Crystal nephropathies are forms of AKI or CKD induced by solid particles of various sizes and shapes composed of atoms, ions, or biomolecules.1,2 Crystal nephropathies can be categorized in three types depending from the localization of the crystals inside afferent blood vessels (type 1), inside the nephron (type 2), or inside the renal pelvis and ureter (type 3).1 Acute oxalosis is a paradigmatic form of a type 2 crystal–induced AKI that occurs upon incident exposures to oxalate-rich food or drinks, vitamin C supplements, and during ethylene glycol poisoning.3–5 Increased intestinal adsorbtion of oxalate, e.g., after bypass surgeries, can contribute to secondary hyperoxaluria.6 In acute oxalosis, calcium oxalate monohydrate and bihydrate (CaOx) crystals form in the distal tubule. They grow to crystal plugs that obstruct the tubular lumen in some nephrons but are also widely taken up into tubular epithelial cells and even reach the interstitial space.1 We recently described that CaOx crystals trigger renal inflammation via the NLRP3 inflammasome- and caspase-1–mediated secretion of IL-1β in intrarenal dendritic cells,7 a process largely contributing to AKI. We subsequently described that crystals also exert a direct cytotoxic effect on tubular cells in acute oxalosis by inducing necroptosis, a receptor-interacting serine-threonine kinase-3 (RIPK3)–dependent and mixed lineage kinase domain-like (MLKL)–dependent form of regulated tubular cell necrosis.8 However, genetic deletion or pharmaceutic inhibition of necroptosis only partially abrogated cytotoxicity of CaOx crystals. We hypothesized that another of the recently identified forms of regulated cell death9 could be involved in CaOx crystal–induced AKI and speculated on mitochondrial permeability transition (MPT)–related cell necrosis,9,10 because a suitable inhibitor was previously shown to limit renal cell death in a rat model of oxalate nephropathy.11 This pathway is characterized by mitochondrial dysfunction involving a peptidylprolyl isomerase F (PPIF; also named cyclophilin D/F)-driven formation and permanent opening of the MPT pore, disruption of the mitochondrial outer membrane potential, mitochondrial swelling, loss of cristae, and potentially the release of reactive oxygen species (ROS) into the cytosol.9,10,12 Indeed, our studies propose MPT in concert with necroptosis promote cytotoxicity of CaOx and numerous other crystals, implying MPT/necroptosis as molecular targets to limit CaOx crystal–induced AKI.
Cell Culture Studies
Murine L929 cells were maintained in DMEM/F12 (GIBCO, Invitrogen, Carlsbad, CA) containing 10% FCS and 1% penicillin-streptomycin. Primary tubular epithelial cells were isolated from kidneys of Ppifd+/+ and Ppifd−/− mice (original strain Ppiftm1Jmol/J, Jackson Laboratories stock no. 009071) and were maintained in DMEM/F12 containing 10% FCS, 1% penicillin-streptomycin, 125 ng/ml PG E1 (Calbiochem, Darmstadt, Germany), 25 ng/ml EGF, 1.8 μg/ml l-thyroxine, 3.38 ng/ml hydrocortisone, and 2.5 mg/ml of insulin-transferrin-sodium selenite supplement (all from Sigma-Aldrich, Munich, Germany unless mentioned). Human renal progenitor cells were prepared from human kidney tissues using standard sieving technique through graded mesh screens (60, 80, 150, and 200 mesh).13 Cells were grown to confluence before use in experiments. Human kidney cells (HK-2) were maintained in RPMI (GIBCO, Invitrogen) containing 10% FCS and 1% penicillin-streptomycin. The cell lines, originally from American Type Culture Collection, were kindly provided by Peter Nelson (HK-2) and Bruno Luckow (L929), both from Ludwig Maximilian University (Munich, Germany). All cell lines used were tested for mycoplasma contamination before use. All cells were stimulated with crystals of CaOx (1000 µg/ml, 1–2 µm size) (Alfa aesar, Karlsruhe, Germany), silica (500 µg/ml, 1–5 µM size) (Alfa aesar), monosodium urate (MSU; 1000 µg/ml, 25–125 nm size) (Invivogen, Toulouse, France), and calcium pyrophosphate dihydrate (CPPD; 500 µg/ml, 25–125 nm size) (Invivogen) in different experiments. Whenever required, 30 minutes before crystal stimulations cells were pretreated with the MPT inhibitor cyclosporine A (Sigma-Aldrich (0.5 and 1 μm), mitochondrial ROS inhibitor (2R, 4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC; Sigma-Aldrich) (100 and 200 μM), phagocytosis (actin-polymerization) inhibitor cytochalasin D (Calbiochem) (5 and 10 μM), and cathepsin inhibitor CA074Me (Calbiochem) (10 and 20 μM). All the cells were cultured in an incubator at 37°C, 5% CO2. Mitochondrial outer membrane potential and ROS production were measured using tetramethylrhodamine ethyl ester (TMRE; Invitrogen), lysotracker (Abcam, Cambridge, UK), and dichlorofluorescin diacetate (DCFDA) dyes (Sigma-Aldrich), respectively, according to the manufacturer’s protocol. Calcein (Invitrogen) was used to identify live cells, whereas propidium iodide (Invitrogen) was used to identify dead cells according to the manufacturer’s protocol. Fluorescence signals were detected using Leica fluorescence microscope (Leica, Wetzlar, Germany), and quantified using ImageJ software. In addition, cell death was also evaluated using lactate dehydrogenase cell cytotoxicity assay kit (Roche, Mannheim, Germany) according to manufacturer’s protocol.
Serial Block Face Scanning Electron Microscopy Data Collection and Reconstruction
Kidney cells were fixed as above but stained using an enhanced staining protocol.14 Stained and dehydrated specimens were embedded into Durcupan ACM resin (Merck, Sigma-Aldrich) that was mixed according to the manufacturer’s recommendations. The embedded tissue was trimmed to a pyramid and mounted onto a pin using conductive epoxy glue (model 2400; CircuitWorks, Kennesaw, GA). Finally, the sides of the pyramid were covered with silver paint (Agar Scientific Ltd., Stansted, UK), and the whole assembly was platinum coated using Quorum Q150TS (Quorum Technologies, Laughton, UK). Serial block face-scanning electron microscopy (SB-EM) datasets were acquired with an FEG-SEM Quanta 250 (Thermo Fisher Scientific, FEI, Hillsboro, OR), using a backscattered electron detector (Gatan Inc., Pleasanton, CA) with 2.5-kV beam voltage, a spot size of 3, and a pressure of 0.2 Torr. The block faces were cut with 30-nm increments and imaged with XY resolution of 22 nm per pixel. The collected 16-bit images were processed for segmentation using an open-source software Microscopy Image Browser15 as follows: (1) individual images were combined into three-dimensional (3D) stacks, (2) the combined 3D stack was aligned, (3) the contrast for the whole stack was normalized, and (4) the images were converted to the eight-bit format. The collected dataset was cropped to include only cells containing crystals. The cropped dataset was filtered using BM3D filter16 and segmented using the semiautomatic graphcut segmentation of MIB. The graphcut segmentation starts with clustering of voxels into 3D supervoxels using the watershed17 (nucleus) or SLIC18 (crystals and cell body) algorithms. An adjacency map of supervoxels with energy barriers required to travel from one supervoxel to another is calculated and these values are used to generate a mathematical graph. Using the provided seeds for background and each aforementioned organelles, the algorithm performs maxflow/mincut split of the graph19 to generate the model. The final visualization of the model was done in Amira (Thermo Fisher Scientific).
Ppifd−/− mice were purchased from The Jackson Laboratories (stock no. 009071) and backcrossed to the C57BL/6J mice background. J. Murphy and W. Alexander (WEHI, Australia) kindly provided Mlkl−/− mice.20 Wild-type mice were purchased from Charles River. All mice were housed in groups of five under specific pathogen-free and 12-hour light/dark cycle conditions with ad libitum access to food and water. To induce acute oxalate nephropathy, 6- to 8-week-old male mice received a single intraperitoneal injection of 100 mg/kg of sodium oxalate (Santa Cruz Biotechnology, Heidelberg, Germany) and 3% sodium oxalate in drinking water and kidneys were harvested after 24 hours (n=5–9 per group).7,8 As a therapeutic strategy, mice received a single dose of either cyclosporine A (2 mg/kg, administered intravenously; Sigma-Aldrich) or necrostatin-1s (1.65 mg/kg, administered intraperitoneally; Millipore, Darmstadt, Germany) or combination before sodium oxalate injections (n=5–9 per group). Mice were euthanized by cervical dislocation after collecting blood and urine. One part of the kidneys was stored in RNA later solution at −20°C for RNA isolation. Another part of the kidney was stored in formalin to be embedded in paraffin for histologic analysis. Samples for electron microscopy were fixed in 4% glutaraldehyde. Mice were assigned different groups with the use of stratified randomization method. The local institutional animal ethics committee and government authorities (Regierung der Oberbayern) approved all experimental procedures. Humane end points, as in the ethical approval for the study, were monitored throughout the study. All animal studies were conducted according to the European equivalent of the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.
A retrospective search for CaOx deposits in a database of 92,000 medical and transplant renal biopsy specimens (Arkana Laboratories, Little Rock, AR) was performed (institutional review board approval number 05–4397–0). Paraffin sections were randomly selected from native kidney biopsy specimens with adequate residual tissue containing CaOx crystals (n=20), and stained with 1:500 rabbit anti pMLKL (#EPR9514; Abcam) and 1:100 rabbit anti CypD (LLC #NBP2–15079; Novus Biologicals, Littleton, CO) as per manufacturer’s instructions. Antigens were retrieved using citrate buffer pH 6.0. Bound primary antibody was visualized with a standard horseradish peroxidase detection system in brown.
Data are presented as mean±SEM. A comparison of groups was performed using paired t test, and one-way ANOVA with post hoc Bonferroni correction was used for multiple comparisons. A value of P<0.05 was considered to indicate statistical significance. We used GraphPad prism 5 software for statistical analysis. Further detailed descriptions on the experimental methods are provided in the supplemental appendix 1.
CaOx Crystal Cytotoxicity Involves MPT
First, we evaluated mitochondrial structure and function upon exposure to CaOx crystals and silica, in vitro. Both induced mitochondrial swelling, loss of cristae, loss of mitochondrial outer membrane potential, and release of ROS with subsequent cell necrosis (Figure 1, A–D). The cyclophilin (and hence MPT) inhibitor cyclosporin A fully reversed the membrane potential, whereas ROS inhibition with APDC did not (Figure 1E), indicating that ROS release is downstream of losing the mitochondrial membrane potential. In support of this finding, cyclosporin A and APDC both abrogated crystal-induced ROS production and cell necrosis (Figure 1, F and G). Similar results were obtained upon PPIF suppression with specific siRNA (Supplemental Figure 1, A and B) in L929 cells, as well as in primary tubular epithelial cells isolated from wild-type and Ppif-deficient mice (Supplemental Figure 1, C and D). Similar results were obtained for crystals of MSU and CPPD (Supplemental Figures 1–3), implying that these effects relate to particle structure rather than to the molecular type of crystal. Thus, crystal-related cell necrosis involves MPT with PPIF-driven loss of the mitochondrial outer membrane potential and ROS release as up- and downstream events, respectively.
Crystal-Induced MPT Involves Crystal Phagocytosis and Lysosomal Leakage of Cathepsins
CaOx crystal phagocytosis, lysosomal destabilization, and leakage of lysosomal proteases such as cathepsins are involved in CaOx crystal–induced activation of the NLRP3 inflammasome and other cytosolic signaling pathways.7,21,22 Indeed, two-dimensional (2D) transmission electron microscopy (TEM) and 3D SB-EM imaging studies documented phagocytosis of large amounts of CaOx crystals into endosomal compartments (Figure 2, A–C, Supplemental Videos 1 and 2). The 2D confocal microscopy documented crystal uptake into lysosomes and loss of TMRE positivity as a marker of an intact mitochondrial outer membrane potential (Figure 2, D and E). Consistently, cytochalasin D, a phagocytosis inhibitor, partially prevented the CaOx crystal–induced loss of the TMRE signal and substantially suppressed ROS production and cell necrosis (Figure 2, F–H). Furthermore, the cathepsin inhibitor CA074Me prevented CaOx crystal–induced loss of mitochondrial outer membrane potential, ROS production, and cell necrosis (Figure 2, F–H). Similar data were obtained for MSU and CPPD crystals (Supplemental Figures 4 and 5). Therefore, we conclude that CaOx crystal uptake via phagocytosis into intracellular lysosomes promotes lysosomal cathepsin leakage as upstream events in crystal-induced MPT-regulated necrosis.
Ppif Deficiency Attenuates CaOx Crystal–Induced AKI
To mimic hyperoxaluria-related AKI, we exposed mice to intraperitoneal sodium oxalate inducing intrarenal CaOx crystal deposition and AKI.7,8,23 CaOx crystal plugs formed within proximal and distal tubules as evident from Pizzolato and hematoxylin and eosin stains, as well as ex vivo kidney microcomputed tomography (Figure 3, A–C, Supplemental Figure 6, A and B, Supplemental Video 3) and induced TUNEL+ cytotoxicity in proximal and distal tubules and sporadically also in collecting ducts (Supplemental Figure 7). The 3D reconstruction of SB-EM sections revealed large luminal crystal plugs and smaller crystals also within epithelial cells of the thick ascending limb of the outer medulla (Figure 3D). In contrast to unaffected tubular segments, crystal-exposed epithelial cells exhibited mitochondrial ballooning (Figure 3, E and F). High-resolution TEM of such cells revealed loss of inner membrane cristae beyond ballooning as ultrastructural signs of MPT, water influx, and mitochondrial dysfunction (Figure 3, G–I). Indeed, advanced mitochondrial abnormalities were often seen in cells undergoing disintegration and detachment from the tubular basement membrane (Supplemental Figure 6C), i.e., acute tubular necrosis. To test whether Ppif and MPT contributes to crystalline AKI, we induced oxalate nephropathy in wild-type and Ppif-deficient mice, which resulted in identical CaOx crystal deposits in both mouse strains (Figure 4A). Functional parameters of crystalline AKI (i.e., plasma creatinine levels) and structural parameters of kidney injury (i.e., tubular necrosis and TUNEL-positive cells) were significantly attenuated in Ppif-deficient mice (Figure 4, B–E). Thus, CaOx crystal–induced cell necrosis involves Ppif-driven MPT-RN also in vivo.
MPT-RN and Necroptosis Both Contribute to CaOx Crystal Cytotoxicity In Vivo
To test for redundancy versus synergy between crystal-induced MPT-RN and necroptosis in oxalate crystal-induced AKI, we induced disease in mice with single versus dual genetic deletion of Ppif and Mlkl. Oxalate exposure induced equal amounts of CaOx crystal deposits in all mouse strains (Figure 4A). Lack of Mlkl showed a similar protection from AKI and tubular injury as compared with lack of Ppif (Figure 4, B–E). Dual gene deficiency for Ppif and Mlkl revealed a significant additive effect on tubular injury compared with the single gene deficiencies; however, this did not translate to other end points such as plasma creatinine levels or the number of TUNEL-positive cells (Figure 4, B–E). We conclude that crystal-induced tissue injury involves PPIF and MLKL, suggesting that both MPT-RN and necroptosis are involved. In addition, their partially redundant roles in crystalline AKI imply a collaborative interaction of these two signaling pathways in vivo.
MPT-RN and Necroptosis-Targeted Pharmaceutical Interventions Abrogate Crystalline Organ Injury and Failure
To test whether PPIF could be a molecular target for therapeutic intervention and to determine the potential of dual MPT-RN/necroptosis blockade, we treated wild-type mice with the MPT inhibitor cyclosporin A, the necroptosis inhibitor necrostatin-1s, or both. Oxalate exposure induced equal amounts of renal CaOx crystal deposits in mice of all groups (Figure 5A). Cyclosporin A and necrostatin-1s had comparable partial renoprotective effects on plasma creatinine levels as a marker of AKI and on tubular injury and the number of TUNEL-positive cells (Figure 5, B–E). Dual blockade was more potent than either monotherapy, especially in terms of AKI and TUNEL positivity (Figure 5, B–E). Thus, dual inhibition of MPT- and MLKL-driven necroptosis can elicit some additive effects on crystalline organ failure, but also this experiment supports functional overlap between the two pathways in vivo.
MPT in Human Tubular Cells and Acute Oxalate Nephropathy
To validate our mouse data in humans, we performed identical experiments on crystal cytotoxicity in HK-2 cells and obtained results identical to those generated with murine cells (Supplemental Figure 8). Then, we used CD24/CD133-positive epithelial progenitor cells isolated from healthy human kidneys, known to contribute to the turnover of tubular epithelial cells and their regeneration upon injury.13,24 For all crystals tested, the results were identical to the murine cells examined before (Figure 6A, Supplemental Figure 9). Next, we searched a database of 92,000 diagnostic kidney biopsies and identified 1262 cases (1.4%) showing CaOx crystals deposits by light microscopy and birefringence under polarized light. A total of 553 of these 1262 specimens (44%, or 0.6% of total) showed CaOx crystals in the context of acute tubular injury. Furthermore, immunostaining of renal tissue from oxalosis-induced AKI revealed diffuse tubular positivity for PPIF and focal positivity for phosphorylated MLKL in injured cells (Figure 6, B–E). Thus, we conclude that crystal-induced cytotoxicities of human and murine kidney cells are similar and that human tubular epithelial cells also express CYPD and activated MLKL in the context of oxalate crystal-induced AKI.
We had previously shown that CaOx crystal cytotoxicity is caspase-independent, and therefore does not involve apoptosis, but rather regulated necrosis.8 However, as interfering with necroptosis had been only partially protective, we had hypothesized that crystal cytotoxicity might involve additional pathways of regulated necrosis.9 Indeed, CaOx crystal cytotoxicity involves phagocytosis and subsequent lysosomal leakage upstream of PPIF-dependent mitochondrial dysfunction, followed by ROS production and necrotic cell death, as previously reported for MPT-RN.9 Consistently, we found PPIF-dependent MPT-RN to contribute to oxalate crystal-induced AKI. However, the lack of robust additive effects with either dual genetic deficiency or inhibition of the both pathways in vivo implies a molecular interference and partial redundancy of these two pathways in vivo in our oxalate-induced AKI. These data further unravel the molecular pathophysiology of hyperoxaluria-related AKI and identify PPIF as another potential molecular target to limit kidney injury in this context.
Mitochondrial proteins in the outer or inner mitochondrial membranes have been ascribed important roles in the regulation of inflammation and cell death.9,25,26 MPT-RN is controlled by PPIF, a mitochondrial matrix protein that is thought to promote the opening of MPT pores. This mechanism drives the breakdown of the mitochondrial outer membrane potential, water influx, mitochondrial swelling, loss of cristae, massive ROS production, and ultimately, mitochondrial and cellular demise.9,25 The biologic significance of this process was documented in several disease contexts by using Ppif-deficient mice and small molecule PPIF inhibitors.12,27,28 In particular, postischemic or radio contrast medium–induced AKI involves PPIF-driven MPT and regulated necrosis,29,30 although the specific triggers of PPIF activation remained elusive. Our data now demonstrate that CaOx crystal phagocytosis exerts a direct PPIF-driven cytotoxic effect, MPT-related regulated necrosis, on tubular epithelial cells in culture. In mice, this process contributes to oxalate crystal-induced acute tubular necrosis and crystalline AKI independent of CaOx crystallization per se, as previously reported.11,31 In acute oxalate nephropathy, this mechanism adds onto MLKL-driven necroptosis and NLRP3-driven interstitial inflammation, i.e., other molecular pathways directly triggered by crystalline particles within the kidney.1,7,8 Thus, hyperoxaluria-related AKI presents numerous molecular targets to attenuate organ failure.
The role of mitochondria in CaOx crystal cytotoxicity raises questions on the causative upstream and downstream signals. Our 2D and 3D analysis revealed that even non-professional phagocytes take up CaOx crystals and other microparticles into intracellular endosomal compartments. Indeed, blocking CaOx crystal uptake prevents the loss of the mitochondrial outer membrane potential, ROS production, and necrosis, suggesting CaOx crystal phagocytosis as an upstream event of MPT-related regulated necrosis. Crystalline microparticles are difficult to digest inside phagolysosomes and may cause lysosomal leakage of proteases, a process well described to contribute to crystal-induced activation of the NLRP3 inflammasome.7,32 In addition, one study indicated that the cysteine cathepsins might cleave RIPK1, an endogenous inhibitor of necroptosis.33 Indeed, we observed that cathepsin inhibition partially prevented CaOx crystal–induced MPT-related regulated necrosis.
The signaling events downstream of MPT are less clear. The release of ROS from mitochondria is considered an essential step in MPT-related regulated necrosis.9,25 However, it is clear that widespread depletion of mitochondria in cell culture does not affect the kinetics of necroptosis.34 However, it is appreciated that data have been published on MLKL-driven necroptosis that involves mitochondrial translocation of pMLKL in certain culture conditions.35–37 As we found that a dual genetic deficiency of Ppif or Mlkl did not give consistent results across all aspects of AKI, we conclude that at least in hyperoxaluria-induced AKI tested here, MPT-related regulated necrosis and necroptosis are not independent and both involve mitochondrial dysfunction in the context of AKI. Indeed, a recent heart transplant study reported MLKL phosphorylation and necroptosis downstream of PPIF activation.38 Although dual deficiency of PPIF or MLKL elicited the most potent protective effect on tubular injury, the combinations of two cell death inhibitors with doubtful specificities did not. These data do not exclude independent roles of the both pathways in acute oxalosis, but are consistent with a significant interference of both pathways in crystal cytotoxicity. This finding would be in line with the concept of some redundancy between signaling pathways of regulated necrosis in AKI.39
In conclusion, crystal cytotoxicity involves mitochondrial dysfunction and MPT-related regulated necrosis as a form of regulated cell death in cell culture and mouse models (Figure 7). Consequently, targeting this pathway, for example, with cyclosporine A, which may have anti-inflammatory capacities beyond inhibition of MPT, may prevent crystal-induced tissue necrosis and organ dysfunction. As interconnections to MLKL-driven necroptosis appear functionally likely, combinations of necrosis inhibitors may exhibit some additive effects, but this needs to be further explored in other crystallopathies.
Dr. Becker and Dr. Anders report grants and personal fees from Alexion Pharmaceuticals, outside the submitted work. Anders reports personal fees from Secarna, personal fees from Inositec, personal fees from Previpharma, outside the submitted work.
We thank the Deutsche Forschungsgemeinschaft (MU3906/1-1 to Mulay; AN372/16-2, 20-1, and 24-1 to Dr. Anders; SFB/TRR57, SFB/TRR219, BO3755/3-1, and BO3755/6-1 to Dr. Boor; CRC1181(C03) to Dr. Herrmann), the Heisenberg-Professorship program (no. 324141047 to Dr. Linkermann), the German Ministry of Education and Research (STOP-FSGS-01GM1518A to Boor), and the Volkswagen-Stiftung (90361 to Dr. Bilyy, Dr. Herrmann, and Dr. Anders) for funding this study.
Mulay and Anders designed the study concept and experiments. Mulay, Honarpisheh, Foresto-Neto, Shi, Desai, Zhao, Marschner, Buhl, Boor, Liapis, Bilyy, Herrmann, Romagnani, Belevich, Jokitalo, Becker, and Anders performed the experiments. Mulay, Anders, Linkermann, and Romagnani interpreted the data and wrote or significantly revised the manuscript.
We gratefully acknowledge the expert technical support of Dan Draganovici and Jana Mandelbaum. We also thank Stefan Krautwald (University of Kiel, Germany) for shipping Mlkl−/− mice, as well as Matias Veikkolainen and Mervi Lindman (University of Helsinki) for their excellent technical assistance in preparation of serial block face-scanning electron microscopy specimens. The authors acknowledge Euro-BioImaging (www.eurobioimaging.eu) for providing access to imaging technologies and services via the Finnish Advanced Light Microscopy Node (Helsinki, Finland); and Moleculart, a science communication initiative (www.moleculart.org), for graphical illustration.
This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018121218/-/DCSupplemental.
Supplemental Figure 1.
Supplemental Figure 2.
Supplemental Figure 3.
Supplemental Figure 4.
Supplemental Figure 5.
Supplemental Figure 6.
Supplemental Figure 7.
Supplemental Figure 8.
Supplemental Figure 9.
Supplemental Video 1.
Supplemental Video 2.
Supplemental Video 3.
Supplemental Appendix 1. Supplemental methods and references.
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