Acute kidney injury (AKI) is a critical illness that is related to increased morbidity and mortality [1
]. Patients with partial AKI develop progressive and persistently aggravated proteinuria and decreased glomerular filtration rate (GFR) [2
]. Previous studies have demonstrated that the recovery of renal function in patients with AKI is often incomplete. Furthermore, AKI is a critical risk factor for the development of chronic kidney disease (CKD) and end-stage renal disease (ESRD) [3
]. AKI increases the risk of CKD by 8.8-fold and the risk for ESRD or kidney transplantation by 3.3-fold [3
]. At present, research on the related mechanisms of AKI progression to CKD has mainly focused on persistent inflammatory response, mitochondrial dysfunction, microvascular endothelial cell injury and the abnormal activation of renal tubular epithelial cells. Moreover, AKI can often cause fibrous tissue hyperplasia, the release of fibrogenic factors and renal fibrosis [5
]. Inflammation and oxidative stress are closely linked, as they generate a bad cycle. Oxidative stress starts inflammation. Inflammation, in turn, induces oxidative stress through the production of reactive oxygen species (ROS). These damaging events cause tissue injury by inducing necrosis, apoptosis and fibrosis [7
]. Therefore, novel drugs or mechanisms that antifibrotic functions and accelerate fibrogenesis in treating the AKI to CKD transition must be developed.
Many recent studies have shown that autophagy plays an important role in several diseases, including kidney disease [8
]. Autophagy is an intracellular degradation process that removes and recycles proteins and damaged organelles to maintain cellular homeostasis [9
]. A previous study demonstrated that ATG5-mediated autophagy in tubules hampered the activation of NF-κB signaling to protect against renal inflammation [12
]. Furthermore, autophagy attenuated G2/M cell cycle arrest in proximal tubular epithelial cells and renal fibrosis [13
]. Our recent study indicated that resveratrol-loaded nanoparticles can be as a strategy to prevent CKD through the autophagy induction and NLRP3 inflammasome attenuation [14
]. Another recent study concluded that overexpression of the SIRT6 gene inhibited apoptosis and induced autophagy, which might be involved in repairing kidney damage in AKI caused by sepsis [15
]. However, whether autophagy plays a key role in the process of the AKI to CKD transition is still unknown.
Lactoferrin is a natural iron-binding glycoprotein originally isolated from milk. Lactoferrin is found notably in milk, mucosal secretions and other bodily fluids [16
]. Previous research has shown that lactoferrin has multipharmacological properties, including antiviral, antibacterial, anti-inflammatory, and antioxidant properties [17
]. In a screening for lactoferrin expression in various organs, kidneys were found to have high levels of lactoferrin mRNA and protein. This indicated that lactoferrin may have important functions in the antioxidant defense and other defense systems protecting kidneys against any other stresses [21
]. In the current study, we found that the mRNA level of lactoferrin was elevated in the renal tissue of AKI and CKD patients compared to healthy individuals. The protective effect of lactoferrin has been assessed in a folic acid-induced AKI to CKD mouse model. We also investigated the roles of autophagy, apoptosis and fibrosis in lactoferrin-treated human kidney cells.
2. Material and Methods
2.1. Microarray Analysis
The microarray datasets (GSE66494 and GSE30718) were downloaded from the Gene Expression Omnibus (GEO) database. The raw data were normalized with GeneSpring software. The differential transcriptional activity of AKI patients and CKD patients was compared with healthy individuals. The results are shown as a boxplot and was produced using SPSS software.
2.2. Cell Culture and Lactoferrin Treatment
The human kidney proximal tubular epithelial cell line HK-2 was purchased from American type culture collection (CRL2190). HK-2 cells were cultured in keratinocyte serum-free (KCSF) medium with 40 μg/mL bovine pituitary extract and 5 ng/mL recombinant epidermal growth factor (Gibco BRL, Grand Island, NY, USA) at 5% CO2 and at 37 °C. For exposure to lactoferrin (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 40 mg/mL fresh stock solutions were prepared. The solution was added to the culture medium and mixed.
2.3. Cell Viability Assay
We used sulforhodamine B (SRB) assay to detect cell viability. Briefly, the cells were fixed with a trichloroacetic acid solution for 1 h and then SRB (Sigma-Aldrich Corp. St Louis, MO, USA) was added for 1 h. After washing, 20 mM of Tris buffer was added. Finally, the absorbance at 562 nm was read by ELISA reader (Molecular Devices, Sunnyvale, CA, USA). The reference value for calculating 100% cell viability is the mean absorbance of the untreated cells.
2.4. Western Blot Analysis
Total protein was prepared from cell lysates using protein extraction buffer. The proteins at 30 μg/lane or TD-PM10315 TOOLS Pre-Stained Protein Marker (10–315 kDa) (BIOTOOLS, New Taipei City, Taiwan) were loaded on a SDS gel, subjected to electrophoresis, blotted, probed using antibodies and detected by a chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Waltham, MA, USA). Anti-Akt, anti-p-Akt, anti-Beclin 1, anti-mTOR, anti-p-mTOR, anti-AMPK, anti-p-AMPK, anti-GAPDH, anti-caspase 3, anti-caspase 9, anti-LC3 and anti-PAI-1 antibodies were purchased from Cell Signaling Technology (Ipswich, MA, USA); anti-lactoferrin antibody was purchased from Biovision Inc. (Mountain View, CA, USA); and anti-Bax, anti-collagen and anti-CTGF antibodies were purchased from Proteintech Group (Chicago, IL, USA).
2.5. Immunofluorescence Microscopy
HK-2 cells were cultured on coverslips. After incubation, the cells were fixed in paraformaldehyde (4%) and blocked with BSA (1%) for 30 min. Then, the cells were incubated with an anti-LC3 antibody (MBL, Japan) for 1 h. The cells were washed in PBS and added with DyLight™ 488-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, PA, USA) for 1 h and stained with 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). Then, the cells were washed in PBS and analyzed with a confocal microscope (Leica TCS SP5, Mannheim, Germany).
2.6. Transfection of siRNA and LTF DNA
The LTF plasmid was constructed by ligating LTF cDNA sequence from pCMV6-XL5-LTF (OriGene Technologies Inc., Rockville, MD, USA). Then, 2 μg LTF DNA was transfected into HK-2 cells in 6-well plate by Lipofectamine® 2000 Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction. The transfection of siRNA was utilized by TransIT-X2® Dynamic Delivery System (Mirus, Madison, WI, USA) according to the manufacturer’s instruction. Beclin 1 siRNA (ID: s16539) was purchased from ThermoFisher Scientific (Waltham, MA, USA). Briefly, Opti-MEM I reduced-serum medium, siRNA solution and TransIT-X2 were mixed gently. The mixed solution was incubated at room temperature for 30 min to form complexes. Then, the complexes were added to cells for 24–72 h.
2.7. Folic Acid Mouse Model
Male C57BL/6 mice (eight weeks old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The animals were housed five per cage with 50% ± 10% relative humidity at 24 ± 2 °C. The animals were acclimatized for 1 week prior to the start of experiments and fed a Purina chow diet with water ad libitum. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Taipei Medical University, Taiwan (approval number: LAC-2018-0362). All animal experiments took place at Laboratory Animal Center of Taipei Medical University. The mice were divided into the following four groups (five mice/group): (1) equivalent volumes of saline administered intraperitoneally (i.p.) two times per week for 5 weeks starting at day 2 (normal group); (2) mice i.p. injected with 250 mg/kg folic acid one time at day 0 (Sigma-Aldrich) (FA group); (3) mice i.p. injected with 250 mg/kg folic acid one time at day 0 and i.p. injected with low-concentration (2 mg/mouse) lactoferrin two times per week for 5 weeks at starting day 2 (FA + LFL group); and (4) mice i.p. injected with 250 mg/kg folic acid one time at day 0 and i.p. injected with high-concentration (4 mg/mouse) lactoferrin two times per week for 5 weeks, starting at day 2 (FA + LFH group). The mice were sacrificed by CO2 exposure, and the kidney tissues were fixed by formalin and paraffin embedded for histopathological and immunohistochemistry (IHC) staining.
2.8. Biochemical Measurements
Whole blood samples of mice were collected by intracardiac puncture. The blood samples were centrifuged at 2000× g for 20 min and were separated from the serum. Biochemistry tests included creatinine and blood urea nitrogen (BUN) levels.
2.9. Histopathological and Immunohistochemical Analysis
The kidneys were fixed in 10% formalin, dehydrated, and embedded in paraffin. Paraffin-embedded kidney tissue sections were dried and rehydrated. The slides were incubated in 3% hydrogen peroxide for 20 min and then were placed in a microwave oven for 15 min in citrate buffer. Tissue sections were stained with hematoxylin and eosin (H+E) for histopathological analysis. For IHC staining, the slides were incubated for 2 h at room temperature with anti-cleaved-caspase 3 (Cell Signaling Technology, Ipswich, MA, USA), anti-α-SMA (Abcam, Cambridge, MA, USA) or anti-LC3 (MBL, Nagoya, Japan) antibodies. The slides were added with a secondary antibody for 1 h and were displayed using a STARR TREK Universal HRP detection kit (Biocare Medical, Concord, CA, USA). Finally, the slides were stained using hematoxylin.
2.10. Masson Staining
Masson trichrome staining was performed according to the protocol (ScyTek Lab., Logan, UT, USA).
2.11. Statistical Analysis
The results are presented as the mean ± standard deviation (SD) between groups using a one-way analysis of variance (ANOVA) followed by a post-hoc Bonferroni test or two-sample t-test. In all statistical tests, differences were considered significant at p < 0.05.
AKI is an important contributor to the increasing risk of developing CKD [3
]. Among the findings to date, irregular repair of AKI may cause CKD through excessive deposition of components of the extracellular matrix, cell death, persistent inflammation and fibrosis [32
]. However, the underlying molecular mechanisms in the AKI to CKD continuum are unclear. In the current study, we analyzed the transcriptional profiles using the microarray dataset in clinical kidney tissues from AKI and CKD patients (Figure 1
). The results found that the mRNA level of LTF was significantly upregulated in the kidney tissues of AKI and CKD patients (Figure 1
B–D). A previous study found the protective effect of lactoferrin on cisplatin-induced nephrotoxicity in rats. Furthermore, lactoferrin ameliorates cisplatin-induced creatinine and BUN in plasma [34
]. Lactoferrin protects the kidney against chromium-induced AKI through antioxidative, antiproliferative and anti-inflammatory effects by the downregulation of IGF-1 and IL-18 [20
]. In addition, lactoferrin inhibits oxidative stress, apoptosis and neuroinflammation through the upregulation of brain-derived neurotrophic factor (BDNF) and hypoxia-inducible factor 1α (HIF-1α) and the inhibition of JNK and P38 in neurons [35
]. Our results showed that lactoferrin suppressed oxidative stress-induced cell death and apoptosis in human kidney tubular epithelial cells (Figure 3
A,B). Moreover, lactoferrin inhibited TGF-β1-induced renal fibrosis by restraining the expression of the profibrogenic genes CTGF, PAI-1 and collagen I (Figure 4
B). In an in vivo study, lactoferrin ameliorated FA-decreased body weights and inhibited FA-induced creatinine and BUN levels. (Figure 6
A–C). In addition, lactoferrin restored FA-induced histopathological alterations in the kidney sections of the mice (Figure 7
A). Fibrosis and apoptosis were attenuated in lactoferrin-treated mice in comparison to fibrosis and apoptosis in FA-treated mice (Figure 7
A previous study found that lactoferrin induces autophagy via low-density lipoprotein receptor-related protein 1 and AMP-activated protein kinase activation [36
]. Autophagy enhances cell survival and maintains cellular and tissue homeostasis [37
]. Current evidence suggests that abnormal autophagy has been implicated in many human diseases, including various kidney diseases (diabetic nephropathies, AKI, polycystic kidney diseases and CKD) [38
]. Autophagy dysfunction can cause a loss of podocytes, glomerulosclerosis and damage to proximal tubular cells. Autophagy also restrained apoptosis of mesangial cells in diabetic nephropathy, through the inhibition of the TGF-1 and PI3K/Akt pathways [41
]. In addition, the LC3B knockout mice showed autophagy deficiency and had severe tubulointerstitial fibrosis after ureter obstruction [42
]. Therefore, the protective mechanisms of autophagy help repair and regenerate damaged kidneys [40
]. In our current study, lactoferrin induced autophagy via the activation of AMPK and the inhibition of the Akt/mTOR pathway in HK-2 cells (Figure 2
). The folic acid mouse model showed that the kidney sections of lactoferrin-treated mice markedly increased autophagy (Figure 7
C). The knockdown of autophagy significantly accelerated H2
-induced cell death (Figure 3
C). These results suggest that lactoferrin inhibits oxidative stress-induced cell death and apoptosis by augmenting autophagy.