Inhibition of IRE1/JNK pathway in HK‐2 cells subjected to hypoxia‐reoxygenation attenuates mesangial cells‐derived extracellular matrix production

Abstract Endoplasmic reticulum (ER) stress and inflammatory responses play active roles in the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). Inositol‐requiring enzyme 1 (IRE1) activates c‐Jun NH2‐terminal kinase (JNK) in ER stress. Tubular epithelial cells (TEC) are the main injury target and source of AKI inflammatory mediators. TEC injury may lead to glomerulosclerosis, however, the underlying mechanism remains unclear. Here, hypoxia/reoxygenation (H/R) HK‐2 cells were used as an AKI model. To determine the partial effects of TEC injury on the glomerulus, HK‐2 cells after H/R were co‐cultured with human renal mesangial cells (HRMC). H/R up‐regulated ER stress, IRE1/JNK pathway, IL‐6 and MCP‐1 in HK‐2 cells. Stimulation of HRMC with IL‐6 enhanced their proliferation and the expression of glomerulosclerosis‐associated fibronectin and collagen IV via signal transducer and activator of transcription 3 (STAT3) activation. Similar responses were observed in HRMC co‐cultured with HK‐2 cells after H/R. IRE1/JNK inhibition reversed these injury responses in HRMC. IRE1/JNK stable knock‐down in HK‐2 cells and shRNA‐mediated STAT3 depletion in HRMC confirmed their role in inflammation/glomerulosclerosis. These findings suggest that IRE1/JNK pathway mediates inflammation in TEC, affecting mesangial cells. The inhibition of this pathway could be a feasible approach to prevent AKI‐CKD transition.


| INTRODUC TI ON
Acute kidney injury (AKI) is a global public health concern characterized by the rapid loss of renal function, associated with high morbidity, mortality and healthcare costs. 1,2 AKI has been recognized as a major risk factor for chronic kidney disease (CKD) and end-stage kidney disease (ESRD). 3 According to a study developed in the USA, Canada, Western Europe and Australia, approximately 1.5 million people suffering from AKI survive each year. Among them, 15%-20% progress to advanced CKD within 24 months, resulting in approximately 300 000 cases of advanced CKD each year. 4 According to a nationwide survey in China, approximately 3 million adult AKI patients are cured in hospitals across the country every year, of note, around 50% develop CKD, resulting in 1.5 million CKD-cases per year. 4 Moreover, another study estimated that 20% of patients experiencing an episode of AKI would develop CKD within 3 years. 5 Even the milder form of AKI can have adverse outcomes and may lead to renal fibrosis, the final pathway to CKD and ESRD. 3 The mechanisms of AKI-CKD transition are extremely complex and include maladaptive repair in renal tubules, endoplasmic reticulum (ER) stress, inflammation, mitochondrial damage, cell cycle arrest, autophagy and senescence. [6][7][8][9][10][11] The ER stress and inflammatory response, as well as maladaptive repair appear to be critical mediators of chronic histological changes. 12,13 Endoplasmic reticulum stress has been implicated in the pathogenesis of both AKI and CKD. [14][15][16][17] In response to cellular stress, unfolded or misfolded proteins accumulate in the ER, triggering the unfolded protein response, which involves three ER stress sensors, double stranded RNA activated protein kinase like ER kinase (PERK), activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1). 1 The activation of IRE1 stimulates the c-Jun NH 2terminal kinase (JNK) pathway, which regulates cell survival and secretion of pro-inflammatory cytokines to promote the characteristic inflammatory milieu. 18 It is reported that specific TEC injury could lead to glomerulosclerosis. The accumulation of extracellular matrix (ECM) in the glomerulus is an important feature of glomerulosclerosis. 3 The main ECM cell-secretors in glomerulus are mesangial cells. Recent studies using db/db mice have shown that the activation of the signal transducer and activator of transcription 3 (STAT3) pathway in mesangial cells by IL-6 leads to the secretion of fibronectin (FN), which, in turn, promotes renal fibrosis and sclerosis. [19][20][21] Therefore, the IL-6/ STAT3 pathway may mediate inflammatory damage in mesangial cells and glomerular ECM, which may promote AKI-CKD transition. 11 However, the mechanism behind remains largely unclear. To study the effect of TEC on mesangial cells in AKI-CKD transition, we stably knocked down IRE1α or JNK1 in human renal proximal tubular epithelial cells (HK-2 cells) and exposed them to hypoxia/reoxygenation (H/R) injury. Afterwards, we silenced STAT3 in human renal mesangial cells (HRMC), stimulated them with IL-6 and co-cultured HRMC with HK-2 cells. Our findings demonstrate that IRE1α or JNK1 silencing in H/R HK-2 cells attenuate chronic injuries in HRMC. Mechanistically, inhibiting the IRE1/JNK pathway weakened inflammation, STAT3 signalling, cell proliferation and ECM production. Thus, the modulation of the IRE1/JNK pathway may prevent glomerulosclerosis and represent a potential therapeutic strategy to impede AKI-CKD transition. supplemented with 10% foetal bovine serum (FBS, Gibco), 100 U/ mL penicillin (Geneview) and 100 µg/mL streptomycin (Geneview) in a humidified incubator with 5% CO 2 at 37°C. The subculture of HK-2 cells was performed using 0.25% Trypsin-EDTA (Gibco). To induce a hypoxia-reoxygenation injury model, HK-2 cells with serum-free DMEM/F12 were exposed to 4 hours of hypoxia (5% CO 2 , 1% O 2 , 94% N 2 at 37°C), followed by 6, 12 or 24 hours of reoxygenation (5% CO 2 , 95% air at 37°C) in normal culture medium. Controls were incubated in a humidified incubator with 5% CO 2 at 37°C. IRE1α or JNK1 knock-down HK-2 cell lines (stable depletion) were generated by Obio Technology Corp. Ltd. The two stable cell lines were incubated with 0.4 µg/mL puromycin (Solarbio) in DMEM/F12 medium. Of note, the knock-down efficiency of both genes was greater than 70%. In some experiments, these cells were subjected to H/R treatment.

| STAT3 knock-down in HRMC
The STAT3 shRNA adenovirus tagged with a GFP fluorescent protein was constructed by the Han Heng Biotechnology Company. STAT3 shRNA adenovirus transfection was carried out to knockdown STAT3 gene expression in HRMC. In brief, the STAT3 shRNA adenovirus (multiplicity of infection, MOI = 100) was added to 50% confluent HRMC cells, and cells were incubated for 6 hours.
Following transfection, the culture media was removed and HRMC were cultured with fresh medium for 24 hours. Thereafter, cells were stimulated with 0.1 ng/mL human IL-6 for another 24 hours.
Control HRMC were transfected with a control adenovirus without STAT3 shRNA using the same method. Cells were subsequently harvested and prepared for further analysis. The top strand of STAT3 shRNA was: AATTCGTGGACAATATCATTGA

| Co-culture system
Trans-well co-culturing was performed using six-well polystyrene plates (Corning, Inc) and inserts with permeable membranes having a pore size of 0.4 µm. HRMC were seeded into the lower chamber, and HK-2 cells in the upper chamber (insert). Stable HK-2 cell lines with control lentivirus or depletion, were cultured in hypoxic conditions for 4 hours, and then were moved into the wells containing HRMC and both cell types were co-cultured in reoxygenation for 24 hours.

| Western blotting
Cells were lysed in radioimmunoprecipitation assay buffer supplemented with protease phosphatase inhibitors. Protein samples were processed for immunoblot analysis as previously reported. 23 The following primary antibodies were used: β-actin

| Detection of cytokines by enzyme-linked immunosorbent assay (ELISA)
HK-2 cell culture supernatants were collected and subsequently centrifuged for 25 minutes at 2500 rpm and 4°C. We used a sandwich ELISA kit for the measurement of IL-6 (R&D Systems) and MCP-1 (R&D Systems), following the manufacturer's instructions.

| Transmission electron microscopy
Following H/R, HK-2 cells were trypsinized and centrifuged at 1000 rpm for 10 minutes. Thereafter, cells were soaked with 2% glutaraldehyde for a minimum of 1 hours at 4°C. Cells were then thoroughly washed with 0.1 mol/L phosphate buffer saline (PBS, without disrupting the cell pellet). Following decanting of the supernatant, 1% osmium acid (in PBS) was added and the mixture left standing for 1.5 hours. Following another washing step, cells were dehydrated with increasing concentrations of acetone (30%, 50%, 70%, 90% and 100%) and embedded in 812 resin (Canemco, 034).
The cell structure and organelles, including the ER were observed using a JEOL JEM-1400 Plus Transmission Electron Microscope (JEOL USA, Inc).

| RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using a commercial RNA Extraction Kit (Qiagen) according to the manufacturer's protocol.

| Immunofluorescence assay
Human renal mesangial cells were cultured onto cover slips in 6-well plates and then exposed as previously described. At the end of exposure, the cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature. Slides were washed in PBS for three times, the target preparation was defined using a liquid blocker pen. Subsequently, the target area was incubated with 3% BSA at room temperature for 30 minutes, and then with primary antibodies FN (ab2413; Abcam) and Col IV (ab6586; Abcam), overnight at 4°C, in a wet box. After three washing steps with PBS (pH 7.4), cell climbing slides were incubated with secondary antibodies, FITC conjugated goat anti-rabbit IgG (GB22303; Servicebio) or Cy3 conjugated goat anti-rabbit IgG (GB21303; Servicebio), at room temperature for 50 minutes. and then cells were stained with 0.2 mg/mL DAPI and analysed using a Zeiss LSM 880 confocal microscope (Carl Zeiss).

| HRMC proliferation assessment using the cell counting kit-8 assay
Human renal mesangial cell were seeded in 96-well tissue culture plates (Costar) at a density of 1 × 10 5 cells/mL in 100 µL MsCM.
Following starvation in serum-free MsCM for 24 hours, cells were washed and stimulated with 0.1 ng/mL human IL-6 for 12, 24 or 36 hours. Controls were incubated with MsCM only. Cell proliferation was assessed using the cell counting kit-8 (CCK-8) (Dojindo).
Briefly, 10 µL of CCK-8 was added to each well and incubated at 37°C for 2 hours. Thereafter, the absorbance was measured at 450 nm using a microplate reader (iMark; Bio-Rad). In the co-culture system, HRMC were co-cultured with H/R HK-2 cells in six-well plates for 24 hours. Subsequently, the method described above was used to detect HRMC proliferation.
The experiments were performed in triplicate and repeated three times. The % cell proliferation was calculated as follows:

| Statistical analyses
Normally distributed data, representative of at least three independent experiments, are expressed as the mean ± standard deviation (SD). For continuous variables, independent-sample t tests and oneway ANOVA tests were performed for comparisons between two, or more than two groups, respectively. A P-value of <.05 was considered significant. Analyses were performed using the SPSS software version 17.0 (SPSS Inc).

| H/R-induce ER stress and inflammatory cytokines in HK-2 cells
Endoplasmic reticulum stress was investigated in HK-2 cells exposed to H/R. As shown in Figure 1A,B, the expression of ER stress markers (GRP78, PERK, ATF6, pIRE1α, pJNK and CHOP) increased significantly at 6, 12 and 24 hours of reoxygenation following 4 hours hypoxia, compared with that in the control (at least P < .05).
Moreover, qRT-PCR analysis revealed a significantly increased expression of CHOP mRNA in HK-2 cells at 6, 12 and 24 hours of reoxygenation following 4 hours of hypoxia (at least P < .05) compared with that in control ( Figure 1C). Importantly, transmission electron microscopy showed that the width of ER in HK-2 cells increased after H/R ( Figure 1D).
Inflammatory cytokines in HK-2 cells also increased after H/R. Figure 1E,F IL-6 and MCP-1 protein and mRNA expression increased significantly in H/R HK-2 cells, compared with that in the control (at least P < .05). Of note, we observed that enhanced expression of IL-6 and MCP-1 in HK-2 cells was directly proportional to reoxygenation time following 4 hours of hypoxia ( Figure 1E,F).

| IL-6 induced activation of STAT3 and ECM production in HRMC
Human renal mesangial cell treated with IL-6 for 12, 24 and 36 hours showed significant increase in expression of p-STAT3, and the main components of ECM (FN and Col IV), compared with those in control cells(at least P < .01 vs control) ( Figure 4A,B). The immunofluorescence analysis of FN and Col IV showed the same results ( Figure 4C).
qRT-PCR analysis also revealed an increase in FN and COL4A1 mRNA expression in HRMC following the stimulation with IL-6 (FN at 36 hours: P < .0001, others: P < .001 vs control) ( Figure 4D).

IL-6 also induced a significant, and time-dependent increase
in the proliferation of HRMC compared with that of control cells (12 hours: P < .01, others: P < .0001 vs control) ( Figure 4E).
To confirm that the effects of IL-6 in HRMC were mediated by Similarly, the IL-6 induced proliferation was also significantly lower in STAT3-depleted HRMC vs vehicle-transfected HRMC (P < .01; Figure 5D).

| Depletion of IRE1/JNK in HK-2 cells decrease STAT3 phosphorylation and ECM deposition in cocultured HRMC, following 24 hour of H/R
The protein expression of pSTAT3, FN and Col IV in HRMC cocultured with vehicle-infected HK-2 cells following 24 hours of H/R was significantly higher than that in HRMC co-cultured with vehicle-infected HK-2 cells not subjected to H/R (at least P < .05).
Moreover, FN and Col IV immunofluorescence revealed the same results ( Figures 6B and 7B). COL4A1 and FN mRNA levels were also significantly increased in HRMC co-cultured with vehicle-infected HK-2 cells in the context of 24 hours of H/R vs no H/R ( Figure 6C and 7C, FN: P < .0001, COL4A1: P < .001).
In addition, protein and mRNA levels of FN and Col IV in HRMC were significantly decreased upon co-culture with IRE1α-or JNK1-depleted HK-2 cells following 24 hours H/R, compared to those in HRMC co-culture with vehicle-infected HK-2 cells following 24 hours of H/R ( Figure 6A-C and 7A-C, at least P < .01).

| D ISCUSS I ON
Acute kidney injury is a pathological condition characterized by the injury and subsequent death of renal tubular epithelial cells. 14,24,25 TEC, a specialized tubular segment adjacent to the glomerulus, is not only the target of injury but also an important mediator of renal fibrosis progression. 26 Several responses to kidney injury are prone to trigger ER stress in AKI. 12 ER stress also contributes critically to the development of chronic kidney pathologies and CKD following AKI. Importantly, inhibition of ER stress may represent a potential findings suggest that targeting IRE1 or JNK may be a useful strategy to treat for glomerulosclerosis and to prevent AKI-CKD transition.

ACK N OWLED G EM ENTS
This work was supported by a grant from the National Natural

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data in this study are openly available in a public repository.