MMP‐7 affects peritoneal ultrafiltration associated with elevated aquaporin‐1 expression via MAPK/ERK pathway in peritoneal mesothelial cells

Abstract Peritoneal membrane dysfunction and the resulting ultrafiltration failure are the major disadvantages of long‐term peritoneal dialysis (PD). It becomes increasingly clear that mesothelial cells play a vital role in the pathophysiological changes of the peritoneal membrane. Matrix metalloproteinases (MMPs) function in the extracellular environment of cells and mediate extracellular matrix turnover during peritoneal membrane homeostasis. We showed here that dialysate MMP‐7 levels markedly increased in the patients with PD, and the elevated MMP‐7 level was negatively associated with peritoneal ultrafiltration volume. Interestingly, MMP‐7 could regulate the cell osmotic pressure and volume of human peritoneal mesothelial cells. Moreover, we provided the evidence that MMP‐7 activated mitogen‐activated protein kinases (MAPKs)‐extracellular signal‐regulated kinase 1/2 (ERK) pathway and subsequently promoted the expression of aquaporin‐1 (AQP‐1) resulting in the change of cell osmotic pressure. Using a specific inhibitor of ERK pathway abrogated the MMP‐7‐mediating AQP‐1 up‐regulation and cellular homeostasis. In summary, all the findings indicate that MMP‐7 could modulate the activity of peritoneal cavity during PD, and dialysate MMP‐7 might be a non‐invasive biomarker and an alternative therapeutic target for PD patients with ultrafiltration failure.


| INTRODUC TI ON
Peritoneal dialysis (PD) is a process that provides patients not only with solute clearance but also with fluid removal, which is called ultrafiltration. Ultrafiltration failure remains a common complication of long-term PD, especially in patients without residual renal function (RRF). 1 Recurrent ultrafiltration failure in PD patients is closely associated with water retention, hypertension resistance, 2 pulmonary oedema 3 and acute or chronic congestive heart failure. 4 Besides interstitial thickening and sclerosis caused by repeated infection or long-term exposure to unphysiological dialysate, water channel proteins damaging in the peritoneal membrane is also an important factor for ultrafiltration failure. 5 Aquaporin-1 (AQP-1), one of the water-specific channel proteins distributed in the endothelium lining the peritoneal capillaries, facilitates the osmotic transport of water across the capillary endothelium, thereby playing an essential role in ultrafiltration during PD. 6 Knocking out AQP-1 gene in mice reduced osmotically driven water transport and approximately 50% of total ultrafiltration, while pharmacological agonist AqF026 enhanced this water transport. 7  and abnormal pathology in the kidney. 9 MMPs are increasingly known to cleave a wide variety of substrates, ranging from cell surface receptors and adhesion molecules to growth factors and cytokines. [9][10][11] Therefore, MMPs promote cell proliferation, migration and differentiation and are also thought to play a significant role in cell apoptosis, angiogenesis, tissue repair and immune response. 10 -12 Among all the MMPs, MMP-7 is the smallest secreted MMP with a broad substrate specificity, consisting of a pro-peptide domain and a catalytic domain. It plays an important role in degrading the extracellular matrix and many other biological activities. 13 Generally, the primary function of MMP-7 is to break down the extracellular matrix by digesting casein, gelatines, fibronectin and proteoglycan. Such an ability of MMP-7 acting on a broad spectrum of substrates indicates its role in controlling numerous biological processes such as tissue remodelling, apoptosis and inflammation. 13,14 MMP-7 has been detected in the serum in patients with disease states, such as chronic inflammatory diseases, some epithelialderived tumours and some acute organic injuries. 15,16 A previous study showed that MMP-7 is a downstream target gene of Wnt/ β-catenin signalling pathway and is involved in kidney fibrosis. 14 Even though the increased serum MMP-7 in chronic kidney disease (CKD) patients has proved to be acted as a non-invasive biomarker of pre-fibrotic signalling, 16 and the urine MMP-7 was a promising predictor for acute kidney injury severity, 13 the distribution and the potential role of MMP-7 in PD patients are still unknown. To test this, we conducted a cross-sectional study in a cohort of 295 PD patients. We observed a higher concentration of both serum and dialysate MMP-7 levels in these patients, and the increased MMP-7 in the peritoneal dialysate was negatively associated with peritoneal ultrafiltration. In human peritoneal mesothelial cells, we found that MMP-7 could regulate the cell osmotic pressure and volume. Furthermore, we provided the evidence that MMP-7 activated mitogen-activated protein kinases (MAPKs)-extracellular signal-regulated kinase 1/2 (ERK) pathway and subsequently promoted the expression of AQP-1, resulting in the change of cell osmotic pressure. These data suggested the possibility of using MMP-7 as a biomarker and an alternative therapeutic target for PD patients with ultrafiltration failure.

| Subjects
Healthy controls (HC, n = 20) and ESRD patients who had been undergoing PD for at least more than 1 month (n = 303) were involved in this cross-sectional study from 1 June 2018 to 31  The intra-and inter-assay variability ranged between 3%-6% and 2%-8% based on blinded replicate samples from study patients, respectively.

| siRNA-mediated RNA interference
Control siRNA (sictrl) and AQP-1-targeting siRNAs (siAQP-1) were purchased from RiboBio Biotechnology. HMrSV5 cells were transfected with the siRNA using the riboFECT CP (RiboBio) reagent according to the standard protocol and incubated for 36 hours before the next assay.

| Extraction of membrane and cytoplasm protein fractions
We used the ProteinExt® mammalian membrane protein extraction kit purchased from TransGen Biotech to separate membrane and cytoplasm protein of cells. 5 × 10 6 cells were washed twice with 1 ml of cold PBS and centrifuged for 3 minutes by 1,000 × g, and then, the supernatant was discarded. 750 μl of membrane protein extraction buffer I (MPEB I) was added to the cell precipitation, and the lysate was mixed well in vortex for 15 seconds. After the lysate was incubated in ice for 10 minutes, the mixture was centrifuged for 15 minutes by 16,000 × g in 4℃. The supernatant including cytoplasmic protein was carefully transferred into a clean EP tube. 150 μl MPEB I was added to the precipitation, and the precipitation was suspended again for 15 seconds, incubated in ice for 30 minutes. 300 μl MPEB I was added into the mixture, and then, the mixture was centrifuged for 15 minutes by 16,000 × g in 4℃. The supernatant including membrane protein was carefully collected into another EP tube. The extracted membrane and plasma protein can be used for further analysis.

| RNA isolation and real-time PCR
Isolated hepatocytes were homogenized in 1 ml TRIzol (Thermo Fisher Scientific), and total RNA was extracted based on the manufacturer's instruction. Total RNA was synthesized to cDNA using TransScript Fly First-Stand cDNA Synthesis SuperMix (TransGen).
Real-time PCR SYBR Green technology was applied to quantify reverse-transcribed mRNAs on an ABI Prism 7500 Sequence Detection System (Thermo Fisher Scientific), according to the following program. The levels of the target gene were normalized with respect to GAPDH gene expression.

| Immunofluorescence assay
HMrSV5 cells were seeded on a 3.5 mm confocal dish and fixed at room temperature using 4% paraformaldehyde, and the cells' membrane was broken with 0.5% Triton-X100 or not. AQP-1 proteins were stained and examined with confocal microscopy equipped with analytical software. The image was observed in three fields for each sample, and the mean fluorescence intensity of GFP and AQP-1 was measured by ImageJ software.

| Cell volume analysis
HMrSV5 cells were trypsinized and resuspended in different mediums (normal saline (NS) or 4.25% glucose peritoneal dialysis).
The cell diameter was measured using the Scepter 2.0 cell counter (Merck Millipore) as previously described. 17 After the resultant cell suspensions had been transferred to 5 ml EP tubes, the cells were counted with a Scepter 2.0 cell counter equipped with a sensor tip, according to the manufacturer's recommendation. Cell counts and cell size distributions were shown as histograms on the monitor of the Scepter 2.0 cell counter, and these data were analysed with the Scepter 2.0 Software Pro computer software. were assessed with either a chi-square test or a Fisher's exact test in patients with or without renal residual function (RRF). Unadjusted and adjusted (with age, sex, history of diabetic mellitus, RRF, peritoneal Kt/V and dialysate glucose concentration) linear regression analyses were performed to determine the relationships between MMP-7 and peritoneal ultrafiltrated volume. P < 0.05 was considered to be statistical significance.

| High-expressed MMP-7 was associated with peritoneal ultrafiltration in PD patients
Because 8 patients had failed to examine MMP-7, 295 patients entered the analysis at last. Among them, the mean age was 43.5 ± 14.6 years, 54.6% were men, and the median dialysis vintage was 3 (1-18) months.
Detailed demographic, clinical and laboratory characteristics between participants with RRF (n = 219) and without RRF (n = 76) are shown in Table 1. Patients without RRF had lower body mass index (BMI), blood pressure, eGFR and longer dialysis vintage than those with RRF. A significantly increased level of MMP-7 in serum was observed in patients with peritoneal dialysis compared with healthy controls (4.28 ± 2.34 ng/ml vs 0.50 ± 0.29 ng/ml, P < 0.0001; Figure 1a and Table S1). Interestingly, both serum and dialysate MMP-7 levels were lower in the patients without RRF than those with RRF ( Figure 1b).
Because the dialysate MMP-7 presented a tightly linear association with serum MMP-7 (r = 0.57, P < 0.001; Figure 1c), we mainly analysed the dialysate MMP-7. As shown in Figure 1d and Table 2, the dialysate MMP-7 level was negatively associated with peritoneal UF volume (P = 0.007). When adjusted with demographics (age, sex), history of diabetic mellitus, RRF, peritoneal dialysis adequacy (peritoneal KT/V) and dialysate glucose concentration, the dialysate MMP-7 still significantly correlated to peritoneal UF volume (β = −76.1, P = 0.039; Table 2). Taken together, these data indicate a negative association of the dialysate MMP-7 level with peritoneal UF volume in PD patients.

| High glucose induced the expression of MMP-7 in peritoneal mesothelial cells
Since glucose-based peritoneal dialysis solutions are still predominantly used dialysate in PD patients in China, and the exposure to glucose might correlate to some unphysiological changes, 18

| Recombinant MMP-7 protein altered cell volume and enhanced AQP-1 expression in peritoneal mesothelial cells
During peritoneal dialysis, the fluid pressure interacts with osmotic pressure to regulate cell volumes. We next investigated whether MMP-7 treatment affected the volume of peritoneal mesothelial cells. Compared to the control group, treatment with MMP-7 recombinant protein significantly altered the volume of HMrSV5 cells, indicating that MMP-7 modulated the cellular volume for maintaining intracellular osmotic pressure (Figure 3a). AQP-1, the universal water channel, is responsible for the rapid response of cell volume. 19 We then addressed whether MMP-7 affects the expression of AQP-1 in HMrSV5 cells. We observed that MMP-7-treated cells displayed

| Ectopic expression of MMP-7 altered cell volume and enhanced AQP-1 expression in peritoneal mesothelial cells
As known, MMPs have a novel proteolytic role in vivo. 20 We, therefore, raised a question of whether ectopic expression of MMP-7 influences the cellular volume in peritoneal mesothelial cells. The      Low level of MMP-7 is expressed in normal healthy people.
However, it is markedly induced in human and animal models of CKD and AKI. Many evidence suggests that the MMP-7 level could serve as a non-invasive biomarker for predicting AKI prognosis and monitoring CKD progression. 21,22 PD is a technique that exposes the patient to glucose, which may result in structural and functional damage of the peritoneal membrane. 18 Of note, our study showed that MMP-7 was significantly increased in the ESRD patients receiving PD. Peritoneal dialysate, which is continuously stayed in the peritoneal and renewed 4 times per day, is a major way for solute and fluid clearance in PD patients. 23 In our cohort study, we did confirm the marked expression of MMP-7 in PD patients, not only in the serum but also in the dialysate, and the dialysate MMP-7 presented the quite well linear association with serum MMP-7. Furthermore, after detailed analyses, we found that dialysate MMP-7 was negatively associated with peritoneal ultrafiltration. These data indicated that MMP-7 might affect the function of the peritoneal membrane, which might play a new pathophysiological role in PD ultrafiltration.
During peritoneal dialysis, peritoneal mesothelial cells are exposed repeatedly to a non-physiological hypertonic environment.
Longer exposure to the hypertonic medium was associated with partial recovery of the reduced mesothelial cell volume. 24 Thus, the cells ceramide pathway and ERK pathway and DNA synthesis. 40 Our current study provided evidence that MMP-7 enhanced the activation of the ERK pathway in peritoneal mesothelial cells. Importantly, the inhibitor of ERK U0126 attenuated MMP-7 inducing AQP-1 production, which suggested that MMP-7 enhanced the cellular osmotic pressure by activating the ERK pathway. Indeed, activation of ERK F I G U R E 5 MMP-7-mediated ERK signalling activation is responsible for the enhancement activity of MMP-7 on cellular osmotic pressure in peritoneal mesothelial cells. The HMrSV5 cells were seeded in the 6-well plate (1 × 10 6 cells/well). (a) HMrSV5 cells were stimulated with 100 ng/ml MMP-7 protein for indicated time points, and the phosphorylation level of ERK, JNK and p38 was detected by Western blotting. (b) HMrSV5 cells were treated with different concentrations of MMP-7 protein for 1 hour, and the phosphorylation level of ERK, JNK and p38 was detected by Western blotting. (c, d) After pre-incubation with U0126 for 2 hours, HMrSV5 cells were stimulated by 100 ng/ ml MMP-7 protein for 24 hours. The mRNA and protein expression level of AQP-1 was detected by quantitative RT-PCR (C) and Western blotting analysis (d), respectively. (e, f) The HMrSV5 cells were infected with negative control lentivirus and MMP-7-overexpressed lentivirus and incubated with U0126 for another 24 hours. The mRNA and protein expression level of AQP-1 was detected by quantitative RT-PCR (e) and Western blotting analysis (f), respectively. (g, h) After pre-incubation with U0126 for 2 hours, the MMP-7-overexpressed HMrSV5 cells were incubated in NS or dialysate with 4.25% glucose for 1 minute. The cell diameter of peritoneal mesothelial cells was evaluated by the Scepter 2.0 cell counter (g). The changes of cell diameter after glucose stimulation were calculated (h). P < 0.05, **P < 0.01, NS, not significant, one of the three independent experiments is shown pathway is essential for cell growth, differentiation and integrin expression. 41 Low et al observed that integrin is a family of multifunctional protein with functions in maintenance and regulation of cell volume. 42 It is of interest to demonstrate whether MMP-7 modulate cell volume through the ERK integrin axis, which is worth further research.
In summary, increased dialysate MMP-7 level was negatively associated with peritoneal ultrafiltration in PD patients, and the MMP-7 could up-regulate cell volume by activating the ERK signalling pathway and subsequently enhancing the expression of AQP-1 in mesothelial cells. Our findings provide an initial mechanistic insight of water re-absorption and the potential MMP-7-targeted therapy in PD ultrafiltration failure.

CO N FLI C T O F I NTE R E S T
The authors have 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 used to support the findings of this study are included within the article.