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. 2015 Jul 10;10(7):e0132724.
doi: 10.1371/journal.pone.0132724. eCollection 2015.

Tacrolimus Protects Podocytes from Injury in Lupus Nephritis Partly by Stabilizing the Cytoskeleton and Inhibiting Podocyte Apoptosis

Ruyi Liao  1 , Qinghua Liu  1 , Zhihua Zheng  1 , Jinjin Fan  1 , Wenxing Peng  1 , Qingyu Kong  1 , Huijuan He  1 , Shicong Yang  2 , Wenfang Chen  2 , Xueqing Tang  1 , Xueqing Yu  1
Affiliations

Tacrolimus Protects Podocytes from Injury in Lupus Nephritis Partly by Stabilizing the Cytoskeleton and Inhibiting Podocyte Apoptosis

Ruyi Liao et al. PLoS One. .

Abstract

Objective: Several studies have reported that tacrolimus (TAC) significantly reduced proteinuria in lupus nephritis (LN) patients and mouse models. However, the mechanism for this effect remains undetermined. This study explored the mechanism of how TAC protects podocytes from injury to identify new targets for protecting renal function.

Methods: MRL/lpr mice were given TAC at a dosage of 0.1 mg/kg per day by intragastric administration for 8 weeks. Urine and blood samples were collected. Kidney sections (2 μm) were stained with hematoxylin-eosin (HE), periodic acid-Schiff base (PAS) and Masson's trichrome stain. Mouse podocyte cells (MPC5) were treated with TAC and/or TGF-β1 for 48 h. The mRNA levels and protein expression of synaptopodin and Wilms' tumor 1 (WT1) were determined by real-time PCR, Western blotting and/or immunofluorescence, respectively. Flow cytometry was used to detect cell apoptosis with annexin V. Podocyte foot processes were observed under transmission electron microscopy. IgG and C3 deposition were assessed with immunofluorescence assays and confocal microscopy.

Results: Synaptopodin expression significantly decreased in MRL/lpr disease control mice, accompanied by increases in 24-h proteinuria, blood urea nitrogen, and serum creatinine. TAC, however, reduced proteinuria, improved renal function, attenuated renal pathology, restored synaptopodin expression and preserved podocyte numbers. In MPC5 cells, TGF-β1 enhanced F-actin damage in podocytes and TAC stabilized it. TAC also decreased TGF-β1-induced podocyte apoptosis in vitro and inhibited foot process fusion in MRL/lpr mice. In addition, our results also showed TAC inhibited glomerular deposition of IgG and C3.

Conclusion: This study demonstrated that TAC reduced proteinuria and preserved renal function in LN through protecting podocytes from injury partly by stabilizing podocyte actin cytoskeleton and inhibiting podocyte apoptosis.

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Conflict of interest statement

Competing Interests: Astellas Pharmaceuticals supplied the tacrolimus used in this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. TAC reduced proteinuria and preserved renal function in MRL/lpr mice.
(A) All animals (starting at 12 weeks of age, 6 per group) were placed in metabolic cages for urine collection before therapy (0 day) and at the end of the study (8 weeks treatment). Urine samples were used to examine the levels of 24 hour urinary protein excretion. (B, C) Blood samples were obtained from MRL/lpr mice (starting at 12 weeks of age, 6 per group) by eye puncture under ether anesthesia to examine the levels of BUN and serum creatinine just before all mice were killed at 0 week and 8 weeks. NC, normal control group; DC, disease control group; Tx, treatment group. *P < 0.05.
Fig 2
Fig 2. TAC attenuated renal pathology damage in MRL/lpr mice.
(A) At the end of the treatment period (8 weeks), kidney tissues from mice (starting at 12 weeks of age) were immersion-fixed in 4% paraformaldehyde/phosphate buffered saline and embedded in paraffin. Sections (2 μm) were stained with HE, PAS and Masson stain. There were 8 and 9 mice in DC 8w and Tx 8w group, respectively; 6 per group in other groups. (B) Thirty glomeruli for each kidney section were digitally quantified. A microscope equipped with a color camera was used and data were analyzed using image analysis software. According to the glomerular, renal tubular and pathology rating criteria [5], we calculated pathological scores in each mouse. NC, normal control group; DC, disease control group; Tx, treatment group. DC 8w vs. NC 8w, P < 0.01; DC 8w vs. Tx 8w, P < 0.05.
Fig 3
Fig 3. TAC restored synaptopodin expression and stabilized podocyte cytoskeleton.
(A) TAC restored synaptopodin expression in MRL/lpr mice (starting at 12 weeks of age, 6 per group), as shown by immunofluorescence. (B) Western blot showing synaptopodin expression in each group (n = 6). (C) Real time-PCR showing synaptopodin mRNA in each group (n = 6). (D) Treatment with TAC attenuated the decrease in F-actin staining (phalloidin staining) induced by TGF-β1 (5ng/ml) in mouse podocytes (MPC5 cells). NC, normal control group; DC, disease control group; Tx, treatment group. * P < 0.05, # P < 0.01.
Fig 4
Fig 4. TAC preserved podocyte number and reduced podocyte apoptosis.
(A) WT1 expression in MRL/lpr mice (starting at 12 weeks of age, 6 per group), as shown by immunoflourescence. (B) Podocyte number per mean glomerular tuft in each group (n = 6). (C) Real time-PCR analysis of WT1-mRNA expression was performed, n = 4. (D, E) Mouse podocyte cells were pre-incubated with TAC (10 μM or 20 μM) followed by treatment with TGF-β1 (5 ng/ml or 10 ng/ml); n = 4. Apoptosis was assessed with annexin V by flow cytometery (FCM). TGF5, TGF-β1 5 ng/ml; TGF10, TGF-β1 10 ng/ml; TAC10, TAC 10 μM; TAC20, TAC 20 μM. NC, normal control group; DC, disease control group; Tx, treatment group. * P < 0.05, # P < 0.01.
Fig 5
Fig 5. TAC prevented the effacement of foot process in MRL/lpr mice.
(A) TAC prevented the effacement of foot process (arrows) and preserved the slit diaphragm in MRL/lpr mice (starting at 12 weeks of age, 6 per group), as shown by transmission electronic microscopy (13,500×). (B) The number of slit pores per 100 μm length of glomerular basement membrane (GBM). FP, foot process. NC, normal control group; DC, disease control group; Tx, treatment group. * P < 0.01.
Fig 6
Fig 6. TAC reduced Glomerular IgG and C3 deposition in MRL/lpr mice.
(A) TAC administration decreases IgG deposition in glomeruli of MRL/lpr mice (starting at 12 weeks of age, 6 per group), as shown by immunoflourescence. (B) The mean fluorescence intensity corresponding to IgG staining/deposition (n = 6). (C) TAC treatment decreases C3 deposition in glomeruli. (D) The mean fluorescence intensity corresponding to C3 staining/deposition (n = 6). Magnification, ×400 in A and C. Nuclei are counterstained with DAPI. NC, normal control group; DC, disease control group; Tx, treatment group. * P < 0.05.
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Grants and funding

This work was supported by grants from National Key Technology Research and Development Program of the Ministry of Science and Technology, China (No. 2011BAI10B05), Department of Science and Technology, Guangzhou City, China (No. 2010U1-E00831), and 5010 clinical Program of Sun Yat-sen University (No. 2007007) to Xueqing Yu. Qinghua Liu was supported by grants from National Natural Science Foundation of China (No. 81370863), The Fundamental Research Funds for the Central Universities (No. 13YKPY17), Research Fund for PhD Program, Ministry of Education, China (No. 20100171120067) and Natural Science Foundation of Guangdong Province (No. 10451008901005957). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Astellas Pharmaceuticals supplied the tacrolimus used in this study, but it played no role in the design and conduct of this study as well as in the analysis and the interpretation of results. Writing and publication of the manuscript were not contingent on the approval of Astellas Pharmaceuticals.

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