Tg mice expressing Pax8–reverse tetracycline transactivator (Pax8–rtTA) in the renal tubules were purchased from Jackson Laboratory (Bar Harbor, ME, USA), and Tg mice expressing tetracycline response element–hCD36 (TRE–hCD36) were obtained from the Einstein Transgenic Facility (New York, NY, USA). Next, Pax8–rtTA Tg mice were mated with TRE–hCD36 Tg mice to generate Pax8–rtTA/TRE–hCD36 Tg mice overexpressing hCD36 under the control of the ‘Tet’O operation regulated with tetracycline. In other words, Tg mice expressing TRE and hCD36 were mated with Tg mice expressing rtTA to generate Pax8–rtTA/TRE–hCD36 Tg mice that overexpressed hCD36 in the proximal tubules under tetracycline feeding. First, 4-week-old Tg mice were fed doxycycline-containing food (Bioserv S3888; Bioserv, San Diego, CA, USA) for two weeks to confirm hCD36 expression. Finally, the Tg mice were analyzed after eating doxycycline-added food for two weeks, and the first and fourth among the four lines showed the highest expression of hCD36 in quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot assay. Immuno-fluorescence analysis of samples obtained from Tg mice showing the highest hCD36 expression also yielded positive results for hCD36. Then, mice of the first and fourth line were mated, and tail genotyping was used to identify these as Tg mice overexpressing hCD36 in the proximal tubule.

Six-week-old mice were intraperitoneally injected with folic acid (250 mg kg⁻¹ dissolved in 300 mM NaHCO₃; Sigma, St. Louis, MO, USA). The mice were divided into the following four groups to compare the effect of hCD36 overexpression on folic acid-induced AKI: (1) folic acid-treated Tg mice (n = 7), (2) folic acid-treated wild-type (WT) mice (n = 7), (3) vehicle-treated Tg mice (n = 7), and (4) vehicle-treated WT mice (n = 7). After 48 hours of folic acid administration, the mouse kidneys were harvested, prepared for histological analysis, and stored at −80°C for mRNA analysis.

The following antibodies were used in this study: anti-hCD36 antibody (Santa Cruz Bio, Paso Robles, CA, USA) and mouse anti-α-smooth muscle actin (α-SMA) antibody (Santa Cruz Bio).

Protein in kidney tissues harvested from Tg mice with hCD36-overexpression and WT mice was extracted for western blot analysis. The proteins were mixed with sodium dodecyl sulfate (SDS) loading buffer, resolved by performing SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The membranes were blocked for nonspecific binding and were incubated overnight at 4°C with anti-hCD36 antibody. Next, the densities of hCD36 were normalized to those of β-actin (Sigma-Aldrich) in protein samples obtained from the kidneys of WT and Tg mice.

Formalin-fixed, paraffin-embedded kidney sections of mice in the four groups were washed twice with 0.01 M phosphate-buffered saline (PBS; pH 7.4) and were treated with 10% BSA/0.3% Triton X-100 in PBS for 1 hours to block nonspecific binding. Immunofluorescence labeling was performed using anti-hCD36 antibody to identify hCD36 overexpression in the renal proximal tubules of Tg mice. Next, the kidney sections were washed and incubated with appropriate secondary antibodies for two hours at room temperature, followed by counterstaining with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma-Aldrich).

Formalin-fixed, paraffin-embedded kidney sections were stained with periodic acid Schiff (PAS) to identify histopathological changes in the renal tubules of hCD36-overexpressing mice. In addition, kidney sections from folic acid-treated mice were incubated overnight at 4°C with anti-α-SMA antibody, followed by treatment with an appropriate biotin-conjugated secondary immuno-globulin G to identify renal fibrosis-associated changes. The tubular injury scoring was quantified into six groups on the basis of percentage of tubular dilatation, epithelial desquamation, and intra-tubular crystal formation in 10 randomly chosen, non-overlapping fields at a magnification of 100× under a light microscope: 0, none; 0.5, < 10%; 1, 10–25%; 2, 25–50%; 3, 50–75%; and 4, > 75%. The positive areas of α-SMA immunostaining in the kidney tissue were measured as percentage of total area in 10 randomly chosen, non-overlapping fields at a magnification of 400× using ImageJ software (http://rsb.info.nih.gov/ij).

At 48 hours after folic acid injection, the mice were placed in metabolic cages to collect 24-hour urine samples for measuring urinary albumin and creatinine excretion. Urinary albumin excretion was measured using Albuwell M ELISA kit (Exocell Inc., Philadelphia, PA, USA), and urinary creatinine excretion was measured using a creatinine ELISA kit (Exocell Inc.). Urinary albumin excretion was expressed as the urinary albumin to creatinine ratio (UACR).

For qRT-PCR, CD36 and Cd36 RNA was isolated from the harvested kidneys using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Next, 1 μg of the RNA was reverse transcribed using a cDNA archival kit (Life Technology, Woburn, MA, USA). qRT-PCR was performed using the ViiA 7 System (Life Technology) with SYBR Green Master Mix (Life Technology) and gene-specific primers. Data obtained were normalized and analyzed using the delta delta CT method. The following primers were used: CD36 forward, 5′-GCTCTGGGGCTACAAAGATG-3′; CD36 reverse, 5′-TAGGGAGAGATATCGGGCCT-3′; Cd36 forward 5′-TG-CTGGGCTGTTATTGGTG-3′; Cd36 reverse, 5′-CATGAGAATGCCTCCA-AACA-3′; Col1a1 forward, 5′-GCTCTTTTTAGATACTGTGG-TGAGGAA-3′; Col1a1 reverse, 5′-GTTTCCACGTCTCACCATTG-3′; Col3a1 forward, 5′-ACAGCTGGTGAACCTGGAAG-3′; Col3a1 reverse, 5′-ACCAGG-AGATCCATCTCGAC-3′; Acta2 forward, 5′-CTGACAGAGGCACC-ACTGAA-3′; Acta2 reverse, 5′-AGAGGCATAGAGGGACAGCA-3′; Fn1 forward, 5′-ACAAGGTTCGGGAAGAGGTT-3′; Fn1 reverse, 5′-CCGTGT-AAGGGTCAAAGCAT-3′; Apaf1 forward, 5′-GGGTGGGTCACCAT-CTATGG-3′; Apaf1 reverse, 5′-TTCCGCAGCTAACACAGACTTG-3′; Bax/Bcl2 forward, 5′-TGGGATGCCTTTGTGGAACT-3′; Bax/Bcl2 reverse, 5′-CAGCCAGGAGAAATCAAACAGA-3′; Cd68 forward, 5′-CTTCC-CACAGGCAGCACAG-3′; Cd68 reverse, 5′-AATGATGAGAGGCAGCAA-GAGG-3′; Mcp1 forward, 5′-TCTCT-CTTCCTCCACCACCATG-3′; and Mcp1 reverse, 5′-GCGTTAACTGCATCTG-GCTGA-3′.

Statistical differences between two groups were determined using the Mann–Whitney U-test, and P < 0.05 was considered statistically significant. All data were analyzed with SAS software, version 9.1.3 (SAS Institute Inc., Cary, NC, USA).

Pax8–rtTA Tg mice expressing rtTA in the renal proximal tubules and TRE–hCD36 Tg mice were mated to generate Pax8–rtTA/TRE–hCD36 Tg mice overexpressing hCD36. The generated Pax8–rtTA/TRE–hCD36 Tg mice overexpressed hCD36 under specific conditions such as tetracycline or doxycycline feeding (Fig. 1).

The four lines of Pax8–rtTA/TRE–hCD36 Tg mice were fed doxycycline-containing food from 4 weeks of age for 2 weeks. Finally, mice from the four lines were obtained at 5 weeks of age after the doxycycline feeding. Western blotting, qRT-PCR, and immunohistochemistry for hCD36 and mCd36 were performed with whole kidneys of all lines to identify hCD36-overespression at molecular levels. The results of qRT-PCR and western blotting showed that mice in the first and fourth lines showed the highest hCD36 expression. In addition, Tg mice showing the highest hCD36 mRNA expression yielded positive results for immunofluorescence analysis of hCD36. Thus, hCD36-overexpressing Tg mice were obtained by mating Pax8–rtTA and TRE–hCD36 Tg mice (Fig. 2). Genotyping of tail samples of the mated mice was also performed to produce definitive Pax8–rtTA/TRE–hCD36 Tg mice. On the basis of genotyping, Tg mice expressing both TRE–hCD36 and Pax8–rtTA were defined as true Pax8–rtTA/ TRE–hCD36 Tg mice, and those not expressing TRE–hCD36 and Pax8–rtTA were defined as WT mice (Fig. 3).

Next, we used these Tg mice overexpressing hCD36 in the renal proximal tubules to investigate the effects of hCD36 on the progression of folic acid-induced AKI.

Folic acid-induced AKI results in the loss or damage of renal tubular epithelial cells. Moreover, severe tubular cell damage aggravates the progression of AKI to CKD [10]. Kidney sections of folic acid-treated mice were stained with PAS for histological analysis of folic acid-induced AKI. Vehicle-treated WT and Tg mice showed relatively normal renal histology (Fig. 4A, B). However, folic acid-treated WT and Tg mice showed marked renal damage, including renal tubular dilatation, inflammatory cell infiltration in the tubulointerstitium, proximal and distal tubular epithelial flattening, and renal tubular cast formation. In addition, folic acid-treated Tg mice showed more severely dilated renal tubules with casts than folic acid-treated WT mice (Fig. 4A, B). Next, the kidney sections of folic acid-treated WT and Tg mice were stained with anti-α-SMA antibody to determine the effects of hCD36 overexpression on folic acid-induced AKI. In the present study, no significant difference was observed between folic acid-treated WT and Tg mice with respect to α-SMA expression (Fig. 4C, D). Fibrosis biomarkers are usually expressed approximately 7 days after the development of folic acid-induced nephropathy. Moreover, the evaluation of samples obtained about 7 days after folic acid-induced AKI may be better for identifying the impact of hCD36-overexpression on progression from AKI to CKD. However, severe AKI is a prominent risk factor of CKD, and preemptive analyses of qRT-PCR for inflammatory and fibrotic biomarkers at the time points (after 2 days and 7 days) in this study showed significant changes only 2 days after folic acid-treatment. Therefore, we performed analyses only 2 days after folic acid-treatment.

A decrease in albuminuria delays the progression of renal disease and improves clinical outcomes, particularly in patients with renal dysfunction [11,12]. Thus, albuminuria may be a risk factor for the progression of renal disease after AKI. In the present study, UACR was measured for all the experimental mice to determine the degree of albuminuria. The UACR was significantly higher for folic acid-treated WT and Tg mice than for vehicle-treated WT mice. Interestingly, albuminuria was significantly higher in folic acid-treated Tg mice than in folic acid-treated WT mice (Fig. 4E).

Thus, the results of histological analysis showed a higher degree of albuminuria and more severe pathological changes in folic acid-treated Tg mice than in folic acid-treated WT mice (Fig. 4D). These results suggest that hCD36 overexpression aggravates the progression of renal diseases after folic acid-induced AKI.

Generally, macrophages are deeply associated with the inflammatory response via various mechanisms. During the inflammatory response, infiltrating macrophages are usually derived from monocytes. Monocyte chemoattractant protein-1 (MCP-1; also referred to as chemokine [C-C motif] ligand 2 [CCL2]) plays an important role in inflammation [13]. CCL2 is a small cytokine associated with proinflammation or inflammation and mainly induces acute inflammatory damage in humans under pathological conditions [14].

Relative Mcp1 mRNA levels were significantly higher in folic acid-treated Tg and WT mice than in vehicle-treated WT mice. Moreover, Mcp1 mRNA levels were significantly higher in folic acid-treated Tg mice than in vehicle-treated Tg mice (P < 0.05). However, Mcp1 mRNA levels were not significantly higher in folic acid-treated Tg mice than in folic acid-treated WT mice.

Cd68 is a transmembrane glycoprotein mainly expressed by macrophages [15]. Like MCP-1, Cd68 functions as a biomarker of acute inflammation or macrophages. Relative Cd68 mRNA levels were significantly higher in folic acid-treated Tg and WT mice compared to vehicle-treated WT mice (P < 0.05). Moreover, relative Cd68 mRNA levels were significantly higher in folic acid-treated Tg mice than in vehicle-treated Tg mice (P < 0.01). However, no significant difference was observed in relative Cd68 mRNA levels between folic acid-treated WT and Tg mice (Fig. 5A, B).

The mRNA levels of typical fibrotic genes, including α-SMA (Acta2), fibronectin (Fn1), collagen 3a1 (Col3a1), and collagen 1a1 (Col1a1), were analyzed to identify the association between hCD36 overexpression and EMT in folic acid-induced AKI.

No significant difference was observed in Acta2 mRNA expression in folic acid- and vehicle-treated WT mice. However, the Acta2 mRNA level was significantly higher in folic acid-treated Tg mice compared to vehicle-treated WT and Tg mice. However, we did not observe a significant difference in Acta2 mRNA expression between folic acid-treated Tg and WT mice (Fig. 5C).

There was a trend toward increased mRNA expression of Fn1 in all folic acid-treated mice compared with vehicle-treated WT mice. In addition, the mRNA expression of Fn1 in folic acid-treated Tg and WT mice was higher than that of vehicle-treated Tg mice. However, no significant difference was observed in Fn1 mRNA expression between folic acid-treated WT and Tg mice (Fig. 5C).

Col1a1 mRNA expression was significantly higher in folic acid-treated Tg and WT mice than in vehicle-treated Tg and WT mice. Moreover, Col3a1 mRNA expression was significantly higher in folic acid-treated Tg and WT mice than in vehicle-treated Tg and WT mice. Interestingly, the mRNA levels of Col1a1 and Col3a1 were prominently increased in folic acid-treated Tg mice compared with folic acid-treated WT mice, unlike the mRNA levels of other fibrotic markers. Moreover, Col3a1 mRNA expression was significantly higher in folic acid-treated Tg mice than in folic acid-treated WT mice (Fig. 5D).

CD36 is associated with apoptosis. CD36 overexpression in renal proximal tubular epithelial cells, particularly under pathological conditions such as diabetic nephropathy and hyperlipidemia, aggravates their apoptosis [8,9]. Therefore, we analyzed the mRNA expression of several apoptotic genes, including apoptotic protease activating factor 1 (Apaf1) and B-cell lymphoma 2-associated X/B-cell lymphoma 2 (Bax/Bcl2), in folic acid-treated Tg mice to identify the relationship between hCD36 overexpression and apoptosis. BAX promotes apoptosis, whereas BCL2 exerts an antiapoptotic effect. Interestingly, the antiapoptotic effect of BCL2 is countered by combination with BAX protein [16].

We found that Apaf1 and Bax/Bcl2 mRNA expression was higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice (folic acid-treated WT mice vs. vehicle-treated WT mice: P < 0.05 and P = 0.07, respectively; folic acid-treated Tg mice vs. vehicle-treated WT mice: P = 0.052 and P < 0.05). Moreover, Bax/Bcl2 mRNA levels were significantly higher in folic acid-treated Tg mice than in vehicle-treated Tg mice. However, Apaf1 and Bax/Bcl2 mRNA levels were not significantly higher in folic acid-treated Tg mice than in folic acid-treated WT mice (Fig. 5E).