Cilastatin protects against tacrolimus-induced nephrotoxicity via anti-oxidative and anti-apoptotic properties

The Animal Care and Use Committee of the Catholic University of Korea approved the experimental protocol (CUMC-2016-0167-02), and all procedures performed in this study were in accordance with ethical guidelines for animal studies. Eight-week-old male Sprague Dawley rats (Charles River Technology, Seoul, Korea) that initially weighed 220–230 g were housed in a pathogen-free facility on a 12-h light/dark schedule and with ad libitum access to food and water at the Catholic University of Korea’s animal care facility. Low-salt diet (0.05% sodium, Teklad Premier, Madison, WI, USA) was fed to rats. Tacrolimus (Prograft, Astellas Pharma Inc., Ibaraki, Japan) was dissolved in olive oil (Sigma, St. Louis, MO, USA) at the concentration of 1 mg/mL. CL was obtained from SH company, Asan, Korea.

After feeding a low-salt diet for 1 week, rats were randomized to six groups containing nine rats. Several numbers are obtained from random number tables. The indicator for randomization is the remainder after the obtained number is divided by six. Rats were treated daily with TAC (1.5 mg/kg, s.c.) or a vehicle (VH, olive oil, 1 mg/ml, s.c.) with or without CL (75 and 150 mg/kg, i.p.) for 4 weeks. The dose and duration of treatment was selected based on previous reports [4, 10, 11].

Rats were monitored and treated for 4 weeks. To measure urine volume and water intake over 24 h, metabolic cages (Tecniplast, Gazzada, Italy) were used individually. Animals were anesthetized with Zoletil (30 mg/kg) and Rompun (10 mg/kg) to minimize suffering. When rat movements to pain stimuli were blunted, a 3 ml syringe was inserted into the internal jugular vein to take blood samples. Animals were euthanized via CO₂ narcosis after sample collection. Kidney cortex tissue was homogenized in lysis buffer. Homogenates were centrifuged at 3000 rpm for 15 min, and Bradford method (Bio-RAD, Hercules, California, United States) was used to determine protein concentration. Kidney tissues were also fixed in a periodate-lysine-paraformaldehyde solution and embedded in wax. After dewaxing, 4-μm sections were processed for further analysis. Urinary excretion of albumin was examined at Samkwang Medical Laboratories (Seoul, Korea) using enzymatic colorimetric methods (Modular DPP system, Roche, Hamburg, Germany). All histologic and immunoblot analysis were examined in blind condition to each group information. The quantitative enzyme colorimetric method (Stanbio Laboratory, Boerne, TX, USA) was used to measure serum creatinine (Scr) and blood urea nitrogen (BUN). Creatinine clearance (CrCl) was calculated using 24 h urine collection and serum creatinine. The TAC concentration in whole blood and kidney was measured using liquid chromatography-tandem mass spectrometry and a General Tacrolimus ELISA Kit (E1207Ge; EIAab Science, Wuhan, China), respectively.

Histological assessment was defined as the development of tubule interstitial fibrosis in trichrome-stained tissue sections, as described previously [12]. Matrix-richexpansion of the interstitium with tubular dilatation, atrophy, and cast formation was primarily defined as tubulointerstitial fibrosis. Epithelial cell sloughing or basement membrane thickening in tubular cells were also considered as tubulointerstitial fibrosis. The extent of fibrosis was estimated by counting the percentage of injured areas per field using a polygon program with a color image analyzer (TDI Scope Eye Version 3.0 for Windows; Seoul, Korea).

Immunohistochemistry (IHC) was performed as described previously [13, 14]. Immunoreactive cells were detected in 4 μm tissue sections. They were incubated with specific antibodies against ED-1 (AbD Serotec, Oxford, UK), osteopontin (OPN, obtained from the Developmental Studies hybridoma bank, University of Iowa, Iowa City, IA, USA), 8-hydroxy-2′-deoxyguanosine (8-OHdG, JaICA, Shizuoka, Japan), 4-hydroxy-2-hexenal (4-HHE, JaICA), and active caspase-3 (Millipore, St. Charles, MO, USA) for 12 h at 4 °C. Each section were examined in 20 fields at 200× magnification using the color image analyzer (TDI Scope Eye).

From tissue lysates from the renal cortex, e-cadherin (BD Biosciences, San Jose, CA, USA), TGFβ-1 (R&D Systems, Minneapolis, MN, USA), MnSOD (Abcam, Cambridge, UK), and β-actin (Sigma) were detected by incubating for 12 h with specific antibodies [15]. Image analyzer (Quantity One version 4.4.0; Bio-Rad, Hercules, CA, USA) were used to analyze the immunoblot.

To identify apoptosis in tissue sections, the ApopTag In Situ Apoptosis Detection Kit (Millipore) was used. The number of TUNEL-positive cells was counted in 20 fields at 200× magnification.

To detect the DNA adduct 8-OHdG as marker of oxidative DNA damage, competitive enzyme-linked immunosorbent assay (Cell Biolabs, San Diego, CA, USA) was used in the serum and 24 h urine.

Human kidney-2 (HK-2) cells was purchased from the American Type Cell Collection and were seeded into 96-well plates at a density of 2.5 × 10⁴ and were pre-incubated for 24 h in an incubator at 37 °C. After 12 h, the culture media was replaced to serum-free media with TAC (50 μg/mL) and CL (250 μg/mL). Cell viability was determined using a Cell Counting Kit-8 assay kit (Dojin Laboratories, Kumamoto, Japan).

Flow cytometry was performed to assess fluorescein isothiocyanate (FITC)-conjugated annexin V (BD Biosciences) production. HK-2 cells were seeded into six-well plates at a density of 2.5 × 10⁵ cells/well and pre-incubated for 12 h at 37 °C in an incubator. Trypsinized cells were treated with 5 μL of FITC- conjugated annexin V (BD Biosciences) in 1× binding buffer (BD Biosciences) for 15 min at room temperature according to the manufacturer’s protocol. Values are expressed as the percentage of fluorescent cells relative to the total cell count.

The data are expressed as means ± SEM of at least three independent experiments. We examined the normality test in all variables using Shapiro-Wilk test (Additional file 1: Table S1–3). Multiple comparisons between groups were performed by one-way ANOVA with the Bonferroni post hoc test, if variables showed normal distrubution (SPSS software version 19.0; IBM, Armonk, NY, USA). Kruskal–Wallis test or Mann-WhitneyU test was used in case of variables without normal distribution. Statistical significance was assumed as P < 0.05.

We performed power calculations to estimate the sample size using standard formulas (α error = 0.05; β error = 0.2). The sample size required to show 6% difference in tubulointerstitial fibrosis between TAC and TAC + CL75 was calculated. The minimal number of rats was nine, if we considered 20% of drop rate.

All animals were analyzed to investigate the effect of cilastatin on TAC-induced nephrotoxicity. Table 1 lists the changes in functional basic parameters in the control and experimental groups. The TAC, TAC + CL75, and TAC + CL150 groups showed lower body weight gain than that by the VH, VH + CL75, and VH + CL150 groups. The TAC-treated group with or without CL had a greater urine volume and larger water intake than the control group. The levels of Scr and BUN and the amount of microalbuminuria were significantly higher in the TAC group than in the control group. Cotreatment with CL reverted these changes. CL treatment improved the rate of CrCl, which was decreased in the TAC group. CL did not affect the trough level of TAC in the whole blood and kidney tissues.

TAC treatment induced extensive interstitial fibrosis, and CL treatment significantly reduced interstitial fibrosis (Fig. 1a). TAC treatment decreased the e-cadherin expression and increased those of TGFβ-1; CL treatment offset these changes of expression levels in TAC-treated kidney (Fig. 1b, c and Additional file 2: Figure S1 and 2).

We investigated the infiltration of ED-1-positive cells and expression of OPN in kidney tissues to find out the effect of CL on inflammatory process. There were rare ED-1-positive cells in the VH and VH + CL groups (Fig. 2a). However, these numbers were significantly increased after TAC treatment, and cotreatment with CL markedly attenuated the infiltration of ED-1 positive cells. Consistently, OPN expression was enhanced in the TAC group, and it was decreased after CL treatment (Fig. 2b).

Figure 3a and b demonstrated the results of immunohistochemistry of 8-OHdG and 4-HHE, which is a marker of oxidative DNA damage. The strong nuclear expression of 8-OHdG and 4-HHE was observed in the TAC group, and these adverse effects were reduced after CL treatment. The serum 8-OHdG level was much higher in the TAC group than in the VH group, and CL administration considerably decreased serum 8-OHdG levels (Fig. 3c). We also evaluated the expression of the antioxidative molecule MnSOD. The expression of MnSOD was decreased in the TAC group as compared with the control groups, and CL treatment recovered its expression (Fig. 3d and Additional file 2: Figure S3).

We evaluated the effects of CL treatment on apoptosis. Greater number of TUNEL-positive cells was observed in the TAC group as compared to those of VH group, and the addition of CL reduced these changes (Fig. 4a). CL cotreatment also exhibited decrements in the active form of caspase-3 in kidney tissues as compared with TAC treatment alone (Fig. 4b).

The viability of HK-2 cells was significantly decreased in the TAC group as compared to the VH group, and CL increased the viability of TAC-treated cells (Fig. 5a). To evaluate the protective effect of CL on apoptosis, we measured the binding activity of FITC-annexin V. TAC treatment increased the number of FITC-annexin V binding cells as compared to the VH group, and CL treatment significantly decreased annexin V positive cells (Fig. 5b).