LncRNA 148400 Promotes the Apoptosis of Renal Tubular Epithelial Cells in Ischemic AKI by Targeting the miR−10b−3p/GRK4 Axis

Although recent studies have reported that long non-coding RNA (lncRNA) is involved in the development of ischemic acute kidney injury (AKI), the exact function and regulatory mechanism of lncRNAs in ischemic AKI remain largely unknown. Herein, we found that ischemic injury promoted the expression of lncRNA 148400 in mouse proximal tubule-derived cell line (BUMPT) and C57BL/6J mice. Furthermore, the lncRNA148400 mediates ischemic injury-induced apoptosis of BUMPT cells. Mechanistically, lncRNA 148400 sponged miR−10b−3p to promote apoptosis via GRK4 upregulation. Finally, knockdown of lncRNA 148400 alleviated the I/R-induced deterioration of renal function, renal tubular injury, and cell apoptosis. In addition, cleaved caspase−3 is increased via targeting the miR−10b−3p/GRK4 axis. Collectively, these results showed that lncRNA 148400/miR−10b−3p/GRK4 axis mediated the development of ischemic AKI.


Introduction
Acute kidney injury (AKI) is a common and severe disease, characterized by a significant increase in serum creatinine level over a short time period and is associated with a sudden decrease in urine volume [1,2]. Patients with severe AKI have a high mortality rate of almost 40-60% [1,3]. Ischemia-reperfusion injury (I/R) is one of the most common causes of AKI [4]. To date, there is no effective method to prevent AKI progression, except for dialysis and renal transplant. Hence, we explored the underlying pathogenesis of AKI.
The present study is the first to report that lncRNA 148400 is induced by ischemic injury in vitro and in vivo. Functionally, lncRNA148400 promotes apoptosis of BUMPT

Ischemic AKI Model
Male C57BL/6J mice aged 8-10 weeks were purchased from Shanghai Animal Center (Shanghai, China) and maintained under a 12-h light/dark cycle with free access to food and water. Before the I/R injury model was established, the lncRNA 148400 siRNA (15 mg/kg per injection) was injected into the tail vein of male C57BL/6J mice twice a day for 1 day [23]. The renal blood supply was blocked for 28 min and then restored for 24 or 48 h [22,24].

Immunoblot Analysis
Western blotting was performed as described previously [25][26][27][28]. Whole cell and renal cortex lysates were detected using SDS/PAGE and transferred to nitrocellulose membrane, blocked in 5% milk, incubated with the corresponding primary and secondary antibodies, and then detected using Western lightning-enhanced chemiluminescence reagent.

Fluorescence In Situ Hybridization (FISH) Analysis
Fluorescent probes of lncRNA 148400 and miR−10b−3p, U6, and 18S were purchased from Ribo. The nuclei of BUMP cells were stained with U6, the cytoplasm was stained with 18S, and lncRNA 148400 was labeled with Cy3. The sections from BUMPT cells and mouse kidneys were hybridized with the corresponding probes overnight and then stained with DAPI. Fluorescence imaging was performed using a laser scanning confocal microscope.

Flow Cytometry (FCM) Analysis of Apoptosis
Apoptosis was examined using annexin V-FITC/PI staining. The BUMPT cells were digested and collected using 0.25% trypsin without EDTA. The cells were washed twice with cold PBS. According to the manufacturer's instructions for Annexin V apoptosis detection kit (Cat. No. 556547; BD Pharmingen, Franklin, NJ, USA), the cells were resuspended with binding buffer and incubated in the dark for 15 min after Annexin V staining. Then, the cells were stained with PI for 5 min. Finally, 200 µL of binding buffer was added to detect cell apoptosis.

Renal Function and Morphology
The levels of serum creatinine and urea nitrogen were used to evaluate blood and renal function, respectively, according to the instructions of renal function examination kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The morphology of renal tissue was assessed using hematoxylin and eosin (H&E) staining [32][33][34]. In addition, TUNEL staining was used to assess apoptosis [35][36][37].

Statistical Analysis
The two groups were compared using two-tailed Student's t-tests. Multiple group comparisons were performed using one-way ANOVA or Two-way ANOVA. Quantitative data are expressed as mean ± SD. Differences with p < 0.05 were statistically significant.

I/R-Induced Expression of lncRNA 148400 in BUMPT Cells and Mice Kidneys
We investigated whether lncRNA 148400 was involved in I/R-induced injury. Before this, we detected the renal function changes at I/R (28 min/24 h, 48 h and 72 h). The data showed that blood urea nitrogen (BUN) and serum creatinine concentrations were increased at 24 h after reperfusion, and then reached a peak at 48 h after reperfusion, and finally declined at 72 h after reperfusion (Supplementary Figure S1A,B). Hence, 48 h after reperfusion was selected as an observation point. First, C57/BL6 mice were subjected to I/R (28 min and 48 h). The serum levels of blood urea nitrogen (BUN) and creatinine were gradually increased at 24 h after reperfusion and reached a peak at 48 h after reperfusion ( Figure 1A,B). Similarly, H&E staining indicated that I/R induced slight and moderate renal tubular injury at 24 and 48 h after reperfusion, respectively, which was further confirmed by the tubular damage score ( Figure 1C,D). Furthermore, RT−qPCR analysis demonstrated that the mRNA level of lncRNA 148400 was gradually increased at the indicated time points ( Figure 1E). The immunoblot analysis showed that the expression of cleaved caspase−3, but not caspase-3, was increased at 24 and 48 h after reperfusion ( Figure 1F,G). Subsequently, RT−qPCR analysis indicated that the mRNA expression of lncRNA 148400 was upregulated at 0 h after oxygen deprivation by antimycin, attained a peak at 2 h afterward, and declined  Figure 1H). The trend of expression level of cleaved caspase−3 was consistent with that of lncRNA 148400 ( Figure 1I,J). Finally, FISH analysis demonstrated that lncRNA 148400 was mainly localized in the cytoplasm of BUMPT cells ( Figure 1K). These data suggest that the expression of lncRNA 148400 was increased under ischemic injury in vivo and in vitro.

LncRNA 148400 siRNA Ameliorates I/R-Induced Apoptosis of BUMPT Cells
Next, we explored the function of lncRNA 148400 in ischemic injury. BUMPT cells were transfected with or without lncRNA 148400 siRNA and then treated with I/R (2 h each). The RT−qPCR showed that the expression level of lncRNA 148400 was markedly suppressed under basic and I/R treatment ( Figure 2A). FCM results confirmed that lncRNA 148400 siRNA significantly suppressed I/R-induced cell apoptosis ( Figure 2B,C). These results were similar to those of immunoblotting analysis for cleaved caspase−3 and caspase-3 ( Figure 2D,E). These results indicated that lncRNA 148400 induces apoptosis in ischemic injury.

LncRNA 148400 Overexpression Enhances I/R-Induced Apoptosis in BUMPT Cells
Although we found that lncRNA 148400 knockdown attenuated the apoptosis caused by I/R, the effect of lncRNA 148400 overexpression on apoptosis remains unclear. The overexpression of lncRNA 148400 not only enhanced the lncRNA 148400mRNA level ( Figure 3A) but also increased the apoptosis and expression of cleaved caspase−3 in

LncRNA 148400 Overexpression Enhances I/R-Induced Apoptosis in BUMPT Cells
Although we found that lncRNA 148400 knockdown attenuated the apoptosis caused by I/R, the effect of lncRNA 148400 overexpression on apoptosis remains unclear. The overexpression of lncRNA 148400 not only enhanced the lncRNA 148400mRNA level ( Figure 3A) but also increased the apoptosis and expression of cleaved caspase−3 in BUMPT cells under standard and I/R conditions ( Figure 3B-E). Our data confirmed that lncRNA 148400 had a pro-apoptotic effect in renal ischemic injury.

miR−10b−3p Was Sponged by the lncRNA 148400
Previous studies reported that lncRNAs act as a competing endogenous RNA (ceRNA) to perform their functions [19,30]. Herein, we predicted that miR−10b−3p was one of the target miRNAs of lncRNA 148400 using the software RegRNA 2.0 ( Figure 4A). Subsequently, dual luciferase reporter (DLR) assay showed that miR−10b−3p mimics suppressed the luciferase activity of lncRNA 148400-WT but not lncRNA148400-MUT ( Figure 4B). FISH analysis showed that lncRNA 148400 and miR−10b−3p co-localized in the cell cytoplasm of both BUMPT cells and mouse kidney tissues under basic and I/R conditions ( Figure 4C). Finally, the RT−qPCR analysis indicated that the expression of miR−10b−3p was reversed by the lncRNA 148400 siRNA. By contrast, this effect was enhanced by the overexpression of lncRNA 148400 under basic and I/R conditions ( Figure 4D,E). Collectively, the data suggest that miR−10b−3p was a target of lncRNA 148400.

GRK4 Was a Target Gene of miR−10b−3p
Although miR−10b−3p suppressed apoptosis, its underlying mechanism is unclear. Using the website mirdb.org, we predicted that GRK4 was one of the target genes of G protein-coupled receptor kinase (GRK4). Figure 6 shows the sequence of complementary and mutated sites of GRK4 and miR−10b−3p ( Figure 6A,B). In addition, miR−10b−3p mimics significantly suppressed the mRNA and protein levels of GRK4 in BUMPT cells ( Figure 6C-E). Finally, we evaluated the function of GRK4. The immunoblot results demonstrated that GRK4 siRNA significantly reduced the I/R-induced expression of cleaved caspase−3 ( Figure 6F,G), which showed that GRK4 was an apoptosis inducer. These data indicated that miR−10b−3p targeted GRK4 to prevent apoptosis.

GRK4 was a Target Gene of miR−10b−3p
Although miR−10b−3p suppressed apoptosis, its underlying mechanism is uncl Using the website mirdb.org, we predicted that GRK4 was one of the target genes o protein-coupled receptor kinase (GRK4). Figure 6 shows the sequence of complement and mutated sites of GRK4 and miR−10b−3p ( Figure 6A,B). In addition, miR−10b mimics significantly suppressed the mRNA and protein levels of GRK4 in BUMPT c ( Figure 6C-E). Finally, we evaluated the function of GRK4. The immunoblot resu demonstrated that GRK4 siRNA significantly reduced the I/R-induced expression cleaved caspase−3 ( Figure 6F,G), which showed that GRK4 was an apoptosis indu These data indicated that miR−10b−3p targeted GRK4 to prevent apoptosis.

LncRNA 148400 siRNA Attenuated I/R-Induced BUMPT Cell Apoptosis, Which Was Reversed by miR−10b−3p Inhibitor
The rescue experiment was used to confirm whether miR−10b−3p mediated the function of lncRNA 148300. The RT−qPCR analysis results showed that lncRNA 148400 siRNA markedly suppressed I/R-induced expression of lncRNA 148400, which was not affected by the miR−10b−3p inhibitor ( Figure 7A). LncRNA 148400 siRNA reversed the I/R-induced downregulation of miR−10b−3p, which was prevented by the miR−10b−3p inhibitor ( Figure 7B). FCM and immunoblot analysis showed that lncRNA 148400 siRNA attenuated the I/R−induced apoptosis as well as the increase in GRK4 and cleaved caspase−3 in BUMPT cells. However, this effect was reversed by the miR−10b−3p inhibitor ( Figure 7C-F). The data provide strong evidence that miR−10b−3p was a key target of lncRNA 148400.

LncRNA 148400 siRNA Attenuates I/R-Induced AKI in Mice
We further evaluated the function of lncRNA 148400 in mice with I/R-induced AKI. LncRNA 148400 siRNA or saline was injected via the tail vein for 12 h, and the mice were

LncRNA 148400 siRNA Attenuates I/R-Induced AKI in Mice
We further evaluated the function of lncRNA 148400 in mice with I/R-induced AKI. LncRNA 148400 siRNA or saline was injected via the tail vein for 12 h, and the mice were subjected to I/R (28 min and 48 h). LncRNA 148400 siRNA significantly reduced the I/Rinduced increase in both serum Cr levels and BUN ( Figure 8A,B). In line with these results, H&E and TUNEL staining showed that lncRNA 148400 siRNA significantly ameliorated the I/R-induced renal tubular damage and apoptosis, respectively ( Figure 8C-F). The RT−qPCR analysis showed that lncRNA 148400 expression was silenced by lncRNA 148400 siRNA. By contrast, miR−10b−3p expression was reversed by lncRNA 148400 siRNA under sham and I/R treatment conditions ( Figure 8G,H). Finally, lncRNA siRNA suppressed the I/R-induced increase in GRK4 and cleaved caspase−3 ( Figure 8I,J). Taken together, these data suggest that the lncRNA 148400/miR−10b−3p/GRK4 axis mediated the progression of ischemic AKI. subjected to I/R (28 min and 48 h). LncRNA 148400 siRNA significantly reduced the I/Rinduced increase in both serum Cr levels and BUN ( Figure 8A,B). In line with these results, H&E and TUNEL staining showed that lncRNA 148400 siRNA significantly ameliorated the I/R-induced renal tubular damage and apoptosis, respectively ( Figure 8C-F). The RT−qPCR analysis showed that lncRNA 148400 expression was silenced by lncRNA 148400 siRNA. By contrast, miR−10b−3p expression was reversed by lncRNA 148400 siRNA under sham and I/R treatment conditions ( Figure 8G,H). Finally, lncRNA siRNA suppressed the I/R-induced increase in GRK4 and cleaved caspase−3 ( Figure 8I,J). Taken together, these data suggest that the lncRNA 148400/miR−10b−3p/GRK4 axis mediated the progression of ischemic AKI.

Discussion
The role of lncRNAs in ischemic AKI remains largely unknown. Herein, we found that lncRNA 148400 promotes renal cell apoptosis in BUMP cells after ischemic injury. Furthermore, we found that lncRNA148400 acted as a ceRNA to suppress miR−10b−3p expression and increased GRK4 expression. Finally, the lncRNA 148400/miR−10b−3p/GRK4 axis mediated the progression of ischemic AKI.
For most lncRNAs, ceRNAs are a key mechanism involved in their function. The prediction using RegRNA 2.0 software and DLR assay demonstrated that lncRNA 148400 directly binds to miR−10b−3p ( Figure 4A,B), which was further confirmed by co-localization of lncRNA 148400 and miR−10b−3p ( Figure 4C). Interestingly, RT−qPCR showed that the expression of miR−10b−3p was negatively regulated by lncRNA 148400 under basic and I/R treatment conditions ( Figure 4D,E). The forementioned data confirmed that miR−10b−3p was a target of lncRNA 148400.
A recent study demonstrated that miR−10b−3p prevented I/R-induced brain cell apoptosis during cerebral injury [38]. In line with this, the present study showed that miR−10b−3p alleviated I/R-induced renal tubular cell apoptosis ( Figure 5). Previous studies reported that both Krüppel-like factor 5 (KLF5) and FOXO3 were downstream factors of miR−10b−3p [38,39]. We found that GRK4, a member of the G protein-coupled receptor kinases, is a target of miR−10b−3p, based on the DLR analysis and regulation experiments of miR−10b−3p and GRK4 ( Figure 6). A recent study reported that GRK4 promoted cardiomyocyte apoptosis [40]. Consistent with this, we also demonstrated that GRK4 knockdown reduced I/R-induced renal tubular cell apoptosis ( Figure 6). Finally, the rescue experiment confirmed that miR−10b−3p/GRK4 was a key downstream factor of lncRNA 148400 (Figure 7), which was further verified by silencing of lncRNA 148400 in mice ischemic AKI (Figure 8).
In conclusion, we found a novel pathogenesis of ischemic AKI, i.e., the lncRNA 148400/miR-378a-3p/Rab10 axis promotes renal cell apoptosis to mediate the progression of ischemic AKI.