Bioenergetic maladaptation and release of HMGB1 in calcineurin inhibitor-mediated nephrotoxicity

Abstract

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.

Keywords: animal models: murine; basic (laboratory) research / science; immunosuppressant - calcineurin inhibitor (CNI); kidney transplantation / nephrology.

FIGURE 1

CsA induces HMGB1 nucleus-to-cytosol translocation and extracellular release from hPTEC. (A) Representative images showing fluorescence patterns of HMGB1 in hPTEC after treatment with CsA (0, 1, 3, or 6 μM) for 24 h. HMGB1—green, nuclei—blue. Arrows indicate increased HMGB1 fluorescence in cytosol. Scale bars 25 µm. (B, C) Representative western blots and optical densitometry indicate HMGB1 levels in culture media from hPTEC exposed to (B) CsA (at the indicated concentrations) for 24 h, or (C) CsA (6 μM) in a time-dependent manner. Data are presented as scatter plot, mean ± SEM, n = 3, *p < .05 (ANOVA). (D) Representative images showing HMGB1 immunofluorescence staining in control hPTEC and after treatment with TAC for 24 h. Scale bars 25 µm. (E, F) HMGB1 levels in culture media of hPTEC that were treated with (E) TAC (dose response) for 24 h, or (F) TAC (30 µM) for indicated time. Immunoblot optical densitometry, mean ± SEM, n = 3, *p < .05 (ANOVA). (G, H) The amounts of total PARP, cleaved PARP and β-actin in lysates from hPTEC. Cells were treated with (G) CsA (0, 6, or 12 µM) or (H) TAC (0, 30, or 60 µM) for 24 h. Data presented as mean ± SEM, n = 3, *p < .05 (ANOVA)

FIGURE 2

HMGB1 nucleus-to-cytosol translocation is associated with mitochondrial fragmentation, ROS formation, and loss cell-to-cell adhesion in CsA-treated hPTEC. (A) Representative images showing HMGB1 fluorescence patterns in control (vehicle) or hPTEC treated with CsA (6 µM) for 24 h. HMGB1—green; nuclei—blue. Scale bars 25 µm. (B, C) Representative images (B) and quantitative analysis of (C) DCF fluorescence in hPTEC treated with or without CsA (6 µM) for 24 h. DCF—green; nuclei—blue. Scale bars 100 µm. Data are presented as fold control of DCF/nuclei fluorescence ratios. Box and whiskers plot (min/max), n = 6–9, *p < .05 (Student’s t-test). (D) Mito-SOX fluorescence intensity in hPTEC treated with CsA (0, 3, or 6 µM) for 24 h. (E) Images depict mitochondrial network in control and CsA-treated hPTEC (6 µM) for 24 h. Dashed boxes indicate regions that are enlarged and displayed on the right side. Mitotracker—red; nuclei—blue. Scale bars 5 µm

FIGURE 3

CsA-mediated mitochondrial dysfunction and a shift toward glycolytic metabolism in PTEC. (A, B) Representative western blots and quantitative analysis of the major mitochondrial ETC subunits from mPTEC-treated with or without (A) CsA (6 µM), or (B) TAC (indicated concentrations) for 48 h. Quantitative analysis is from CsA (6 µM) or TAC (30 µM)-treated cells for 48 h. Scatter plot; mean ± SEM, fold control, n = 3, *p < .05 (ANOVA). (C-E) The effects of CsA (6 µM, 48 h) on (C) oxygen consumption rates (OCR), including basal, maximal, ATP-linked, reserve capacity, proton leak, and nonmitochondrial OCR; (D) mitochondrial ETC complex I, II and IV activity; and (E) extracellular acidification rates (ECAR). Mean ± SEM, n = 3–4, *p < .05 (ANOVA). (F) OCR and ECAR analysis of CsA (0–9 µM)-treated mPTEC for 48 h. Mean ± SEM, n = 5 per indicated groups. Red arrows indicate a shift toward glycolytic metabolism

FIGURE 4

CsA and TAC stimulate Erk1/2-dependent HMGB1 nucleus-to-cytosol translocation and extracellular release from PTEC. (A, B) Representative western blots and optical densitometry showing HMGB1 levels in culture media, while total and phospho Thr202/Tyr204-Erk, as well as GAPDH were determined in cell lysates. Cells were treated with Erk1/2 inhibitor PD0325901 (0 or 10 nM) for 60 minutes prior to inclusion of (A) CsA (0 or 6 μM) or (B) TAC (0 or 30 µM) for additional 60 minutes. Data presented as scatter plot, mean ± SEM, n = 3, *p < .05 (ANOVA). (C) Images show HMGB1 fluorescence patterns in hPTEC. Cells were incubated with CsA or TAC and inhibitors as depicted in A and B, respectively. Arrows indicate HMGB1 immunofluorescence in cytosol. HMGB1—green; nuclei—blue. Scale bars 25 μm

FIGURE 5

Pro-inflammatory and profibrogenic stimuli promote HMGB1 release from kidney epithelial cells. (A) Representative images show HMGB1 fluorescence patterns in control (vehicle) or mIMCD3 treated with TGFβ1 (10 ng/ml), TNFα (40 ng/ml), IFN-γ (40 ng/ml), or IL-1β (40 ng/ml) for 24 h. HMGB1—green; nuclei—blue. Scale bars 25 μm. (B-D) Representative western blots and optical densitometry of HMGB1 in culture media from (B) mIMDC3, (C) mPTEC, and (D) hPTEC. Cells were treated as depicted in (A). Data presented as scatter plot, mean ± SEM, n = 3–4, *p < .05 (ANOVA)

FIGURE 6

CNI-mediated nephrotoxicity is associated with HMGB1 release from kidney epithelial cells. (A) Treatment outline for CsA or TAC administration in mice. (B) Periodic acid Schiff (PAS) staining of kidney sections from control (vehicle) or mice treated with CsA (60 mg/kg; i.p.) or TAC (1 mg/kg; i.p.), daily for a total of 2 or 4 weeks. Scale bars 100 μm. Indices of injury include vacuolized tubules [v], tubular atrophy areas [a], and tubular dilatation [h]. (C, D) Indices of CsA- or TAC-mediated nephrotoxicity. Panel (C) depicts the tubular injury score (%), and (D) serum creatinine. Data are presented as box and whiskers plot, (min/max), n = 5 mice/group (morphometric analysis from 5 images/mouse), or n = 5 mice/group for serum creatinine, *p < .05 (ANOVA). (E) HMGB1 fluorescence in kidney cortex and medulla from control (vehicle) or mice treated with CsA or TAC for 4 weeks. Representative images are shown and white arrows indicate HMGB1 release. Scale bars 100 μm. HMGB1—green. (F, G) HMGB1 levels in urine from control (vehicle) and (F) CsA- or (G) TAC-treated mice for 2 and 4 weeks, as depicted in (A). Data are presented as box and whiskers plot (min/max), n = 4–5 mice urine, *p < .05 (ANOVA)

FIGURE 7

Inhibition of Erk1/2 signaling diminished CNI-mediated nephrotoxicity in mice. (A) Therapeutic administration of Erk1/2 inhibitor (PD 2.5 mg/kg; i.p.) in mice subjected to CsA (60 mg/kg; i.p.) or TAC (1 mg/kg; i.p.), each day for 2 weeks. (B) Kidney PAS staining showing a reduced kidney injury in mice treated with Erk1/2 inhibitor combined with CsA or TAC. Vacuolized tubules [v], tubular atrophy areas [a], and tubular dilatation [h] are indicated. (C) Tubular injury score (%) and serum creatinine in indicated groups of mice. Box and whiskers plot (min/max), n = 5 mice/group for morphometric analysis using 5 images/mouse, or n = 5 mice/group for serum creatinine, *p < .05 (ANOVA). (D) HMGB1 and nuclei fluorescence patterns in kidney sections from indicated groups of mice. Dashed boxes indicate regions that are magnified and displayed in lower panels. Arrows depict HMGB1 nucleus-to-cytosol translocation in kidney epithelial cells. (E, F) HMGB1 levels in urine from control (vehicle) mice or subjected to CsA, TAC, and PD alone, as well as CsA or TAC combined treatment with PD. Western blots and optical densitometry are shown. Data presented as box and whiskers plot (min/max), n = 5, *p < .05 (ANOVA)

FIGURE 8

Proposed paradigm for CNI-mediated nephrotoxicity. Under nonapoptotic conditions, CNI-induced mitochondrial dysfunction, oxidative stress, and metabolic maladaptation, that while not compromising cell viability, result in loss of epithelial phenotype and HMGB1 release is mediated by ERK signaling. At apoptotic conditions, HMGB1 is actively released