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. 2015 Mar 18;7(279):279ra36.
doi: 10.1126/scitranslmed.3010755.

MG53-mediated cell membrane repair protects against acute kidney injury

Pu Duann #  1 , Haichang Li #  1 , Peihui Lin  1 , Tao Tan  1 , Zhen Wang  2 , Ken Chen  2 , Xinyu Zhou  1 , Kristyn Gumpper  1 , Hua Zhu  1 , Thomas Ludwig  3 , Peter J Mohler  4   5 , Brad Rovin  5 , William T Abraham  5 , Chunyu Zeng  2 , Jianjie Ma  1   3
Affiliations

MG53-mediated cell membrane repair protects against acute kidney injury

Pu Duann et al. Sci Transl Med. .

Abstract

Injury to the renal proximal tubular epithelium (PTE) represents the underlying consequence of acute kidney injury (AKI) after exposure to various stressors, including nephrotoxins and ischemia/reperfusion (I/R). Although the kidney has the ability to repair itself after mild injury, insufficient repair of PTE cells may trigger inflammatory and fibrotic responses, leading to chronic renal failure. We report that MG53, a member of the TRIM family of proteins, participates in repair of injured PTE cells and protects against the development of AKI. We show that MG53 translocates to acute injury sites on PTE cells and forms a repair patch. Ablation of MG53 leads to defective membrane repair. MG53-deficient mice develop pronounced tubulointerstitial injury and increased susceptibility to I/R-induced AKI compared to wild-type mice. Recombinant human MG53 (rhMG53) protein can _target injury sites on PTE cells to facilitate repair after I/R injury or nephrotoxin exposure. Moreover, in animal studies, intravenous delivery of rhMG53 ameliorates cisplatin-induced AKI without affecting the tumor suppressor efficacy of cisplatin. These findings identify MG53 as a vital component of reno-protection, and _targeting MG53-mediated repair of PTE cells represents a potential approach to prevention and treatment of AKI.

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Figures

Fig. 1
Fig. 1. MG53 deficiency impairs renal function
(A and B) Mg53−/− mice develop proteinuria as they age (20-week versus 10-week ages), as shown by colloidal blue–stained SDS–polyacrylamide gel electrophoresis (SDS-PAGE) of urine (A), and Up/Uc ratios (B). **P < 0.001. Bovine serum albumin (BSA) was used as a loading control (10 and 3 μg). (C) Mg53−/− animals display impaired kidney function with an increase in SCr compared with littermate wild-type (WT) controls (**P < 0.001). (D) Compared with WT kidney, Mg53−/− kidney shows pathology at the inner cortex with pronounced vacuolization (red arrows) and disorganized cisternae (yellow arrow). Scale bar, 1 mm. (E) H&E staining shows widening of the interstitial compartment in the Mg53−/− kidney. Scale bar, 100 μm. (F) Transmission electron micrographs reveal disorganized microvilli and brush border at the apical surface of PTE cells derived from the Mg53−/− kidney. Scale bar, 2 μm. (G) The intertubular space was ~2.5-fold larger in the Mg53−/− kidney than that in the WT kidney (averaged from a total of 12 images; **P < 0.001).
Fig. 2
Fig. 2. MG53 mediates membrane repair in proximal tubular epithelial cells
(A) Western blot of lysates (50 μg) from total kidney or isolated cortex and medulla derived from WT (+/+) or Mg53 knockout (−/−) mice. One-tenth of the amount (5 μg) of WT skeletal muscle lysates was used for comparison. Purified rhMG53 was used as a positive control. (B) MG53 protein is detected in PTE cells from WT mice and rats, but not in glomeruli isolated from rats or in PTE cells from Mg53−/− mice. The identities of PTE cells or glomeruli were verified by the expression of E-cadherin or nephrin, respectively. (C) Total tissue lysates (50 μg) from human kidney and bladder were immunoblotted with anti-MG53 antibody. (D) GFP-MG53 expressed in Mg53−/− PTE cells translocates to the area of acute mechanical injury after microelectrode penetration. Scale bar, 10 μm. (E) The WT PTE cells survive after acute mechanical injury, whereas the Mg53−/− PTE cells always die within 10 s of micro-electrode penetration. (F) GFP-MG53 overexpression rescues survival of Mg53−/− PTE cells after microelectrode-induced membrane damage. P values for comparisons with Mg53−/− group are all <0.001.
Fig. 3
Fig. 3. MG53 deficiency aggravates I/R-induced AKI
(A and B) H&E and PAS stains were used to evaluate the pathological changes in the kidneys of WT (A) and Mg53−/− mice (B) upon sham treatment (top panels) or I/R-induced AKI (bottom panels). Scale bar, 100 μm. Mg53−/− kidneys are more susceptible to I/R-induced injury. (C and D) Time-dependent urinary protein excretion (C) and SCr concentration at 5 days after I/R-induced AKI (D) show significant differences between WT and Mg53−/− mice. *P < 0.01, **P < 0.001.
Fig. 4
Fig. 4. rhMG53 colocalizes with annexin V at the plasma membrane of PTE cells after A/R injury
PTE cells were treated with rhodamine-labeled rhMG53 (0.1 mg/ml) or rhodamine-labeled BSA (0.1 mg/ml, as control). Immunostaining was performed with fluorescein isothiocyanate (FITC)–annexin V for labeling of PS exposed at the plasma membrane. Control PTE cells are negative for staining with rhMG53 or annexin V. PTE cells exposed to A/R show positive staining with rhMG53 and annexin V (bottom panels). In addition to localization at the plasma membrane (overlapping pattern with annexin V), a portion of rhMG53 could enter the PTE cells after exposure to A/R. As control, cells incubated with BSA (middle panels) showed neither plasma membrane _targeting nor intracellular localization of BSA. Scale bar, 10 μm.
Fig. 5
Fig. 5. rhMG53 protein ameliorates I/R-induced AKI in rat model
(A and B) Kidney function assessed by Ualb/Uc (A) or SCr (B) demonstrates the beneficial effects of rhMG53 in prevention of I/R-induced AKI. (C) IHC staining with anti–KIM-1 reveals reduced kidney pathology in rhMG53-treated animals 5 days after I/R injury. (D) H&E staining shows that rhMG53 treatment improved kidney histopathology 5 days after I/R-induced AKI. Scale bar, 100 μm. (E) Injury scores based on quantitative analysis of KIM-1 [shown in (C)] reveal diminished tubular injury in I/R-injured rats that receive rhMG53 (n = 4 to 9 per group; *P < 0.01, **P < 0.001). Veh, vehicle.
Fig. 6
Fig. 6. Cisplatin-induced injury of PTE cells leads to colocalization of rhMG53 and annexin V at the plasma membrane
PTE cells were treated with cisplatin (50 μg/ml) for 3 hours. Rhodamine-labeled rhMG53 (0.1 mg/ml) or rhodamine-labeled BSA (0.1 mg/ml, as control) was then added to the cells. Immunostaining was performed with FITC–annexin V for labeling of exposed PS on the plasma membrane. Control PTE cells are negative for staining with rhMG53 or annexin V. PTE cells exposed to cisplatin show positive staining with rhMG53 and annexin V (bottom panels). Cells incubated with BSA show neither plasma membrane _targeting nor intracellular localization of BSA, under control conditions or after cisplatin treatment. Scale bar, 10 μm.
Fig. 7
Fig. 7. rhMG53 protects against cisplatin-induced AKI in mice
(A) H&E and PAS staining show kidney pathology in WT mice 5 days after cisplatin treatment (30 mg/kg, intraperitoneal). Mice were given one intravenous dose of either rhMG53 (2 mg/kg, left) or vehicle (right) 10 min before cisplatin treatment. Kidney histology showed less severe tubular injury after rhMG53 administration. Scale bar, 100 μm. (B) Summary data of urinary protein measurements (Up/Uc) for mice treated with different combinations (vehicle, cisplatin, or cisplatin + rhMG53). (C) Mice that received rhMG53 display reduced SCr concentrations at 5 days after cisplatin treatment. *P < 0.01, ** P < 0.001. (D) MTT assay shows that rhMG53 does not alter IC50 for cisplatin-induced cell death in murine pancreatic cancer cells (KPC-Brca1). n = 4 per group. (E) rhMG53 does not alter the efficacy of cisplatin suppression of tumor growth. KPC-Brca1 pancreatic tumor cells were injected subcutaneously into both flanks of nude mice and allowed to grow for 5 days before initiating treatment. Arrows indicate when the mice received injections of cisplatin (6 mg/kg, intraperitoneal) together with rhMG53 (2 mg/kg, intravenous) or saline as vehicle control. Mice demonstrated a similar extent of tumor regression with or without rhMG53 administration (n = 10 for each group).
Fig. 8
Fig. 8. Repetitive intravenous administration of rhMG53 in beagle dogs
(A) rhMG53 (1 mg/kg body weight) was administered to beagle dogs by intravenous injection every other day for a total of seven doses. Histological (H&E staining) analyses did not reveal gross abnormality within major vital organs. Scale bar, 200 μm. (B) ELISA determination of serum levels of rhMG53 shows that the pharmacokinetic properties for MG53 remained unchanged between the beginning (first dose) and the end of repeated intravenous administration (seventh dose), with a half-lifetime of ~1.4 hours.
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