Secreted Neutrophil Gelatinase-Associated Lipocalin Shows Stronger Ability To Inhibit Cyst Enlargement Of ADPKD Cells Compared With Nonsecreted Form Ⅳ

4. Discussion

In this study, we demonstrated that the addition and/or overexpression of secreted mNGAL protein decreased 2L3 cell proliferation and inhibited cyst growth in vitro by upregulating apoptosis and autophagy and downregulating proliferation-related signaling pathways. To identify whether M-1 cells, established from similar loci of mouse renal tubules as 2L3 cells, could be used as wild-type control cells for 2L3 cells, lectin staining of M-1 and 2L3 cells with LTL and DBA was performed (Figure 1D). LTL and DBA were used to identify the differentiated proximal tubules and collecting ducts of mouse kidneys, respectively [45,46]. The findings indicate that M-1 cells have similar morphology (Figure 1C) and origins of renal tubules (Figure 1D) to 2L3 cells; therefore, the M-1 cell line was selected as the wild-type control for 2L3 cells.

NEW HERBS FOR ADPKD(POLYCYSTIC KIDNEY DISEASE)

In most clinical cases, ADPKD patients show higher levels of NGAL in their urine than normal people [19,47–49]. The ADPKD mouse model used in this study also has higher NGAL and NGAL-R expression levels [31]. Both RT–qPCR and western blot results showed that 2L3 cells expressed higher levels of NGAL than the M-1 wild-type cell line (Figure 1F, H), whereas no significant difference in NGAL-R expression level was identified between the two cell lines (Figure 1G, H), which is inconsistent with the in vivo results [31]. This difference might be due to the complex interaction within the mouse tissues, which does not occur in the much simpler cell system.

The western blot results showed that direct administration and/or genetic overexpression of mNGAL in 2L3 inhibited the ERK pathway. As shown in Figures 4A and 8A, the signal of p-ERK1 (44 kDa) was weaker than p-ERK2 (42 kDa), while ERK1 (44 kDa) was more intense than ERK2 (42 kDa), causing a marked increase in p-ERK2/ERK2 compared with p-ERK1/ERK1 in all experimental groups. ERK1 and ERK2 are two isoforms of ERKs that have been suggested to have different effects on cell proliferation [50]. ERK1 has been identified to induce cell survival and growth; however, ERK2 promotes cell differentiation and blocks cell growth [51]. In our study, the ratio of p-ERK2/ERK2 was notably higher than that of p-ERK1/ERK1 in all groups, which indicated that cells tended to differentiate and reduce growth. However, both the addition and overexpression of mNGAL dramatically lowered the ratio of both p-ERK1/ERK1 and p-ERK2/ERK2 compared with the control group and led to a significantly lower ratio of p-ERK/ERK, which indicated the mNGAL effect in inhibition of cell proliferation.

To establish a 3D culture platform for the characterization and treatment evaluation of cyst formation in 2L3 cells, several conditions were attempted. It has been demonstrated that the concentration of Matrigel for 3D culture influences the percentage of structural cysts and tubules formed in vitro [25,52,53]. In our study, we found that 40% Matrigel was the best formula to show the optimal cyst structure formation of 2L3 cells. There were several conditions, including embedded patterns and on-top patterns for 3D cultures [54]. We chose the on-top method (sandwich-like pattern) for a better and easier way to analyze the cysts (Figure 6). However, our experience showed that the cysts of 2L3 cells were not sufficiently large when cultured without any inducer in 3D culture until Day 14. Forskolin is commonly used to induce cyst formation by activating adenylyl cyclase (AC) to increase cyclic adenosine monophosphate (cAMP) [11,55]. Therefore, in our study, we used forskolin (20 µM) to induce cyst formation in 2L3 cells before mental treatment.

Our previous study showed that overexpression of kidney-specific NGAL in 2L3 mice could slow ADPKD progression by decreasing the cyst size but not the cyst number in 2L3 mice [31]. In the present study, the addition of mental significantly inhibited cyst enlargement of 2L3 cells by suppressing the cyst diameter, area, and percentage of large cysts (Figure 6F, G, I), while there was no significant difference in the increasing rate of total cyst number (Figure 6H). The results of 2L3 cyst progression in 3D culture were consistent with the in vivo results. However, mNGAL treatment inhibited cyst growth of 2L3 cells in 3D culture, while we used a higher concentration of mNGAL (2 mg/mL) (Figure 6) than in the previous 2D experiments (600 µg/mL) for the MTT assay to examine the effect of mNGAL on cell viability (Figure 3). We speculated that recombinant mNGAL purified from E. coli lacked posttranslational modification [56,57] and resulted in a low bioactivity of recombinant mNGAL. Overexpression of NGAL in 2L3 cells by transfection of pN + LS and pN − LS constructs could help elucidate this question.

To determine whether a higher concentration of mNGAL (2 mg/mL) used in 3D culture to suppress cyst enlargement of 2L3 cells (Figure 6) resulted from the lack of posttranslational modification of mNGAL and to test whether NGAL had to be secreted and interact with NGAL-R to induce apoptosis and further inhibit cyst progression, pN + LS and pN − LS constructs were transfected into 2L3 cells to overexpress the secreted and nonsecreted NGAL proteins (Figure 7). Pooled transfected cells were harvested one week after blasticidin selection, and both pN + LS and pN − LS transfection highly increased mRNA expression levels of Lcn2 (total, including both endogenous and exogenous NGAL) and Lcn2-c-myc (exogenous NGAL) compared with the control group (Figure 7F, G) and there was no significant difference in Lcn2 and Lcn2-c-myc between pN + LS and pN − LS groups (Figure 7F, G). The expression of Lcn2-myc in pN + LS and pN − LS groups was approximately 8500-fold greater than that in 2L3 cells (Figure 7G); however, the expression of Lcn2 in pN + LS and pN − LS was approximately 1.5-fold greater than that in 2L3 cells (Figure 7F). In theory, Lcn2-myc was not present in 2L3 cells, leading to low expression levels (background) by RT–QPCR. As a result, the relative ratio of Lcn2-myc in pN + LS and pN − LS cells was markedly higher (approximately 8500 times) than that in the 2L3 control group, resulting in the large difference between the relative ratio of Lcn2 and Lcn2-myc. In addition, the expression ratio of Lcn2 in pN + LS and pN − LS cells (pN + LS was 0.91-fold that of pN − LS) was similar to that of Lcn2-c-myc (pN + LS was 1.01-fold that of pN − LS) (Figure 7F, G). Intracellular NGAL protein expression in pN + LS and pN − LS cells was increased compared with that in the control group (Figure 7I), and there was no significant difference between the pN + LS and pN − LS groups. The western blot results for total NGAL protein expression in the pN + LS and pN − LS groups (pN + LS was 0.92-fold that of pN − LS) (Figure 7I) were consistent with the RT–qPCR results (pN + LS was 0.91-fold that of pN − LS) (Figure 7F). According to these results, the levels of NGAL (both total and exogenous) proteins in pN + LS and pN − LS cells were similar, which indicated that the two groups should be compared in further experiments. 

We predicted that overexpression of NGAL without a signal peptide would not increase NGAL secretion despite the high level of intracellular NGAL expression. However, the ELISA results using the supernatant from the culture showed that both pN + LS and pN − LS cells had significantly higher levels of secreted NGAL than the 2L3 control cells (Figure 7J), while the amount of NGAL secreted by pN − LS cells was significantly lower than that of pN + LS cells (Figure 7J). These results revealed that overexpression of NGAL without signal peptide (pN − LS) did limit the secretion of NGAL, although high levels of intracellular NGAL mRNA and protein were identified within the cells (Figure 7F, I). The BrdU incorporation results showed significantly higher proliferation of pN − LS cells than pN + LS cells (Figure 7K).

The intracellular and secretion amounts of NGAL were approximately 1.6-fold and 2.2-fold higher in pN + LS than in 2L3 cells, respectively (Figure 7I, J), which could lead to the inhibition of pN + LS compared with 2L3 cell proliferation (Figure 7K). However, intracellular and secreted amounts of NGAL in 2L3 cells were approximately 4-fold and 5.3-fold higher than those in M-1 cells, respectively (Figure 1H, I), while higher cell proliferation was observed in 2L3 than in M-1 cells (Figure 2C). We suspect that increased NGAL expression and secretion, which play a protective role in 2L3 cells, is insufficient to inhibit the cell proliferation resulting from Pkd1 knockout, but the increased NGAL expression and secretion in pN + LS provides supplemental NGAL to achieve proliferation suppression. In our previous study, Pkd1L3/L3 mice had significantly greater renal levels of NGAL protein and proliferating cell nuclear antigen (PCNA) than Pkd1+/+ mice [31]. Nevertheless, the renal level of NGAL protein in Pkd1L3/L3 × NGALTg/Tg mice was approximately 2-fold of that in Pkd1L3/L3 × NGAL+/+ mice, and overexpression of NGAL led to markedly lower levels of PCNA in Pkd1L3/L3 × NGALTg/Tg than in Pkd1L3/L3 × NGAL+/+ mice and further inhibited cyst growth and prolonged survival days [31]. NGAL seems to play a protective role in inhibiting the increased proliferation in ADPKD models, whereas endogenous NGAL expression is insufficient, and supplemental exogenous NGAL can help.

According to the 3D culture results (Figure 10), both the pN + LS and pN − LS groups inhibited cyst growth by lowering the diameter (Figure 10C), area (Figure 10D), and percentage of large cysts (diameter > 100 µm) (Figure 10F), but not the numbers (Figure 10E). This finding is inconsistent with our suspicion that only pN + LS but not pN − LS inhibits cyst enlargement because secretion of mNGAL from 2L3 cells is required for cell proliferation inhibition. However, inhibition of cyst enlargement by pN − LS was poorer than the pN + LS (Figure 10B–D), which revealed that overexpression of secreted mNGAL had a superior ability to inhibit cyst enlargement of 2L3 cells in 3D culture than that of nonsecreted mNGAL. Although we see stronger suppression of cyst enlargement in the pN + LS group compared with the pN − LS group in 3D culture, it is hard to see the better effect of pN + LS than pN − LS in p-AKT/AKT (Figure 8B), p-CREB/CREB (Figure 8C) and p62 (Figure 9D). It has been demonstrated that NGAL and NGAL-R coupling can sequester intracellular iron and further induce apoptosis [29], which is associated with an iron-depletion strategy of the innate immune system against bacterial infection [30]. We proposed that secreted mNGAL induced apoptosis in 2L3 cells through interacting with NGAL-R, whereas nonsecreted mNGAL could not interact with NGAL-R to induce apoptosis (Figure 9A, B). It has been demonstrated that the Ras/B-Raf/MEK/ERK [10,11] and AKT/mTOR [38] pathways are upregulated in ADPKD cells, which leads to increased cell proliferation and cyst enlargement. Our results showed that secreted mNGAL inhibited ERK (Figure 8A) and AKT (Figure 8B) pathways and nonsecreted mNGAL inactivated ERK pathway (Figure 8A). It suggests that mental inactivation the ERK pathway may not be necessary through NGAL-R. Knockout of NGAL-R in 2L3 cells will be performed to explore the question

In addition, overexpression of mNGAL in 2L3 cells inhibited cell cyst enlargement (Figure 10) and occurred with a relatively lower secretion of NGAL (2.27 ng/mL) (Figure 7J) compared with the effect with a high concentration of mNGAL (2 mg/mL) applied in previous 3D culture experiments (Figure 6). This result might be due to the lack of post-translational modification of mNGAL from E. coli that was used in previous experiments, which required a high dose of mNGAL to inhibit cyst enlargement (Figure 6). To further examine whether a posttranslational modification of mNGAL is the factor responsible for the requirement of more recombinant mNGAL from E. coli, a conditioned medium including mNGAL secreted from a mouse cell line will be used in further studies. 

In the present study, pN + LS and pN − LS cells used for RT–qPCR, western blotting, ELISA, BrdU incorporation, and 3D culture were obtained from a mixed polyclonal pool, which had varied transgene expression levels in the mixed population. Identification of the mechanism of mNGAL in ADPKD 2L3 cells by the above-described methods using clonal cells should aid in finding strategies for the future clinical treatment of ADPKD. 

5. Conclusions

In this study, we explored the underlying mechanism of reduced cyst progression in the presence of NGAL using an immortalized ADPKD cell line. The addition of recombinant mNGAL inhibited cell proliferation in ADPKD cells. Pathological analysis showed that mNGAL decreased proliferation and induced apoptosis and autophagy pathways in ADPKD cells. In addition, a 3D cell culture platform was established to identify cyst progression and showed that the addition of recombinant mNGAL inhibited cyst enlargement. Furthermore, overexpression of secreted mNGAL (pN + LS) had a stronger effect at inhibiting cyst enlargement of ADPKD cells than that of nonsecreted mNGAL (pN − LS), although both forms lowered the cyst diameter, the area and percentage of large size cysts, as well as their effects on proliferation, apoptosis, and autophagy. We conclude that secreted mNGAL has a more pronounced and consistent ability to inhibit cyst enlargement than that of nonsecreted mNGAL in ADPKD cells. Our work could help identify strategies for the future clinical treatment of ADPKD. 

Supplementary Materials: Supplementary videos to this article can be found online (1) Video S1: Construction of a cyst in 3D culture: https://reurl.cc/Kjy8rq (accessed on accessed on 26 November 2021); (2) Video S2: Z-stack sections of a cyst in 3D culture: https://reurl.cc/VXGY5y (accessed on accessed on 26 November 2021). Supplementary graph: negative controls for Fig 1D (leaving out LTL and DBA staining) can be found online at https://reurl.cc/LpMmje (accessed on accessed on 26 November 2021). Author Contributions: Conceptualization, W.-Y.J., H.M.H.-L. and Y.-Y.C.; data curation, H.-Y.C.; formal analysis, H.-Y.C.; funding acquisition, H.M.H.-L. and Y.-Y.C.; methodology, H.-Y.C., W.-Y.J., E.W., C.-M.H., H.M.H.-L. and Y.-Y.C.; project administration, H.-Y.C. and H.M.H.-L.; resources, W.-Y.J., S.-T.J. and Y.-Y.C.; supervision, H.M.H.-L.; visualization, H.-Y.C.; writing-original draft, H.-Y.C. and H.M.H.-L.; writing-review and editing, H.-Y.C. and H.M.H.-L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants 107-2622-B-006-005-CC2 and 108-2622-B-006-002- CC2 from the Ministry of Science and Technology (MOST), and the Headquarters of University Advancement from National Cheng Kung University, Tainan, Taiwan (Yuan-Yow Chiou). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data generated during the study are available from the corresponding author upon request. Acknowledgments: The authors are thankful for the assistance of the Molecular Imaging Core Facility of the National Taiwan Normal University under the auspices of the MOST. We thank the National RNAi Core Facility at Academia Sinica in Taiwan for providing virus particles, plasmids, and related services. We thank Shu-Wen Lo for assisting with graphic editing. Conflicts of Interest: The authors declare no conflict of interest. 

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