Cabin1 localizes in glomerular podocyte and undergoes nuclear translocation during podocyte injury

Abstract

Context: Podocyte injury is related to increasing proteinuria and contributes to the progression of kidney disease. Calcineurin binding protein 1 (Cabin1) is a repressor of myocyte enhancer factor 2 (MEF2) and calcineurin-mediated transcription in the immune system. Moreover, Cabin1 interacts with p53 and negatively regulates p53 in tumor cells. However, its function in kidney is unknown. Objective: To explore the exact localization of Cabin1 in glomeruli, as well as the relationship between Cabin1 and podocyte injury. Methods: Sprague–Dawley rats were sacrificed to observe the localization and protein expression of Cabin1 in the kidney. Cabin1 localization and protein expression were detected by immunofluorescence staining and western blot, respectively. Mouse podocytes were cultivated at 33 °C to propagate, then cells were transferred to an incubator at 37 °C to allow differentiation. Differentiated podocytes were stimulated by angiotensin II (AngII) or AngII plus tacrolimus. Cells were harvested to detect the localization and protein expression of Cabin1. Cytoplasmic and nuclear protein were separated by protein extraction kit. Results: Cabin1 mainly localized in the nuclei of glomerular innate cells, it colocalized with WT-1 in podocytes nuclei. Western bolt showed Cabin1 protein remarkably expressed in renal cortex. AngII-induced Cabin1 nuclear protein significantly increased, accompanied by cytoskeleton disruption in cultured mouse podocytes. Conclusion: Cabin1 localizes in glomerular podocytes. AngII induces nuclear translocation of Cabin1 in cultured podocytes.

• Angiotensin II
• Cabin1
• calcineurin
• glomeruli
• podocyte
• p53

Introduction

Podocyte is the essential structure to maintain the normal function of glomerular filtration barrier (GBM). The slit diaphragm (SD) is the final layer of GBM, which plays an important role in restraining serum proteins from discharging to urine.1 Recently, a great deal of SD molecules, which relates to podocyte injury have been recognized, including nephrin, podocin, CD2AP, and TRPC6, etc.2^,3

Angiotensin-II (AngII) is an important risk factor in podocyte injury, researchers found it promoted podocyte apoptosis both in vitro and in vivo4–7 AngII binds to cell surface G protein-coupled receptors (GPCR), which couple to heterotrimeric G proteins of the Gq/11 family.8 Gq enhances podocyte apoptosis in a calcineurin (CaN)-dependent fashion, while the activation of CaN could be blocked by CaN inhibitor.9 However, administration of CaN-specific inhibitor is associated with significant side effects in patients.

As a natural selective inhibitor, calcineurin binding protein 1 (Cabin1) is an ideal candidate to restrain those side effects. Cabin1 extensively expresses in brain, heart, and other organs.10^,11 Cabin1 plays an important role in regulating cell functions by inhibiting CaN via its C-terminus. Down-regulating Cabin1 expression sensitized human nasopharyngeal carcinoma cells to genotoxic drugs.12 Overexpression of human Cabin1 peptide in arthritic fibroblast-like synoviocytes reduced the production of IL-6 and MMP-2.13 Moreover, Cabin1 interacts with p53 via its N terminus, it renders p53 transcriptionally inactive.14 However, the localization and function of Cabin1 in kidney is unclear.

To demonstrate the exact localization of Cabin1 in glomeruli as well as the relationship between Cabin1 and podocyte injury, we detected the expression of Cabin1 in the kidney of rats and established AngII injured podocytes model in vitro.

Materials and methods

The expression of Cabin1 in the kidney of rats

With the approval of the Animal Ethics Committee (Guangdong Province), experiments were conducted on Sprague–Dawley rats (Medical Laboratory Animal Center of Guangdong Province, Guangzhou, China). In this study, we strictly followed the principles of laboratory animal care. The experiments were performed with male Sprague–Dawley rats weighing 200–250 g. Rats were sacrificed to observe the localization and protein expression of Cabin1 in the kidney. Cabin1 localization and protein expression were detected by immunofluorescence staining and western blot, respectively.

Podocytes culture and treatment

Conditionally immortalized mouse podocyte clone (MPC5) was cultured at 33 °C to propagate in RPMI1640 medium (Gibco, Gaithersburg, MD) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD), 100 U/mL of penicillin-streptomycin (BioWhittaker, East Rutherford, NJ) and 10 U/mL of recombinant mouse γ-interferon (PeproTech, London, UK). To induce differentiation, podocytes were cultured at 37 °C without interferon for 10–14 days. Differentiated cells were stimulated with 10⁻⁶M AngII (Sigma, St. Louis, MO) alone, or 10⁻⁶M AngII plus 10 nM tacrolimus simultaneously, tacrolimus for injection was obtained from the pharmacy at the second affiliated hospital of Guangzhou Medical University (Guangzhou, China). Different doses of AngII (10⁻⁶M, 10⁻⁷M,10⁻⁸M,10⁻⁹M) were used to stimulate podocytes in preliminary experiment. We found 10⁻⁶M AngII-induced significant cytoskeleton disruption, which was restrained by tacrolimus. Cells were harvested at 48 h for immunofluorescence staining and western blot.

Immunofluorescence staining

The sections of paraffin-embedded renal tissues were rehydrated and subjected to pressure tank. Podocytes cultured on the cover slips were washed with PBS and fixed for 10 min in 4% paraformaldehyde. Renal sections or cultured podocytes were permeabilized with 0.3% Triton-100 for 5 min, followed by being blocked with 5% bovine serum albumin in PBS for 60 min, then were incubated with primary antibody (Cabin1, Novus, Littleton, CO; WT-1, Abcam, Hong Kong, China) overnight at 4 °C. Excessive primary antibody was removed by washing with PBS, followed by incubation with goat anti-rabbit IgG or goat anti-mouse IgG (Cell Signaling Technology, Boston, MA) for 1 h, and DAPI (Cell Signaling Technology, Boston, MA) was directly used to stain the nuclei. Finally, the renal sections or cover slips were mounted on glass slides with Mounting Medium (R&D Systems, Minneapolis, MN), and viewed by using confocal fluorescence microscopy (Zeiss, Oberkochen, Germany).

Western blotting

Renal cortex samples were collected and lysed in lysis buffer (Cell Signaling Technology, Danvers, MA). Cytoplasmic and nuclear protein of cultured podocytes were separated by protein extraction kit (Pierce Biotechnology, Rockford, IL). Equal amounts of protein loadings were separated by SDS-PAGE on 8% gels and electrophoretically transferred to a nitrocellulose membrane. Non-specific binding sites were blocked with 5% nonfat milk powder in PBS for 1 h. Membranes were incubated in primary antibody (Cabin1, Novus, Littleton, CO; β-actin, Cell Signaling Technology, Boston, MA; GAPDH, Kangchen, China; Fibrillarin, Abcam, Hong Kong, China) overnight at 4 °C. Finally, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. An enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect the bound antibodies. The specific protein bands were scanned.

Statistical analysis

The data were presented as the mean ± SD. All experiments for statistical calculation were carried out in triplicate. For comparisons between three groups, statistical analysis was performed using one-way ANOVA (analysis of variance). p < 0.05 was considered statistically significant. Statistical analyses were performed with SPSS software, version 16.0 (SPSS, Inc., Chicago, IL).

Results

Cabin1 mainly localized in the nuclei of glomerular innate cells

To examine the exact localization of Cabin1 in glomeruli, renal cortex sections were detected by confocal fluorescence microscope after indirect immunofluorescence staining. Cabin1 mainly localized in the nuclei of glomerular innate cells in normal rats (Figure 1A); WT-1 is the specific podocyte nuclear protein, it localized in the nuclei of podocytes (Figure 1B); Cabin1 and WT-1 colocalized in podocytes nuclei (Figure 1E and F). Western blot result showed Cabin1 protein remarkably expressed in renal cortex (Figure 2).

Figure 1. Cabin1 localized in the nuclei of glomerular innate cells of normal rats. Notes: A–E:original magnification: ×400; F: ×1500) (A) Cabin1 mainly localized in the nuclei of glomerular innate cells; (B) WT-1 localized in podocytes nuclei; (C) nuclei; (D) Differential interference contrast (DIC); (E and F) Cabin1 and WT-1 colocalized in podocytes nuclei.

Figure 2. Cabin1 expressed in renal cortex of normal rats. Note: In normal SD rats, western blot showed Cabin1 protein remarkably expressed in renal cortex.

AngII-induced actin cytoskeleton disruption in cultured podocytes

Phalloidin staining displayed fine and long bundles of intracellular actin filaments in untreated cells (Figure 3A). AngII induced the loss of the cytoplasmic cytoskeleton and the reorganized the actin cytoskeleton at 24 h (Figure 3B). Tacrolimus inhibited the injury of AngII, maintained well-defined actin stress fibers (Figure 3C).

Figure 3. AngII-induced actin cytoskeleton disruption. Notes: Fluorescence microscope ×400. (A) Untreated podocytes, white arrow showed the fine connection of podocyte foot processes; (B) AngII-treated podocytes induced the loss of the cytoplasmic cytoskeleton and the reorganized of actin cytoskeleton at 24 h, white arrow showed the disruption of podocyte foot processes connection; (C) Tacrolimus inhibited the injury of AngII, maintained well-defined actin stress fibers, white arrow showed the maintain of podocyte foot processes connection.

The localization of Cabin1 in AngII injured podocytes

In normal cultured mouse podocytes, Cabin1 presented strong staining in cytoplasm yet weak staining in the nuclei (Figure 4A). In AngII injured podocytes, Cabin1 distribution in the nuclei became much more obvious at 48 h (Figure 4D). Tacrolimus mildly inhibited the strong staining of Cabin1 in the nuclei (Figure 4G).

Figure 4. The localization of Cabin1 in AngII injuried podocytes. Notes: Fluorescence microscope ×400). (A and C) In normal podocytes, Cabin1 presented strong staining in cytoplasm yet weak staining in the nuclei; (B, E and H) Immunofluorescence staining of nuclei; (D and F) AngII-induced strong staining of Cabin1 in podocytes nuclei; (G and I) Tacrolimus mildly inhibited the strong staining of Cabin1 in the nuclei.

AngII-induced Cabin1 protein increased in the nuclei of cultured podocytes

Compared to the control group, western blot showed that AngII-induced Cabin1 nuclear protein content significantly increased in podocytes at 48 h (p < 0.05). This result indicated AngII induced nuclear translocation of Cabin1 in cultured podocytes. Tacrolimus plus AngII did not significantly stabilized Cabin1 expression (Figure 5).

Figure 5. AngII-induced Cabin1 nuclear protein increased in cultured podocytes. Notes: (A) Western blot showed AngII-induced Cabin1 nuclear protein expression significantly increased at 48 h; (B) Quantification of Cabin1 nuclear and cytoplasmic protein expression. *p < 0.05 versus normal control group. N: normal control.

Disscussion

Increased recognition of Cabin1, as a natural selective inhibitor of calcineurin and a negative regulator of p53, has highlighted the importance of Cabin1. Our experiment indicated Cabin1 localized in the nuclei of glomerular innate cells. As WT-1 is the specific podocyte nuclear protein, colocalization of Cabin1 and WT-1 confirmed Cabin1 localized in podocytes. Western blot result also indicated Cabin1 expressed in renal cortex.

To study the relationship between Cabin1 and podocyte injury, cultured podocytes were stimulated by AngII. AngII is one of the crucial factors in chronic kidney disease and podocyte injury.6^,7 We found AngII-induced cytoskeleton disruption and nuclear translocation of Cabin1 in cultured podocytes. These results indicated Cabin1 undergone nuclear translocation during podocyte injury. Fan et al.15 demonstrated calmodulin-dependent protein kinase IV regulated nuclear export of Cabin1 during T-cell activation. Whether nuclear translocation of Cabin1 inhibits podocyte injury need to be confirmed by further study.

It is reported that AngII enhanced podocyte apoptosis in a CaN-dependent fashion. CaN activation promoted podocyte apoptosis via nuclear factor of activated T cells (NFAT)-dependent gene transcription.9 Cabin1 is a natural selective inhibitor of CaN, it plays an important role in regulating cell functions by inhibiting CaN via its C-terminus.10 As nuclear translocation of Cabin1 inhibited calcineurin-NFAT pathway during T-cell activation,15 Cabin1 is likely translocate to nuclei in order to restrain calcineurin-NFAT pathway during podocyte injury. Targeted inhibition of CaN by Cabin1 has been showed in many studies. Overexpression of human Cabin1 peptide in arthritic fibroblast-like synoviocytes reduced the production of IL-6 and MMP-2.13 Overexpression of human Cabin1 in rheumatoid arthritis mice promoted apoptosis of fibroblast-like synoviocytes and attenuated inflammation.16 On the contrary, Chang et al.12 found down-regulating Cabin1 expression was sensitized human nasopharyngeal carcinoma cells to genotoxic drugs. Kim et al.17 illustrated calpain-dependent cleavage of Cabin1-activated CaN to mediate calcium-triggered human T lymphoma cells death. In patients with congestive heart failure, calpain upregulation was associated with increased cleavage of Cabin1 and activation of CaN.11 Overexpression of Cabin1 in podocyte should restore the damage of podocyte.

CaN promoted podocyte apoptosis could be blocked by CaN-specific inhibitor, such as cyclosporine A and tacrolimus.9^,18 Tacrolimus effectively released proteinuria of patients, who suffered from minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS).19 Tacrolimus restricted CaN-induced synaptopodin dephosphorylation, avoided podocyte from skeleton degradation.20 However, administration of CaN-specific inhibitor is associated with significant side effects. Tacrolimus may induce hypertension, nephrotoxicity and hyperlipidemia during the treatment.21–23 Whether Cabin1 is an ideal candidate to avoid those side effects need to be further investigated.

It has been illuminated that Cabin1 interacted with p53 and negatively regulated p53 in the nuclei of HCT116 cells.14 Ying et al.24 indicated Cabin1 promoted cancer cells apoptosis by regulating p53. Choi et al.25 found genotoxic stress-induced phosphorylation and ubiquitination-dependent degradation of Cabin1, released p53 for transcriptional activation.

P53 involves in many cellular functions, such as cell growth, cell cycle progression, DNA repair and death.26–28 Furthermore, p53 is a key protein that activates p53 target pro-apoptotic genes, such as BAX, Noxa and PUMA, which promote podocyte apoptosis.29 Cabin1 probably translocates to podocyte nucleus and inhibits podocyte apoptosis by regulating p53.

In conclusion, Cabin1 expressed in glomerular podocyte and undergone nuclear translocation during podocyte injury.

Acknowledgments

Conditionally immortalized mouse podocyte clone (MPC5) is a kind gift from Prof. Peter Mundel and Ding Jie.

Declaration of interest

The authors declare no competing interests. This study was supported by the Science and Technology Planning Project of Guangdong Province, China (grant no. 2011B031800307) and Doctoral Program Foundation of Guangzhou Medical University, China (grant no. 2014C33).