the kidney is an important cause of morbidity and mortality in hospitalized intensive care unit patients. Renal ischemia is a major cause of ARF, initiating a complex and interrelated sequence of events, resulting in injury and the eventual death of renal cells (
Long noncoding RNA (lncRNA) is a group of non-coding RNAs with a length longer than 200 nucleotides (
The current study aimed to explore the expression and function of np_5318 in renal I/R and investigate the potential interaction between the np_5318 and TGF-β/Smad signaling pathway in this disease. In the present study, the roles of np_5318 in renal I/R were explored by establishing I/R animal and cell models and the related mechnism was also verified using transfection experiments. It was demonstrated that lncRNA np_5318 may participate in the development of renal I/R injury through the TGF-β/Smad signaling pathway.
Specific pathogen free grade female Balb/C mice (n=20; age, 3 weeks; weight, 20±5 g) were obtained from Jinan Jinfeng Experimental Animal Breeding Co., Ltd., [license number: SCXK (lu) 2014-0006]. Animals were reared under specific pathogen-free conditions (12 h dark/light cycles; 25˚C) with 95% humidity with access to food and water
Serum creatinine (Scr) level and blood urea nitrogen (BUN) were detected using automatic biochemical test (Hitachi 7180; Hitachi, Ltd., Tokyo, Japan) at 4, 24 and 48 h after reperfusion. Renal tissue was collected for hematoxylin and eosin (HE) staining at room temperature for 24 h after reperfusion to observe the histopathological changes. A light microscope was used to observe the staining.
Tissues were fixed at 4˚C in fresh 4% (w/v) formaldehyde solution for 12 h and paraffin-embedding was performed. A total of 5 paraffin-embedded sections (12 µm) were selected at each time point. Tissue sections were dewaxed and then washed with PBS. Tissue sections were blocked in blocking fluid at room temperature for 20 min to reduce nonspecific background staining caused by endogenous peroxidase. Tissue sections were then blocked with 10% serum (Roche Diagnostics) for 10 min at room temperature, followed by incubation with corresponding primary antibodies (eBioscience; Thermo Fisher Scientific, Inc.) over night at 4˚C. After washing with PBS, tissue sections were incubated with fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin (Ig) G secondary antibody (Chemicon international; Thermo Fisher Scientific, Inc.) at room temperature for 30 min. After washing with PBS again, tissue sections were incubated with streptavidin-peroxidase solution at room temperature for half an hour. After washing with PBS, DAB color development was performed. After washing with distilled water and counter staining, the slides were sealed. A total of 5 fields of view were selected under a light microscope (x400) and positive cells were counted, and the average value was calculated to represent microvessel density.
Primary human renal cells (PCS-400-012) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in complete renal epithelial cell growth media (ATCC) at 37˚C in an incubator with 95% air and 5% CO2 (
Vector pc-np_5318 for overexpression of lncRNA np_5318 in I/R cell was constructed by inserting the coding oligonucleotides of lncRNA np_5318 into a pcDNA3.1 vector (Invitrogen: Thermo Fisher Scientific, Inc.). Small interfering (si)RNAs targeting lncRNA np_5318 (si-np_5318) were constructed for inhibition of lncRNA np_5318. For cell transfection, I/R cells as mentioned before were cultured in a six-well plates (105 cells per well) for 24 h and transfected with 10 nM pc-np_5318, si-np_5318 (5'-CCUGUGCACGUUCGAUUCAUA-3'), and their corresponding controls [pc-NC and si-NC (5'-GCGUGACGUACGUACGUACGA-3') respectively] using Lipofectamine 2000® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Cells continued to be incubated for another 48 h before subsequent experiments.
Using an MTT colorimetric assay, cell viability was assessed. In brief, different transfected cells at logarithmic stage were grown into a 96-well plate. After 24, 48 and 72 h of transfection, 20 ml MTT was added into each well to incubate cells for another 4 h. Then 150 ml dimethylsulfoxide was added into each well to dissolve the formazan precipitates for 10 min. The absorbance (490 nm) was measured under an absorption spectrophotometer (Olympus Corporation, Tokyo, Japan). Each experiment was repeated 3 times.
Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.). RNA concertation were determined using NanoDrop™ 2000 Spectrophotometers (Thermo Fisher Scientific, Inc.) and only the ones with a ratio of A260/A280 between 1.8 and 2.0 were used for reverse transcription to synthesize cDNA using ploy (T) as primer and SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific, Inc.) through following conditions: 55˚C for 15 min and 75˚C for 10 min. SYBR® Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, Inc.) was used to prepare the PCR reaction system. Following primers were used: 5'-AACTCGCCACAGAAATCCAC-3' (forward) and 5'-ACAACCCCAAACAAGCTGTC-3' (reverse) for np_5318; 5'-TGCTGAGTATGTCGTGGAGTCTA-3' (forward) and 5'-AGTGGGAGTTGCTGTTGAAATC-3' (reverse) for GAPDH. PCR reaction conditions were: 95˚C for 40 sec, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 45 sec. Cq values were processed using 2-ΔΔCq method (
ChIP was performed using Diagenode's iDeal ChIP-seq kit for Transcription Factors (Diagenode SA, Seraing, Belgium) according to the manufacturer's protocol. Mouse embryonic fibroblasts (MEFs) were purchased from Sigma-Aldrich; Merck KGaA and were cultivated under conditions described in manufacturer's protocol. Briefly, Cross-linking was performed at 37˚C for 10 min and was quenched with glycine. DNA fragments ranged from 300-600 bp were generated through sonication using a Bioruptor (Diagenode SA). Then an antibody against Smad3 (cat. no. 06-920, 1:1200; EMD Millipore) was used for immunoprecipitation with normal IgG as control. The antibody against IgG was also purchased from EMD Millipore (1:1200; cat. no. PP64). The following primers were used in PCR to detect the precipitated DNAs: Smad binding site (SBS) for np_5318, 5'-CTCTCTCAAACAGCCTGTGG-3' and 5'-GAAATTTGGAGGTGCAATCAA-3'.
Total protein extraction was performed using a Qproteome Mammalian Protein Prep kit (Qiagen GmbH, Hilden, German) according to the manufacturer's protocol. Protein concentration was measured by bicinchoninic acid method. Protein samples were denatured and 50 µg of protein was subjected to 10% SDS-PAGE gel electrophoresis, followed by transmembrane to PVDF membrane. After blocking with 5% skimmed milk at room temperature for 2 h, membranes were washed with Tween buffered TBS (TBST; 01% Tween 20). Membranes were then incubated with primary antibodies overnight at 4˚C. Primary antibodies used were rabbit anti TGF-β1 (1:2,000; cat. no. ab92486; Abcam), Smad3 (1:2,000; cat. no. ab28379; Abcam), p-Smad3 (1:2000; cat. no. ab52903; Abcam) and α-tubulin (1:2,000; cat. no. ab18251; Abcam). After that, membranes were washed three times with TBST. Membranes were then incubated with secondary antibody at room temperature for 2 h. The secondary antibody was HRP-Goat Anti-Rabbit (IgG) secondary antibody (1:2000; cat. no. ab6721; Abcam). After washing twice with TBST, 15 min for each time, ECL™ Blotting Reagents GE Healthcare (Sigma-Aldrich; Merck KGaA) was added to detect the signals. Images were processed using Bandscan 5.0 software (Nuohebio) to calculate the relative expression level of each protein.
SPSS 10.0 was used for all statistical analysis (SPSS, Inc., Chicago, IL, USA). Experimental data were expressed as the mean ± standard deviation. Comparisons within a group were performed using single factor analysis of variance and comparisons between two groups were performed using independent t test. Multiple comparison corrections were performed using Bonferroni multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.
Compared with the levels of SCr and BUN in the sham group (24.56±5.94 and 7.44±3.99 µmol/l, respectively), levels of SCr and BUN were increased in I/R group at 4 (41.61±4.79 and 14.45±0.83 µmol/l, respectively), 24 (94.42±53.89 and 39.35±15.88 µmol/l, respectively) and 72 h (36.34±4.78 and 17.42±11.48 µmol/l, respectively) after reperfusion. However, significant differences were only found at 24 h after reperfusion (P<0.05). Those data suggest that I/R can increase the levels of SCr and BUN in mice, which in turn impairs renal function (
As shown in
Cluster of differentiation (CD)31 mediates the penetration of leukocytes into blood vessel walls to cause tissue damage and compensatory hyperplasia (
To investigate the level of np_5318 in the mice, RT-qPCR was used in the sham and I/R groups with GAPDH as endogenous control. As shown in
As shown in
To determine TGF-β1 and p-Smad3 expression in the kidney, immunohistochemical staining was performed. The sham operated controls did not show any TGF-β1 and p-Smad3 expression. However, there was significant increase in TGF-β1 and p-Smad3 expression in I/R model (P<0.01) compared with the sham mice (
Then levels of TGF-β1, Smad3 and p-Smad3 in cells were detected by western blotting, including I/R, I/R+si-NC, I/R+si-np_5318, I/R+pc-NC, I/R+pc-np_5318, and the control groups as shown in
Renal IR is a process of the reduced blood supply to kidneys followed by re-oxygenation and restoration of blood flow (
Numerous studies have shown that the onset, development and progression of ischemia-reperfusion injury are closely related to the function of different lncRNAs (
The TGF-β/Smad signaling pathway plays pivotal roles in various renal diseases. The activation of TGF-β can regulate a variety of cellular functions including proliferation, differentiation, apoptosis and inflammation (
In conclusion, expression level of lncRNA np_5318 was increased in mice with renal I/R and I/R cells. np_5318 could regulate the expression of Smad3 by binding to its promoter region. In addition, levels of TGF-β1, Smad3 and p-Smad3 were increased by renal I/R. Therefore, lncRNA np_5318 may participate in the development of renal I/R injury through the TGF-β/Smad signaling pathway. The present study provides new insights into the mechanism and treatment of I/R injury. However, the present study is still limited by the small sample size. Further studies with bigger sample size are still needed to verify the conclusions in this study.
Not applicable.
No funding received.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
JS conceived and designed the study. JL and JM performed the experiments. JL and JM wrote the paper. JL and JS reviewed and edited the manuscript. All authors read and approved the final manuscript.
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Yucheng People's Hospital.
Not applicable.
The authors declare that they have no competing interests.
Representative results of hematoxylin and eosin staining in kidney tissue. I/R, ischaemia/reperfusion.
Increased expression level of np_5318 is confirmed in I/R groups and np_5318 may promote renal I/R injury. (A) The expression of np_5318 in the mice was determined by RT-qPCR in the sham and I/R groups. (B) The expression of np_5318 in these cell groups (I/R, I/R+si-NC, I/R+si-np_5318, I/R+pc-NC, I/R+pc-np_5318 and control groups) was confirmed by RT-qPCR. The transfection efficiency is shown below. Si-np_5318 significantly decreased np_5318 level and pc-np_5318 significantly increase np_5318 level. (C) Relative cell viability at 24, 48, 72 h in these groups (I/R, I/R+si-NC, I/R+si-np_5318, I/R+pc-NC, I/R+pc-np_5318 and control groups) were investigated by MTT assay. *P<0.05 and **P<0.01. si, small interfering; I/R, ischaemia/reperfusion; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; Ctrl, control; lnc, long noncoding.
LncRNA np_5318 can bind to Smad3. (A) DNA sequence alignments of SBS in the promoter regions of np_5318. Bold and underlined sequences indicate the location of SBSs. (B) Chromatin immunoprecipitation assays for Smad3 were performed with chromatin from mouse embryonic fibroblast cells treated with TGF-β1. Precipitated DNA was amplified with oligonucleotides spanning SBS regions. Total inputs are indicated. p-Smad3, phosphorylated-mothers against decapentaplegic homolog; TGF, transforming growth factor; lnc, long noncoding; Ig, immunoglobulin; SBS, Smad binding site.
Renal I/R injury group shows increased TGF-β1 and p-Smad3 expression. (A) Representative results of immunohistochemical staining. (B) Normalized expression levels of TGF-β and p-Smad3. **P<0.01 vs. the sham group. p-Smad3, phosphorylated-mothers against decapentaplegic homolog; TGF, transforming growth factor; I/R, ischaemia/reperfusion; NC, negative control.
Levels of TGF-β1, Smad3 and p-Smad3 are determined
Comparison of levels of SCr and BUN among groups.
Groups | SCr (µmol/l) | BUN (µmol/l) |
---|---|---|
Sham | 24.56±5.94 | 7.44±3.99 |
I/R 4 h | 41.61±4.79 | 14.45±0.83 |
I/R 24 h | 94.42±53.89 |
39.35±15.88 |
I/R 72 h | 36.34±4.78 | 17.42±11.48 |
aP<0.05 vs. the sham group. BUN, blood urea nitrogen; SCr, serum creatinine; I/R, ischaemia/reperfusion.
Comparison of positive rate of CD31 among groups
Groups | CD31 |
---|---|
Sham | 12.04±0.78 |
I/R 24 h | 21.43±0.22 |
I/R 72 h | 23.51±0.69 |
aP<0.05 vs. the sham group. CD, cluster of differentiation; I/R, ischaemia/reperfusion.