Accelerated receptor shedding inhibits kidney injury molecule-1 (KIM-1)-mediated efferocytosis

Abstract

Efficient clearance of apoptotic cells (efferocytosis) prevents inflammation and permits repair following tissue injury. Kidney injury molecule-1 (KIM-1) is a receptor for phosphatidylserine, an "eat-me" signal exposed on the surface of apoptotic cells that marks them for phagocytic clearance. KIM-1 is upregulated on proximal tubule epithelial cells (PTECs) during ischemic acute kidney injury (AKI), enabling efferocytosis by surviving PTECs. KIM-1 is spontaneously cleaved at its ectodomain region to generate a soluble fragment that serves a sensitive and specific biomarker for AKI, but the biological relevance of KIM-1 shedding is unknown. Here, we sought to determine how KIM-1 shedding might regulate efferocytosis. Using cells that endogenously and exogenously express KIM-1, we found that hydrogen peroxide-mediated oxidative injury or PMA treatment accelerated KIM-1 shedding in a dose-dependent manner. KIM-1 shedding was also accelerated when apoptotic cells were added. Accelerated shedding or the presence of excess soluble KIM-1 in the extracellular milieu significantly inhibited efferocytosis. We also identified that TNF-α-converting enzyme (TACE or ADAM17) mediates both the spontaneous and PMA-accelerated shedding of KIM-1. While accelerated shedding inhibited efferocytosis, we found that spontaneous KIM-1 cleavage does not affect the phagocytic efficiency of PTECs. Our results suggest that KIM-1 shedding is accelerated by worsening cellular injury, and excess soluble KIM-1 competitively inhibits efferocytosis. These findings may be important in AKI when there is severe cellular injury.

Keywords: efferocytosis; kidney injury molecule-1 (KIM-1); proximal tubule epithelial cells (PTECs); receptor; shedding.

Fig. 1.

H₂O₂ and excess apoptotic cells accelerate Kidney injury molecule-1 (KIM-1) shedding. A: primary mouse proximal tubule epithelial cells (PTECs) were treated with increasing concentrations of H₂O₂, and both conditioned media and total cell lysates were collected. Soluble mouse KIM-1 was detected by SDS-PAGE and Western blotting. Soluble KIM-1 relative to total KIM-1 was quantified by densitometry. B: 769-P cells were exposed to H₂O (1 μl) or H₂O₂ (1 mM) for 30 min in serum-free DMEM and the total cell lysates were collected and analyzed by Western blotting for cleaved and total KIM-1. The graph summarizes cleaved KIM-1 relative to total KIM-1 as determined by densitometry from 3 independent experiments (right). C: confluent monolayers of 769-P cells plated in 6-well tissue culture plates were exposed to H₂O (1 μl) or various concentrations of H₂O₂ (0.01, 0.1, 1.0, and 10 mM) for 30 min in serum-free DMEM before analysis of the conditioned medium for soluble KIM-1. D: H₂O₂-induced KIM-1 shedding occurs independently of changes in KIM-1 mRNA expression. 769-P cells were exposed to H₂O (1 μl) or H₂O₂ (1 mM) for 30 min in serum-free DMEM. Total cellular RNA was harvested, and relative KIM-1 mRNA expression was quantified using quantitative RT-PCR. E: UV-induced apoptotic(AC) or live mouse thymocytes that were stained with annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry. F: confluent monolayers of 769-P cells were not exposed or exposed to 10⁷ apoptotic cells for 1 h. Relative shedding as a fraction of total KIM-1 was quantified by densitometry. A–E: soluble KIM-1 was detected from media samples with AKG antibody. Cleaved and total KIM-1 were detected in the total cellular lysates with 195 antibody. Actin was used as a loading control. The Western blot result is representative of 3 independent experiments. G: after being not fed or fed 5-(and 6)-carboxyfluorescein di-acetate succinimidyl ester (CFSE)-labeled apoptotic cells (AC) as in D, single-cell suspensions of the 769-P cells were analyzed for change in surface KIM-1 expression (Alexa 633-KIM-1) after staining with anti-KIM-1 primary antibody (AKG) and Alexa 633-conjugated secondary antibody. NS, not significant. *P < 0.05.

Fig. 2.

PMA and pervanadate (PV) accelerate KIM-1 shedding. A: confluent monolayers of 769-P cells were incubated in serum-free DMEM with DMSO (control) or PMA, (1 μM) for 1 h or PV (50 μM) for 30 min. A graph summarizing soluble KIM-1 relative to total KIM-1 in 769-P cells as determined by densitometry from 3 independent experiments is shown. B: 769-P cells were pretreated with PMA at various concentrations (0, 10, and 100 nM, and 5 μM) for 30 min (left) and for various durations of time (30 min, 1 h, or 2 h) at a concentration of 1 μM (right). C: PMA-induced KIM-1 shedding occurs independently of mRNA synthesis. 769-P cells were exposed to DMSO (control) or PMA for 1 h in serum-free DMEM. Total cellular RNA was harvested, and relative KIM-1 mRNA expression was analyzed using quantitative RT-PCR. D: 769-P cells were incubated for 30 min with or without PMA (1 μM) or PV (50 μM) before detection of surface KIM-1 expression by flow cytometry using AKG antibody and Alexa Fluoro 633-conjugated as secondary antibodies. KIM-1 expression is displayed in the form of a single-parameter (Alex 633-KIM-1) histogram against the % maximum mean fluorescent intensity. E: PMA-induced KIM-1 shedding occurs independently of protein synthesis. 769-P cells were preincubated for 30 min with cycloheximide (CHX; 25 μg/ml) in DMEM medium and then, with DMSO (control) or PMA (1 μM) for an additional 1 h before detection of soluble, cleaved, and total KIM-1 by Western blotting. F: KIM-1-PK1 cells were incubated in DMEM with DMSO (control) or PMA (1 μM) for 1 h, or with or without PV (50 μM) for 30 min. Graph summarizing cleaved KIM-1 relative total KIM-1 in KIM-1-PK-1 cells as determined by densitometry from 3 independent experiments is shown. G: HK-2 cells were incubated in DMEM with DMSO (control) or PMA (1 μM) for 1 h, or PV (50 μM) for 30 min. A–G: soluble KIM-1 was detected from media samples with AKG antibody. Cleaved and total KIM-1 were detected in the total cellular lysates with 195 antibody. Actin was used as a loading control. Cleaved KIM-1 relative to total KIM-1 was quantified by densitometry. Western blot results described above are representative of results obtained from 3 independent experiments. *P < 0.05.

Fig. 3.

Constitutive and accelerated KIM-1 shedding is inhibited by TNF-α-converting enzyme (TACE) inhibitors and short hairpin (sh) RNA-mediated knockdown of TACE. A: confluent monolayers of 769-P renal cell carcinoma (RCC) cells were first pretreated with DMSO (control), GM6001 (1 μM), or TAPI-0 (10 μM) for 30 min following exposure to H₂O (1 μl) or H₂O₂ (1 mM) for 30 min. Shed KIM-1 was detected in media with AKG antibody. Cleaved and total KIM-1 was detected in total cellular lysate with 195 antibody. B and C: experiment in A was repeated with PMA (1 μM) for 1 h and PV (50 μM) for 30 min. A and B: soluble KIM-1 relative to total KIM-1 was quantified by densitometry. Lines between the bands represent any lanes that were rearranged from the original blot. Cleaved KIM-1 was detected using a 15% gel, while total KIM-1 and actin were detected using a 10% gel. D: cell lysates of 786-O RCC cells stably expressing a vector encoding shRNA targeting TACE (shTACE) or the empty vector alone (pSilencer) were analyzed by Western blotting with anti-TACE antibody. A band corresponding to TACE is shown. E: confluent monolayers shTACE and pSilencer cells were preincubated with PMA for 30 min, and KIM-1 shedding was assessed by Western blot analysis. Actin was used as a loading control. The Western blot data are representative of the results obtained from 3 independent experiments. F: 769-P cells were incubated with H₂O (1 μl), H₂O₂ (1 mM), DMSO, or PMA (1 μM) for 30 and 60 min, respectively, before detection of surface TACE expression by flow cytometry using a TACE antibody. Surface TACE expression is displayed in the form of a histogram against the % maximum mean fluorescent intensity and as a graph representative of data from 3 independent experiments. *P < 0.05.

Fig. 4.

Accelerated KIM-1 shedding inhibits efferocytosis in KIM-1-expressing PTECs. A: PCDNA-PK1 or KIM-1-PK1 cells were incubated with 10⁶ or 10⁷ CFSE-labeled apoptotic cells(AC) in 6-well plates for 90 min, and the phagocytic uptake of apoptotic cells was analyzed by flow cytometry. Data re depicted in the form of a representative single-parameter (CFSE-labeled AC) histogram determined from 3 independent experiments. B: KIM-1-PK1 cells were untreated or pretreated with H₂O (control) or H₂O₂ (1 mM) for 30 min in 6-well plates followed by incubation with 10⁷ CFSE-labeled apoptotic cells for 90 min before measurement of phagocytic uptake of apoptotic cells by flow cytometry. C: KIM-1-PK1 cells were pretreated with DMSO (control), PV (50 μM) for 30 min, or PMA (1 μM) for 1 h in 6-well plates followed by incubation with 10⁷ CFSE-labeled apoptotic cells for 90 min. Samples were analyzed by flow cytometry as in A. D: data from 3 independent experiments in C were graphed to show percent phagocytosis with error bars representing SD. E: change in surface KIM-1- expression in KIM-1-PK1 cells after addition of DMSO (vehicle), PMA, and PV as determined by flow cytometry using AKG primary antibody and Alexa Fluoro 633-conjugated anti-mouse IgG secondary antibody. F: 769-P cells were first pretreated with TAPI-0 (10 μM) for 30 min and then with H₂O₂ (1 mM) for 30 min before incubation with 10⁶ CFSE-labeled apoptotic cells for an additional 90 min. Percent phagocytosis was determined as described in A from 3 independent experiments. G: KIM-1-PK1 cells were first pretreated with TAPI-0 (10 μM) for 30 min and then with DMSO (−PV) or PV (+PV) for another 30 min before incubation with 10⁶ CFSE-labeled apoptotic cells for an additional 90 min. Percent phagocytosis was determined as described in A. H: shTACE and pSilencer cells were pretreated with and without PMA (1 μM) for 1 h followed by incubation with apoptotic cells for 90 min. Flow cytometry analysis was done as in A. *P < 0.05.

Fig. 5.

Soluble KIM-1 competitively inhibits apoptotic cell engulfment by KIM-1-expressing PTECs. A: CFSE-labeled apoptotic cells were incubated on ice for 30 min with increasing fractions of conditioned medium from 769-P cells cultured for 3 days. Thereafter, the conditioned medium containing the apoptotic cells was added to confluent monolayers of KIM-1-PK1 cells for 90 min before measurement of phagocytic uptake by flow cytometry. B: Western blot detecting soluble KIM-1 in the conditioned medium collected from KIM1-Tet-off MDCKII cells either treated with doxycycline (100 ng/ml) to inhibit KIM-1 expression (−sKIM-1) or no doxycycline for 5 days, permitting high-level expression of KIM-1 (+sKIM-1). C: CFSE-labeled apoptotic cells were incubated on ice for 30 min with conditioned medium from KIM1-Tet-off MDCKII cells either treated with doxycycline (−sKIM-1) or no doxycycline (+sKIM-1). Thereafter, the conditioned media containing the apoptotic cells were added to monolayers of KIM-1-PK1 cells for 90 min before measurement of phagocytic uptake by flow cytometry. D: relative phagocytic uptake of labeled apoptotic cells by KIM-1-PK1 cells incubated in the absence or presence of recombinant KIM-1-Fc protein (n = 3) or nonspecific IgG at the concentration indicated. C and D: results are expressed as percent relative phagocytosis of untreated control. A–D: fresh culture medium was used as the control. E: soluble KIM-1 does not confer on PTECs the ability to engulf apoptotic cells in the absence of KIM-1 expression. CFSE-labeled apoptotic cells were incubated on ice for 30 min with the conditioned (−sKIM-1 or +sKIM-1) medium from KIM1-Tet-off MDCKII cells, and thereafter the conditioned media containing the apoptotic cells were added to the PCDNA-PK1 cells for an additional 90 min before measurement of phagocytic uptake by flow cytometry. *P < 0.05.

Fig. 6.

Phagocytic uptake of apoptotic cells is impaired in PTECs expressing a cleavage-mutant of KIM-1. A: LLC-PK₁ cells stably expressing wild-type (KIM-1-PK1) or a cleavage-mutant of KIM-1 (Δ278–283-PK1) were treated with PMA (1 μM) or vehicle control (DMSO) for 30 min. Soluble and cleaved KIM-1 were detected from media and cell lysate samples, respectively. Actin was used as a loading control. Soluble and cleaved KIM-1 relative to total KIM-1 was quantified by densitometry. B: surface expression of KIM-1 in KIM-1-PK1 and Δ278–283-PK1 cells was evaluated by flow cytometry using both AKG or control antibody and Alexa Fluoro 633-conjugated secondary without permeabilization. C: KIM-1-PK1 and Δ278–283-PK1 cells were incubated with 10⁶ CFSE-labeled apoptotic cells for 90 min, and phagocytosis was analyzed by flow cytometry. Data are depicted in the form of a representative single-parameter histogram (left) and a graph summarizing the relative (%) phagocytosis determined from 3 independent experiments (right). D: binding of pHrodo-labeled apoptotic cells(AC) to confluent monolayers of KIM-1-PK1 and Δ278–283-PK1 cells at 4°C as observed by confocal microscopy. Images were captured at 400× magnification, and the scale bar represents 10 μm. E: percent bound apoptotic cells relative to the total number of nuclei determined from 3 independent experiments in D. F: KIM-1-PK1 cells were pretreated with DMSO (control) or GM6001 (1 μM) for 30 min followed by incubation with 10⁷ CFSE-labeled apoptotic cells (AC) for 90 min. Flow cytometric analysis was used to determine the percent phagocytosis from 3 independent experiments. G: KIM-1-PK1 and Δ278–283-PK1 cells were pretreated with DMSO (control) or PMA (1 μM) for 1 h in 6-well plates followed by incubation with 10⁷ CFSE-labeled apoptotic cells for 90 min. Percent phagocytosis was determined by flow cytometry from 3 independent experiments. *P < 0.05.

Fig. 7.

Hypothetical model showing the regulation of KIM-1-mediated efferocytosis by receptor shedding. A: spontaneous KIM-1 shedding does not affect the efficiency of KIM-1-dependent efferocytosis. B: competitive inhibition of efferocytosis by decoy (soluble KIM-1) receptors resulting from excess KIM-1 shedding due to exaggerated cellular injury (e.g., H₂O₂).