Upregulated long noncoding RNA LOC105375913 induces tubulointerstitial fibrosis in focal segmental glomerulosclerosis

Tubulointerstitial fibrosis impacts renal prognosis of focal segmental glomerulosclerosis (FSGS). Based on transcriptomic analysis, we found that the level of LOC105375913 was increased in tubular cells of FSGS patients. C3a induced the expression of LOC105375913, which promoted the expression of fibronectin and collagen I in tubular cells. Silence of snail reversed the level of fibronectin and collagen I in cells overexpressing LOC105375913. MiR-27b was predicted and confirmed to regulate the expression of snail in tubular cells, and LOC105375913 contained the response element of miR-27b. The competitive binding between LOC105375913 and miR-27b increased the level of snail and promoted fibrogenesis in tubular cells. Upstream, p38 and XBP-1s regulated the expression of LOC105375913. Inhibition of p38 or silence of XBP-1s decreased the level of LOC105375913, and suppressed the expression of snail, fibronectin and collagen I in tubular cells treated with C3a. Overexpression of LOC105375913 decreased the level of miR-27b, increased the level of snail and caused tubulointerstitial fibrosis in mice. In conclusion, the activation of C3a/p38/XBP-1s pathway induces the expression of LOC105375913 in tubular cells, and LOC105375913 increases the level of snail and induces tubulointerstitial fibrosis through competitive binding of miR-27b in tubular cells of FSGS patients.

Focal segmental glomerulosclerosis (FSGS) accounts 40% of cases of nephrotic syndrome in adults 1 . Tubulointerstitial fibrosis is an independent risk factor of renal function decline in FSGS patients 2,3 . We completed a transcriptome analysis of tubulointerstitial tissues in 5 patients with FSGS and 5 normal controls. Among the differentially expressed long noncoding RNAs (lncRNAs), the level of LOC105375913 showed the maximum increase in tubulointerstitial tissues of FSGS patients.
LncRNAs are a kind of noncoding RNAs longer than approximately 200 nucleotides with no protein-encoding capacity 4 . Emerging evidence suggests that lncRNAs are involved in the regulation of renal fibrosis. Sun J et al. reported that 24 lncRNAs were up-regulated in the renal tissues of rats with unilateral ureteral obstruction, and 19 of them were predicted containing Smad3 binding motifs in the promoter 5 . Wang M et al. reported that lncRNA CYP4B1-PS1-001 was downregulated in early diabetic nephropathy, and overexpression of CYP4B1-PS1-001 inhibited proliferation and fibrosis of mesangial cells under high glucose conditions 6 . So far, the role of lncRNAs in renal fibrosis of FSGS patients have not been reported.
During nephrotic syndrome, plasma protein leakage into the urinary space directly contributes to local tissue injury. Deposits of the complement component 3 protein (C3) were detected on the proximal tubules of kidneys from nephrotic patients 3 . We noted that the level of C3 mRNA was also upregulated in the tubulointerstitial tissues of FSGS patients. Therefore, both systemic and local complement synthesis contribute to intrarenal complement activation. Tang Z et al. reported that the kidneys of C3aR-deficient mice had less interstitial collagen I and alpha-smooth muscle actin in the adriamycin-induced proteinuria model 7  Based on our findings, we believe that the increase in LOC105375913 may be associated with the abnormal exposure of renal tubular cells to complement components in FSGS patients. In this study, experiments were further conducted to investigate the underlying mechanism of LOC105375913 expression and investigate the role of LOC105375913 in the tubular cells of FSGS patients.

Results
LOC105375913 is increased in tubular cells and relates to tubulointerstitial fibrosis in FSGS patients. Renal tubulointerstitial tissues from 5 patients with FSGS and 5 normal controls were micro-dissected, and an Affymetrix HTA 2.0 microarray was used to perform a global analysis of the gene expression patterns in the tissues. Among the differentially expressed lncRNAs, the level of LOC105375913 showed the maximum increase in patients with FSGS (Fig. 1a). PCR analysis of another set of patients confirmed that the level of LOC105375913 was significantly increased in the tubulointerstitial tissues of FSGS patients, and was correlated with the score of tubulointerstitial fibrosis in patients with FSGS ( Fig. 1b-d). ISH staining showed that LOC105375913 expression was upregulated in the tubular cells of FSGS patients (Fig. 1e). HK-2 cells were then cultured and treated in vitro. Overexpression of LOC105375913 obviously increased the mRNA and protein levels of fibronectin (FN) and collagen I (Col I) in HK-2 cells (Fig. 1f,g).
C3a induces the expression of LOC105375913 in tubular cells. Treatment with patients' serum significantly induced the expression of LOC105375913 in a time-and dose-dependent manner (Fig. 2a,b). The level of LOC105375913 was significantly increased in HK-2 cells treated with 10% or 20% serum for more than 24 h.
Inhibition of C3aR but not C5b-9 inhibition prevented the increase of LOC105375913 in tubular cells treated with patients' serum ( Fig. 2c). Immunohistochemistry staining (IHC) showed that massive C3a bound to the tubular cells in FSGS patients (Fig. 2d). Treatment with C3a alone induced the overexpression of LOC105375913 in tubular cells (Fig. 2e). C3a treatment also increased the levels of FN and Col I in tubular cells, which was suppressed in cells transfected with LOC105375913 siRNA (Fig. 2f,g).

LOC105375913 overexpression increases the expression of snail in tubular cells. Snail is a key
transcription factor which involved in tubulointerstitial fibrosis in renal disease. IHC analysis showed that, the expression of snail was increased in the tubular cells of FSGS patients, compared to normal controls (Fig. 3a). Treatment with C3a or transfection of LOC105375913 plasmid increased the expression of snail in tubular cells (Fig. 3b). LOC105375913 upregulates the level of snail by competitive binding of miR-27b in tubular cells. Among the most enriched miRNAs in renal tissues, computational analysis detected a miR-27b binding sequence in the 3′UTR of snail mRNA (Fig. 4a) 9 . MiR-27b mimics significantly inhibited the reporter activity of Luc-snail 3′UTR. Site-directed mutations rescued the miR-27b-mediated inhibition of Luc-snail 3′UTR (Fig. 4b).
Transfection with miR-27b mimics also reversed the expression of snail in tubular cells treated with C3a or transfected with LOC105375913 plasmid (Fig. 4c,d). Conversely, transfection with miR-27b antisense oligonucleotide (ASO) increased the level of snail in HK-2 cells (Fig. 4e). PCR analysis showed that the level of miR-27b but not pri-miR-27b was decreased in the tubulointerstitial tissues of FSGS patients and in tubular cells treated with C3a, suggesting a post-transcriptional mechanism involved in the regulation of miR-27b in tubular cells of FSGS (Fig. 4f,g).
Bioinformatic analysis with RNAHybrid software showed that, LOC105375913 contains the response element of miR-27b (Fig. 4h). RNA pull-down and PCR analysis showed that miR-27b directly bound to LOC105375913 in tubulointerstitial tissues, and this binding was significantly increased in the tissues of FSGS patients compared to normal controls (Fig. 4i). Treatment with C3a increased the binding between LOC105375913 and miR-27b, which was prevented by LOC105375913 siRNA (Fig. 4j). Silence of LOC105375913 also decreased the level of snail in C3a-treated tubular cells (Fig. 4k).
C3a induces the expression of LOC105375913 by activating p38 MAPK pathway in tubular cells. Protein kinase B (AKT), p38 and extracellular signal-regulated kinase (ERK) function downstream of C3aR. The p38 inhibitor SB203580 but not the AKT inhibitor MK2206 or the ERK inhibitor PD098059 significantly prevented the upexpression of LOC105375913 in tubular cells treated with C3a (Fig. 5a). The p38 inhibitor SB203580 also suppressed the binding between LOC105375913 and miR-27b, and decreased the level of snail, FN and Col I in tubular cells treated with C3a (Fig. 5b,c). Treatment with C3a induced the phosphorylation of p38 in tubular cells, while overexpression of LOC105375913 didn't change the level of p-p38 in tubular cells (Fig. 5d,e). Conversely, treatment with p38 activator induced the expression of LOC105375913, promoted the binding between LOC105375913 and miR-27b, and increased the level of snail, FN and Col I in tubular cells ( Fig. 5f-h).
Transcription factor XBP-1s regulates the expression of LOC105375913 in tubular cells. The PROMO (TRANSFAC 8.3) software was applied to explore the transcription factors that may bind to the LOC105375913 promoter region. Among the predicted transcriptional factors, 5 factors have been reported to be the downstream substrates of p38 MAPK in the literature 10 . We knocked down the expression level by siRNAs, and found that knockdown of XBP-1s significantly inhibited the increase of LOC105375913 in HK-2 cells treated with C3a (Fig. 6a). The phosphorylation of XBP-1s was increased in tubular cells treated with C3a, and inhibition of p38 prevented the C3a-induced phosphorylation of XBP-1s (Fig. 6b).
ChIP analysis confirmed that XBP-1s bound to the LOC105375913 promoter in tubular cells, which was enhanced by C3a treatment (Fig. 6c). Overexpression of XBP-1s increased the expression of luciferase reporter construct containing the binding sequence. Site-directed mutations rescued the XBP-1s-mediated upregulation of the LOC105375913 promoter-luciferase reporter plasmid (Fig. 6e). XBP-1s overexpression also increased the level of endogenous LOC105375913, promoted the binding between LOC105375913 and miR-27b, and increased the expression of snail, FN and Col I in tubular cells (Fig. 6f-h). Conversely, knockdown of XBP-1s suppressed C3a-induced the binding between LOC105375913 and miR-27b, and decreased the expression of snail, FN and Col I in tubular cells (Fig. 6i,j).

Overexpression of LOC105375913 induces tubulointerstitial fibrosis in mice. LOC105375913
was not conserved in mouse. To analyze the pathogenic role of LOC105375913 in vivo, we elevated renal LOC105375913 expression by hydrodynamic-based delivery of LOC105375913-expressing plasmid in mice. After 8 weeks of treatment, LOC105375913 was obviously expressed in the tubular cells of mice (Fig. 7a,b). Mice overexpressing LOC105375913 caused obvious tubulointerstitial fibrosis and an increase in serum creatinine (Fig. 7c,d). Overexpression of LOC105375913 didn't change the level of pri-miR-27b, instead it decreased the level of miR-27b in the tubulointerstitial tissues of mice (Fig. 7e). RNA pull-down and RT-PCR analysis showed that the binding between LOC105375913 and miR-27b was increased in the tubulointerstitial tissues of mice overexpressing LOC105375913 (Fig. 7f). Meanwhile, overexpression of LOC105375913 increased the expression of snail in tubulointerstitial tissues of mice (Fig. 7g). As a result, the level of FN and Col I were obviously increased in the mice overexpressing LOC105375913 (Fig. 7h,i).

Discussion
FSGS accounts for about 40% of nephrotic syndrome in adult 1 . Most of the patients suffer non selective proteinuria, hypertension, renal impairment with tubular dysfunction at onset. In pathology, patients with FSGS often show severe renal tubulointerstitial lesions 11 . The prognosis of FSGS patients is poor, with renal survival rate only 43.8% at 10 years after the biopsy. By multivariate proportional analysis, chronic tubulointerstitial injury was one of the independent predictors of end-stage renal disease in FSGS patients 2 .
LncRNA refers to noncoding RNA which has more than 200 nucleotides 12 . LncRNAs plays an important role in the development of renal disease. Puthanveetil P et al. reported that lncRNA MALAT1 regulates glucose-induced up-regulation of inflammatory mediators IL-6 and TNF-α through activation of SAA3 in the endothelial cells 13 . Sun SF et al. showed that lncRNA Erbb4-IR promotes renal fibrosis by suppressing miR-29b in db/db mice 14 . We completed a tubulointerstitial transcriptional analysis of 5 cases of FSGS and 5 normal controls, the level of LOC105375913 showed the maximum increase in the tubulointerstitial tissues of FSGS patients, and overexpression of LOC105375913 induced the expression of FN and Col I in tubular cells.
Complement activation plays an important role in the progression of chronic kidney disease. In patients with FSGS, the permeability of glomerular filtration membrane increased, and plasma complement protein leaked into the renal tubule lumen. In our transcriptome analysis, the level of C3 mRNA was also increased in tubular tissues of FSGS patients. Therefore, both systemic and local complement synthesis contribute to intrarenal complement protein activation in FSGS patients. Zaferani A et al. found that tubular heparan sulfate serves as a docking platform for the alternative complement component properdin in proteinuric renal disease 15 . Properdin stabilizes the alternative pathway convertase that lead to the formation of C3a and C5b-9. Tang Z et al. reported that C3a induces tubular epithelial to mesenchymal transition in proteinuric nephropathy 7 . Our results indicate that C3a induces the upexpression of LOC105375913 in tubular cells of FSGS. For statistical analysis, one-way ANOVA with Tukey's post hoc test was used for (a,b and g) and a two-tailed Student's t-test was used for (f). *P < 0.05 compared with control. # P < 0.05 compared with C3a-treated cells. Snail is a zinc finger protein, which promotes the epithelial to mesenchymal transition in renal tubular cells 16 . The level of snail expression was upregulated in unilateral ureteral obstruction rat model and TGF-β1-treated renal tubular cells 17   reported that lncRNA PFAL promotes lung fibrosis through CTGF by competitively binding miR-18a 22 . Among the 50 miRNAs enriched in renal tissues, miR-27b was predicted and confirmed to target the 3′ UTR region of snail mRNA 9 . Previously, Graham JR et al. reported that TGF-β1 promoted fibrosis process by inhibiting miR-27b expression in alveolar epithelial cells 23 . Our analysis showed that miR-27b response element existed in the sequence of LOC105375913. Overexpression of LOC105375913 decreased the level of miR-27b, increased the level of snail and caused tubulointerstitial fibrosis in mice. Activation of p38 MAPK has been reported to be involved in renal fibrosis by independent studies 24,25 . Our experiments showed that inhibition of p38 MAPK reversed the upregulation of LOC105375913 in C3a-treated tubular cells. We explored and confirmed that, the activation of p38 phosphorylated XBP-1s, which bound to the promoter and increased the expression of LOC105375913 in tubular cells. Inhibition of p38 or silence of XBP-1s decreased the level of LOC105375913, suppressed the binding between LOC105375913 and miR-27b, decreased the level of snail, FN and Col I in tubular cells treated with C3a. In consistent with our findings, Zhao S et al. reported that XBP-1s is involved in the lipid synthesis in renal tubular epithelial cells treated with high glucose 26 . Mo XT et al. reported that XBP-1s participates in snail expression in alveolar epithelial cells 27 .
In conclusion, C3a/p38/XBP-1s pathway induces the expression of LOC105375913 in renal tubular cells, and LOC105375913 promotes the expression of snail and induce fibrogenesis through competitive binding of miR-27b in tubular cells of FSGS patients.  Gene expression profile analysis. Transcriptomic analysis of microdissected tubulointerstitial tissues were performed with Affymetrix HTA2.0 microarrays in according to standard procedures as described by Affymetrix. The accession number for the microarray data reported in this paper is GEO: GSE121211.
Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in phosphate buffered saline (PBS) for 30 min. The sections were incubated for 1 h at room temperature with primary antibody diluted in 1% BSA in PBS (Supplementary Table S2). The staining was visualized with Polyvalent HRP/DAB detection kit (ab64264, Abcam, Cambridge, USA). Negative controls were obtained by omission of the primary antibody from the staining procedure.
Western blot analysis. Western blots were performed as previously described 32 . Tissues or cells were lysed in RIPA buffer supplemented with protease inhibitors. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Sigma, St. Louis, MO). 25 μg of the total proteins was loaded into the wells of 10% SDS-PAGE along with the molecular weight markers. After running gel for 1 hour, the proteins were transferred