Loss of Function of von Hippel-Lindau Trigger Lipocalin 2-Dependent Inflammatory Responses in Cultured and Primary Renal Tubular Cells

Previous studies have shown that mutations in the tumor suppressor gene von Hippel-Lindau (VHL) can result in the overproduction of reactive oxygen species (ROS) and chronic inflammation and are a significant predisposing factor for the development of clear-cell renal cell carcinoma (ccRCC). To study VHL's role in ccRCC formation, we previously developed a novel conditional knockout mouse model that mimicked the features of kidney inflammation and fibrosis that lead to cyst formation and hyperplasia. However, due to VHL's complex cellular functions, the mechanism of this phenomenon remains unclear. Here, we used the HK-2 cells and mouse primary renal tubule cells (mRTCs) carrying VHL mutations as models to study the effects and underlying molecular mechanisms of ROS accumulation. We also studied the role of lipocalin 2 (LCN2) in regulating macrophage recruitment by HK-2 cells. We measured the level of ROS in HK-2 cells in the presence or absence of LCN2 knockdown and found that the VHL mutation caused ROS overproduction, but an LCN2 knockdown could attenuate the process. VHL was also found to mediate the in vitro and in vivo expression and secretion of LCN2. Thus, VHL likely affects ROS production in an LCN2-dependent manner. Our findings also suggest that LCN2 sensitizes the inflammatory response of HK-2 cells and the chemotactic abilities of macrophage RAW264.7 cells. By demonstrating that the loss of function of von Hippel-Lindau triggers lipocalin 2-dependent inflammatory responses in cultured and primary renal tubular cells, our results offer novel insights into a potential therapeutic approach for interfering with the development of ccRCC.


Introduction
Mutations in von Hippel-Lindau (VHL) play a critical role in developing clear-cell renal cell carcinoma (ccRCC) [1,2]. Our previous study showed that, in the kidney lesions in Vhl knockout mice, there was a marked increase in inflammation and fibrosis with substantial collagen fiber deposition accumulated within clusters of distorted tubules [3]. We have previously demonstrated that the inflammation and fibrosis in Vhl knockout mouse lesions can be alleviated after treatment with an inhibitor of serine/threonine protein kinase/endoribonuclease (IRE1α) [4]. Inflammation may be caused by changes in proteostasis due to the overproduction of inflammatory cytokines [5,6]. Notably, it has been hypothesized that inflammation plays a critical role in carcinogenesis [7], and VHL mutation-associated ccRCC likely results from chronic inflammation [3,8].
Also, VHL is known to regulate many other genes involved in the inflammatory response. For example, VHL regulates the expression of MKI67, a HIF1Α target gene [9], and the marker of ccRCC [10], CA9, whose expression in kidney lesions may also be involved in the pathogenesis of ccRCC.
We further elucidated the inflammatory response in the Vhl knockout kidney by performing a microarray to profile the genes in the whole-kidney extract (Gene Expression Omnibus accession number GSE116326). We used the whole-kidney extract instead of the isolated tubule cell extract to examine the changes in the reactive microenvironment in the Vhl mutant cells. The microarray data showed that one of the candidate genes, LCN2, which encoded lipocalin 2, was highly overexpressed in the Vhl mutant kidney.
This study sought to investigate the potential role of LCN2 in mediating inflammatory response in VHL-mutated HK-2 cells. Furthermore, we tested the hypothesis that LCN2 was related to VHL mutation-sensitized macrophage migration and mediated inflammation via LCN2-ROS-dependent pathways.
The murine monocyte/macrophage cell line RAW264.7 (Bioresource Collection and Research Center, Taiwan) was cultured in T75 flasks in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate, and penicillin-streptomycin (50 U/mL; Sigma, MO, USA) at 37°C in a 5% CO 2 incubator. The fresh culture medium was replaced every other day. Once the cells reached 50-60% confluence, they were trypsinized for the following experiments.  Figure 1: Effect of VHL on ROS production in HK-2 cells. HK-2 cells were transfected with a vector expressing a scrambled shRNA (SC) or shRNA specific for VHL (shVHL1) (V1) (4) by electroporation with the Gemini X2 System and incubated for 48 hours. (a) After transfection, the intracellular ROS levels were determined using DCFDA. (b) The fluorescence intensity was detected using FACSCalibur analysis. All data were presented as the mean ± SD. * p < 0:05 as compared to the scrambled vector. 2 Oxidative Medicine and Cellular Longevity 2.3. Culture of Primary Renal Tubular Epithelial Cells. Mouse primary proximal tubule cells (mRTCs) were isolated from wild-type Hoxb7-Cre-GFP/+ (W) or mutant Hoxb7-Cre-GFP/+; Vhl fl/fl (M) mice using a previously described method [20][21][22], with some modifications. Briefly, mice were sacrificed by cervical dislocation. The kidneys were immediately removed and placed in 15 mL conical tubes with ice-cold Hank's balanced salt solution (Biological Industries, CT, USA). The renal capsule, cortex, and excess fat were removed, and the remaining cortical tissue was added into the dunce using two razors. Next, the plunger was pushed down to the bottom of the glass five times to break up the  The level of LCN2 was quantified using ImageJ. All data are presented as the mean ± SD. * * p < 0:01.
3 Oxidative Medicine and Cellular Longevity tissue. Then, the tissues were transferred into a 50 mL conical tube on ice. The tubular tissues were centrifuged in a swinging bucket rotor at 500 rpm and 4°C for 2 minutes. Then, the supernatant was aspirated and discarded while leaving the pelleted tissue intact.
Afterward, the tube was filled with warm digestion medium containing Collagenase I (Worthington, NJ, USA) at 140 units/mL and 15 mg Soybean Trypsin Inhibitor (Sigma, MO, USA) in 20 mL HBSS and incubated on an orbital shaker at 70 rpm for 15 minutes at 37°C. The tubule suspension was mixed with a 10 mL pipet and returned to the incubator every 5 minutes. After digestion, the tubule suspension was incubated with 20 mL of cold horse serum to inactivate the enzymes and enrich the tubules; the tube was inverted until the suspension became uniform. Then, the tubules were allowed to settle for one minute. The supernatant containing the proximal tubules was transferred to a 50 mL conical tube and centrifuged for 2 minutes at 500 rpm in a swinging bucket rotor. The tubules were washed with 10 mL of HBSS and centrifuged at 500 rpm for 2 minutes. A volume of 1-2 mL of culture medium was added to mix gently with the pellet using a sterile pipet. The cortical tubule suspension was gently layered onto a preformed 40% Percoll/60% culture medium gradient. Then, the tubules were centrifuged at 400 × g for 10 min at 4°C. The proximal renal tubules in the largest band were transferred to a 50  Oxidative Medicine and Cellular Longevity mL conical tube containing 20 mL of culture medium and centrifuged at 500 rpm for 2 minutes. Finally, the tubule pellet was resuspend in 20 mL of MRPTC Culture Medium, which consisted of DMEM/F-12 culture media (Gibco, NY, USA) with insulin/transferrin/selenium (5 μg/mL, 5 μg/mL, and 5 ng/mL, respectively, Sigma), 0.05 μM hydrocortisone (Sigma), 50 μM L-ascorbic acid-2-phosphate (Wako, Tokyo, Japan), and 1% antibiotic/antimycotic solution (10,000 units/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 μg/mL amphotericin B, Biological Industries, CT, USA).
Dilutional studies were performed to determine the volume of the resuspended tubules needed for different plates to ensure optimal growth. The tubules were plated on 12well (1-1.5 mL/well) or 6-well (3 mL/well) Nunclon-treated tissue culture plates (Nalgene/Nunc International, Rochester, NY) and incubated at 37°C with 5% CO 2 . The media was replaced with MRPTC culture media every day. Confluence can be achieved in 5 to 12 days.    Figure 4. Their cell lysates were collected and separated into cytosolic and nuclear fractions. The subcellular localization of NF-κB was detected by Western blot using an antibody against NF-κB's subunit p65. β-Actin was used as a cytosolic marker and fibrillarin as a nuclear marker. (b) NF-κB's cytosolic localization was decreased in VHL knockdown (V3) cells; such decrease was attenuated in the VHL (V3) and LCN2 (L1) double knockdown cells. The level of cytosolic p65 (cyto p65) was quantified using ImageJ. (c) NF-κB's nuclear localization was increased in VHL knockdown (V3) cells; however, such an increase was attenuated in VHL (V3) and LCN2 (L1) double knockdown cells. The expression of nuclear p65 (nuclear p65) was quantified using ImageJ. (d) The HK-2 cells were cultured and treated, as described in Figure 4. The cell lysates were collected and separated into cytosolic and nuclear fractions. The subcellular localization of NF-κB was detected with Western blot using an antibody against the NF-κB subunit p65. β-Actin was used as a cytosolic marker and fibrillarin as a nuclear marker. (e) NF-κB's cytosolic localization was decreased in VHL knockdown (V1) cells; however, the decrease was attenuated in VHL (V1) and LCN2 (L1) double knockdown cells. The level of cytosolic p65 (cyto p65) was quantified using ImageJ. (f) NF-κB's nuclear localization was increased in VHL knockdown (V1) cells; the increase was attenuated in VHL (V1) and LCN2 (L1) double knockdown cells. The level of nuclear p65 (nuclear p65) was quantified using ImageJ. All data are presented as the mean ± SD. * p < 0:05 and * * p < 0:01.   7 Oxidative Medicine and Cellular Longevity RAW274.7 cells. HK-2 cells were added into the lower chamber and transfected with a vector carrying a scrambled or the shVHL [24] sequence by electroporation. After incubation for 48 hours, the cells were treated with the indicated inhibitors for 24 hours in a serum-free medium supplemented with 5% bovine serum albumin (BIONOVAS, Toronto, Canada). Then, 1 × 10 5 RAW264.7 cells were added to the upper chamber with an uncoated membrane in a serum-free medium supplemented with 5% bovine serum albumin (BIONOVAS, Toronto, Canada). In separate experiments, RAW264.7 cells were exposed to recombinant human lipocalin 2/NGAL protein (rhLCN2) (Novus Biologicals, CO, USA) added to the serum-free medium and their migration measured. After 24 hours, the migratory cells on the underside of the membrane of the upper chamber were stained with 0.1% crystal violet for 5 minutes, washed with H 2 O, and scanned using an EPSON V750 PRO scanner. The cells were destained for crystal violet with methanol for 15 minutes and measured at OD570 with the Synergy HT (BioTek, VT, USA).
2.9. Statistical Analysis. The data were expressed as means ± SEM. The groups were compared using a one-way or two-way ANOVA followed by the Bonferroni post hoc analysis; p < 0:05 was considered statistically significant.

VHL Mutation-Induced ROS Production in an LCN2-Dependent Manner.
We investigated the effect of VHL deficiency on ROS production by measuring the level of ROS in VHL knockdown HK-2 cells using a previously described protocol [25][26][27]. We found that VHL knockdown caused ROS overproduction in HK-2 cells (Figure 1). LCN2 is one of the candidate genes predominantly related to immune response; however, its role in precancer-ous kidney cells is still unclear. We hypothesized that VHL deficiency caused inflammation and immune cell infiltration by overexpressing LCN2 in HK-2 cells and mouse primary renal tubule cells (mRTCs). VHL knockdown was found to induce LCN2 secretion in HK-2 cells (Figures 2(a) and 2(b)). Meanwhile, the level of LCN2 was elevated in Vhl mutant mRTCs (Figure 3). Interestingly, the enhanced level of LCN2 was detected in the urine of Vhl conditional knockout mice (Figure 2(d)). The presence of LCN2 in urine indicates acute kidney injury (AKI) [28]. These findings demonstrated that VHL deficiency induced expression and secretion of LCN2 in vitro and in vivo.
We further studied the role of LCN2 on ROS production by examining the effect of LCN2 knockdown on ROS levels. The increase in ROS levels was attenuated in LCN2 knockdown in VHL-deficient (knockdown) HK-2 cells (Figure 4).

Vhl Mutant Caused Inflammatory Response and
Sensitized RAW264.7 Cell Chemotaxis in a LCN2-Dependent Manner. We dissected the role of LCN2 in inflammation by investigating the effect of LCN2 knockdown on the phosphorylation of JNK and the nuclear translocation of NF-κB's p65 subunit in HK-2 cells and chemotaxis of RAW264.7 cells. LCN2 knockdown was found to significantly attenuate the increase in p-JNK expression activated by Figure 5. LCN2 knockdown also diminished the nuclear translocation of p65 in the VHL knockdown HK-2 cells, as demonstrated by Western blotting (Figure 6).
On the other hand, Vhl mutant was found to induce the chemotaxis of the RAW264.7 cells (Figure 7, V3/SC compared with SC/SC), and LCN2 knockdown could reduce the Transwell migration of RAW264.7 induced by VHL knockdown HK-2 cells (Figure 7, V3/L1 compared with V3/SC). This suggests that LCN2 is required for the chemotactic function of VHL-deficient HK-2 cells. We confirm the chemotactic effect of LCN2 by comparing the relative migration of RAW264.7 cells in the absence and presence (100 ng/mL) of recombinant human LCN2 (rhLCN2). We found that LCN2 significantly induced RAW264.7 cell migration after the rhLCN2 treatment ( Figure 8). In summary, our findings suggested that LCN2 sensitizes the inflammatory response of HK-2 cells and chemotactic abilities of macrophage RAW264.7 cells.

Discussion
The mechanism underlying the inflammatory response to VHL inactivation in kidney tubule cells presents an intriguing pathophysiological question and a potential therapeutic target. It has been shown that VHL mutant cells are involved in increased protein synthesis and ROS accumulation [4], likely due to the increased mTOR signaling in the mutant cells [29,30]. Moreover, the significantly elevated activation of immune cells likely contributes substantially to tissue damage during inflammatory diseases.
The diverse functional roles of LCN2 were exemplified in the central nervous systems [45]. Therefore, emerging evidence suggests that LCN2 has protective as well as pathogenic activities [46][47][48]. We propose that the dual biological effects of LCN2 may be considered during the design of therapeutics against ccRCC.

Conclusion
Our results indicated that VHL deficiency caused overproduction of reactive oxygen species (ROS), but an LCN2 knockdown could reverse this process. VHL deficiency was also found to increase in vitro and in vivo LCN2 expression and secretion. Our findings reveal that the regulatory effect of VHL on chronic inflammation in ccRCC progression is likely mediated, at least in part, via the LCN2-ROS pathway ( Figure 9). Our study offers novel insights into the therapeutic target and strategy for attenuating the development of ccRCC.

Data Availability
The original data used to support the findings of this study are included in the article.

Conflicts of Interest
The authors declare no conflict of interest.