Background
Polycystic kidney disease (PKD) is the most common genetic disorder of the kidneys and is characterized by the progressive formation and enlargement of fluid-filled cysts. These cysts compress the normal adjacent parenchyma, ultimately leading to end-stage renal disease that requires dialysis and renal transplantation [
1,
2]. The two major inherited forms of PKD are Autosomal Dominant PKD (ADPKD) and Autosomal Recessive PKD (ARPKD). ADPKD is caused by a mutation in either the
PKD1 or
PKD2 genes and is manifested in adulthood with an incidence of 1:1000. ARPKD is caused by mutation in
PKHD1 gene and is responsible for childhood cystic disease with an incidence of 1:20,000 [
3,
4]. Although remarkable progress has been made in understanding the function of the PKD genes, the molecular and cellular mechanisms that lead to cyst formation and enlargement in PKD kidneys are still not fully understood.
Growing evidence suggests that abnormal extracellular matrix (ECM) remodeling may represent a common pathway for cyst formation and development in ADPKD and ARPKD kidneys. Normal ECM remodeling is tightly regulated by dynamic synthesis and degradation of its components. This tight regulation is essential for proper morphogenesis during embryo development and for maintaining tissue and organ structure homeostasis in the adult [
5,
6]. During kidney development, ECM undergoes active remodeling and the levels of both, collagens, the main components of ECM, and MMPs, the main enzymes that degrade ECM components, are dynamically regulated. In particular, MMP2, MMP9, and MT1-MMP have been shown to play an important role in regulating local ECM remodeling during ureteric bud (UB) branching morphogenesis in kidney development [
7,
8].
Studies have shown that renal interstitial fibrosis, which is caused by excessive accumulation and deposition of collagens, is a characteristic feature of both ADPKD and ARPKD. There is a strong correlation between the progression of interstitial fibrosis and the progression of cystic disease [
9,
10]. In addition, overexpression of ECM collagens has been observed in cultured PKD cells as well as in PKD kidneys of human patients and animal models [
11‐
14]. Finally, gene expression profiling of cystic kidney tissues from both human PKD patients and animal PKD models has shown that a large portion of upregulated genes encode for ECM proteins [
15,
16].
MMPs are key enzymes in ECM remodeling and their overexpression is another characteristic feature of PKD kidneys. Among the MMP family proteins, MMP2, MMP9 and MT1-MMP are the main collagen degradation enzymes. It has been shown that the expressions of these specific MMPs are increased in both PKD patients and PKD animal models [
17‐
19]. MT1-MMP has been shown to play a unique role in modifying pericellular collagen microenvironment and in regulating pro-MMP2 and pro-MMP9 activation. In addition to its collagen degradation activity, MT1-MMP has also been shown to be a potent mitogen for cell proliferation [
20‐
22]. Increased cell proliferation and dynamic remodeling of the microenvironment is a prerequisite for cyst enlargement, suggesting that MT1-MMP may be a major contributor in cyst growth.
In the present study, we investigated the role of collagen I and MT1-MMP in cyst formation and growth using both in vitro 3D collagen I culture system and in vivo PCK rat model of PKD. First, our studies reveal that the initiation of cyst structures requires higher collagen levels than that required for the formation of tubule structures. Secondly, we find that MT1-MMP stimulates cell proliferation and cyst enlargement. Finally, we show that the treatment of PCK rats with doxycycline, an FDA approved pan-MMPs inhibitor, significantly inhibited cystic disease progression in polycystic kidneys. Taken together, our results suggest that increased collagen expression and MMP activity in PKD kidneys may play a crucial role in renal cyst formation and enlargement.
Methods
PCK rat model
3 to 4 weeks old male PCK rats were purchased from Charles River (Wilmington, MA). The protocol for animal studies has been approved by the Texas Tech University Health Sciences Center Animal Care and Use Committee. Rats had free access to tap water and standard rat chow. Male PCK rats were treated at 4 weeks of age daily with either doxycycline (Sigma, St. Louis, MO) at 30 mg/kg or vehicle (PBS) subcutaneously for 5 weeks.
Immunohistochemical staining
OCT-embedded kidney sections (5 μm) were used for the immunohistochemical studies. For antigen retrieval, the sections were first immersed in heated (95°C to 100°C) 0.1 M citrate buffer, pH 6.0 for 10 min and then allowed to cool to room temperature. The sections were then incubated with the antibodies against collagen I (Santa Cruz Biotechnology, Santa Cruz, CA), MMP2 (Millipore, Temecula, CA), MMP9 (Santa Cruz Biotechnology, Santa Cruz, CA) or MT1-MMP (Thermo Fisher Scientific, Fremont, CA), respectively. After extensive wash, the sections were incubated with horseradish peroxidase (HRP)-conjugated polymer (DAKO EnVision System, Carpinteria, CA) for 1 h, and the antigen sites were visualized by incubating with the substrate - DAB.
Cell culture and transfections
MDCK cells were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (50:50) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin (Mediatech, Manassas, VA). pMSCVGFP-MT1-MMP expression vector is a gift from Dr. Zonghan Dai (Texas Tech University HSC). Transfections of MDCK cells with pMSCVGFP-MT1-MMP or pMSCVGFP control vector were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), as described by manufacturer’s procedure and the stably transfected clones were selected by 5 μg/ml puromycin.
RNA isolation and RT-PCR
Total RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA), as described in the manufacturer’s instructions. cDNA was synthesized using SuperScript III First-strand Synthesis System (Invitrogen, Carlsbad, CA). The RT-PCR was performed using the following primers: MT1-MMP forward primer: 5′-GGATACCCAATGCCCATTGGCCA-3′, reverse primer: 5′-CCATTGGGCATCCAGAAGAGAGC-3′; 28S RNA forward primer: 5′-TTGAAAATCCGGGGGAGAG-3′, and reverse primer: 5′-ACATTGTTCCACATGCCAG-3′.
3D collagen gel culture and treatment
Collagen I was obtained from BD Biosciences (San Jose, CA). Collagen I concentration was determined using Modified Lowry Protein Assay Kit (Thermo Scientific, Pittsburgh, PA). The collagen I solutions with desired concentration in DMEM containing 100 U/ml penicillin, 100 U/ml streptomycin, 10 mM Hepes (pH 7.2) and 1.2 mg/ml NaHCO3 were prepared on ice, and then mixed with cells to make a cell-collagen suspension at a density of 1 × 104 cells/ml. Aliquots of 0.4 ml of the cell-collagen suspension were plated in a 24-well plate and incubated for 30 min in a 37°C, 5% CO2 incubator to allow collagen solidified. Complete culture medium was then added on the top of collagen gel and cultured for 9 days. To test the effect of MMPs on cyst growth, the cultures were treated with doxycycline at a concentration of 12.5ug/ml. To view the histological structure of the cysts and tubules, the 3D cultures were embedded in OCT at the end of experiment.
Western blotting analysis
Lysates from 2D cultures were prepared as described previously [
23]. To prepare lysates from 3D cultures, the collagen gels were washed with PBS for 3 times and then solubilized in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 20 mM Tris–HCl, pH 7.4, 0.16 M NaCl, 1 mM EGTA, 1 mM EDTA, and 15 mM sodium fluoride) containing protease inhibitor cocktails (Sigma, St. Louis, MO). The mixtures were kept on ice for 1 h with periodically vortexing and then, centrifuged at 6000 rpm for 10 min at 4°C. The supernatants were collected as cell lysates. Because the 3D culture lysates prepared by this method may contain collagen I, we first normalized these lysates for β-actin contents by Western blotting analysis. The lysates containing equal amount of β-actin were then separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The membranes were probed with the antibodies against GFP (Chemicon, Temecula, CA) and PCNA (Santa Cruz Biotechnology), respectively. To confirm equal loading, the membranes were also probed with anti-β-actin antibody (Sigma, St. Louis, MO). After incubated with HRP-conjugated secondary antibodies, the immunoreactive proteins were visualized by ECL detection system (Amersham Biosciences Piscataway, NJ).
Cyst and tubule structure measurement and statistical analysis
Images of cysts and tubules in 3D collagen gels were captured and analyzed using an inverted microscope (Nikon Eclipse TE 2000-U). This was performed by a reviewer who was blinded to the identity of the treatment modality. At least ten random areas were captured for each treatment condition. The diameters of the cysts were measured using NIS-Elements BR 2.30 software and the mean and standard deviation were determined using Excel software. Comparison of the means was performed with the Student’s t-test. The P value < 0.05 was considered statistically significant. Values are expressed as mean ± STDEV.
Cyst volume density and kidney function
Hematoxylin-eosin-stained kidney sections cut longitudinally through the midline were used to determine the cyst volume density by point counting stereology [
24]. The entire kidney section was counted to guard against field selection variation. Serum creatinine levels were measured using Creatinine Assay kit (BioVison, Mountain View, CA). Blood urea nitrogenBlood urea nitrogen levels were measured using MaxDiscovery
™ Blood Urea Nitrogen Enzymatic Assay Kit (B100 Scientific Corporation, Austin, TX).
Discussion
Studies in human PKD patients and several PKD animal models including Han: SPRD rats and pcy mice showed that abnormal ECM remodeling, such as increased collagen I level and MT1-MMP expression, are the characteristic feature of PKD kidneys [
15,
16]. However, the direct link of increased ECM collagen and MT1-MMP on cyst formation and enlargement in PKD kidneys has not been well described.
In this study, we investigated the role of increased collagen I and the collagenase MMPs on cystic kidney disease progression using both
in vivo PCK rats and
in vitro 3D collagen I cyst culture systems. PCK rats are orthologous model of human ARPKD and have been successfully used for testing several potential therapeutic agents, therefore provide one of the best animal models for studies of human PKD [
25,
26]. A recent study has shown that there is an epithelial-to-mesenchymal transition in cyst-lining cells of PCK rats, suggesting that increased ECM production may occur in the kidneys [
27]. However, the expression of ECM collagen and MMPs in PKD kidneys of PCK rats has not been previously described. We show here that the expression of collagen I, MMP2, MMP9 and MT1-MMP in PKD kidneys of 9 week old PCK rats are all significantly increased compared to the age-matched normal control rats. This finding further supports the proposition that abnormal ECM metabolism is a common feature of PKD. We found that increased collagen and MMPs expression not only occurred in cyst-lining epithelial cells, but also in the interstitial area in PKD kidneys. These observations are in agreement with the studies of pcy mice model by Okada et al, showing that both cystic epithelial cells and interstitial fibroblasts express higher levels of collagens compared to normal controls [
10].
3D collagen I gel is a well-established culture system for studies of epithelial cell tubule and cyst morphogenesis. However, past studies have primarily focused on the role of signaling molecules, such as HGF and cAMP on cyst and tubule structure development [
28,
29]. No studies have been conducted to directly examine the effect of collagen concentrations and MMPs activities on cyst and tubule morphogenesis of cultured cells. We found that the formation of cyst structure requires a collagen concentration higher than that required for the formation of tubular structure. Given that the increased collagen expression has been detected in cystic epithelial cells and adjacent fibroblasts, our results suggest that this locally increased collagen production in PKD kidneys may play a critical role in the initiation of cyst formation.
Finally, we assessed the effects of MMPs inhibition by doxycycline on cystic disease progression in a PKD animal model, PCK rat. Doxycycline, also known as Periostat, is currently the only FDA approved MMPs inhibitor. Doxycycline is used to treat several disorders that are mediated by increased MMP activities such as cancer and periodontal disease. It has been shown to be potent in inhibiting the activities of a large number of MMPs, including MMP2, MMP9, and MT1-MMP [
30]. We show that doxycycline significantly inhibited MT1-MMP, MMP9 and MMP2 expression and slowed cyst disease progression in PCK rats. We also demonstrate that doxycycline inhibits cyst growth in 3D collagen gel. Increased cell proliferation is prerequisite to cyst expansion and MT1-MMP has been shown to stimulate cell proliferation in both
in vivo and
in vitro studies. In this study, we showed that doxycycline inhibits MT1-MMP expression and cell proliferation in PKD kidneys. The effect of MMPs inhibition on PKD progression has been previously studied by Obermuller et al in Han:SPRD PKD rats using batimastat, a synthetic inhibitor of MMPs [
31]. Their study show that the batimastat treatment significantly decreased cyst number but not the total cyst volume density in PKD kidneys. They proposed that this is likely due to that the dosage of batimastat used in their study is too low to effectively block the high amounts of MT1-MMP being expressed in large cysts and therefore, to inhibit large cyst expansion. They also indicate that the application of higher doses of batimastat may increase side effect, in particular, renal dysfunction. In our study, we show that the dosage of doxycycline used in our experiments not only efficiently inhibits overall cyst volume density in PKD kidney of PCK rats, but also avoid any apparent kidney function damage and other side effects.
Conclusions
Our in vivo and in vitro data suggest that abnormal increase in collagen and collagenase MMPs expression may play an important role in cyst formation and enlargement in PKD kidneys. The finding that doxycycline markedly inhibits cyst growth in PCK rats suggests a potential use of this drug in therapeutic intervention of the cystic disease progression. As doxycycline has been used successfully in treating human patients with several disorders that are mediated by increased MMP activities, we believe that it may provide a promising drug candidate for the treatment of human PKD patients.
Acknowledgments
We thank Dr. Candace Myers and Yue Dai for critical review of the manuscript. This work was supported by NIH/NIDDK grant K01 DK067191 (Y. Tao).
Author details
1Internal Medicine, Texas Tech University Health Sciences Center, 79106 Amarillo, TX, USA. 2Institute of Life Science and Biology, Hunan University, Hunan, People’s Republic of China. 3Henan Provincial Centers for Disease Control and Prevention, Zhengzhou, People’s Republic of China.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BL, CL, and ZL carried out experiments and analyzed data. ZD analyzed and interpreted data and involved writing of the manuscript. YT designed the study and wrote the paper. BL and CL contributed equally to this work. All authors had final approval of the submitted and published versions.