The calcineurin inhibitor tacrolimus reduces proteinuria in membranous nephropathy accompanied by a decrease in angiopoietin-like-4

Abstract

Tacrolimus is an anticalcineurinic agent with potent immunosuppressive activity that has recently been shown to have the added benefit of reducing proteinuria in membranous nephropathy (MN) patients. However, its potential mechanisms remain unknown. To reveal the mechanism, rat cohorts were administered tacrolimus or vehicle from days 7 to 28 after the induction of passive Heymann nephritis (PHN). PHN induction resulted in heavy proteinuria and increased expression of desmin, a marker of injured podocytes. We also showed that the glomerular expression of angiopoietin-like-4 (Angptl4) was markedly upregulated in PHN rats and human MN followed by an increase in urine Angptl4 excretion. In addition, increased Angptl4 expression may be related to podocyte injury and proteinuria. Furthermore, upregulated Angptl4 expression primarily colocalized with podocytes rather than endothelial or mesangial cells, indicating that podocytes may be the source of Angptl4, which then gradually migrated to the glomerular basement membrane over time. However, tacrolimus treatment markedly reduced glomerular and urinary Angptl4, accompanied by a reduction in the established proteinuria and the promotion of podocyte repair. Additionally, glomerular immune deposits and circulating IgG levels induced by PHN clearly decreased following tacrolimus treatment. In conclusion, this is the first demonstration that the calcineurin inhibitor tacrolimus can reduce Angptl4 in podocytes accompanied by a decrease in established proteinuria and promotion of podocyte repair in MN.

Figure 1. Tacrolimus reduced 24-hour urine protein and ameliorated serum albumin and triglyceride in passive Heymann nephritis (PHN) rats.

(A) Tacrolimus reduced 24-hour urinary protein excretion in PHN rats. (B) Tacrolimus ameliorated serum albumin levels in PHN rats. (C) Tacrolimus ameliorated serum triglyceride levels in PHN rats. Con, normal controls; Untreated, PHN rats without treatment; TAC, PHN rats with tacrolimus treatment. Arrows indicate that tacrolimus treatment started on day 7. *P<0.01 vs. normal controls, #P<0.05 vs. PHN. (n = 10 for every group at each time point).

Figure 2. Tacrolimus diminished glomerular subepithelial immune deposits and serum IgG levels and ameliorated foot process effacement in passive Heymann nephritis (PHN) rats.

(A) PASM staining of tacrolimus-treated and untreated PHN rats on day 21 (magnification, x400). Tacrolimus reversed swollen podocytes to nearly normal shapes and ameliorated the broadened GBM in PHN rats. Black arrows, subepithelial immune depositions. Red arrows, prominent podocytes. (B and C) Transmission electron microscopy of normal rats and of tacrolimus-treated and untreated PHN rats. Tacrolimus reduced subepithelial immune deposits and reversed severe foot process effacement and the disappearance of slit diaphragms in PHN rats. Subepithelial immune deposits are indicated with black arrows. Foot process effacement is shown with asterisks. (D and E) Immunofluorescence of glomerular rat IgG in normal rats and in tacrolimus-treated and untreated PHN rats (magnification, x400). (F) Quantification of the intensity of fluorescent staining for glomerular rat IgG. *P<0.01 vs. normal controls; #P<0.01 vs. PHN. Tacrolimus markedly reduced glomerular IgG levels. (G) Circulating rat anti-rabbit IgG antibodies. *P<0.01 vs. normal controls; #P<0.01 vs. PHN. Tacrolimus markedly reduced serum IgG levels. (H and I) Immunofluorescence of glomerular C5b-9 in normal rats and in tacrolimus-treated and untreated PHN rats (magnification, x400). (J) Quantification of the fluorescent staining intensity of glomerular C5b-9. *P<0.01 vs. normal controls; #P<0.01 vs. PHN. Tacrolimus markedly reduced glomerular C5b-9 deposits.

Figure 3. Tacrolimus diminished glomerular Angptl4 in passive Heymann nephritis (PHN) rats.

(A and B) Immunofluorescence of glomerular Angptl4 in normal rats and in tacrolimus-treated and untreated PHN rats (magnification, x400). (C) Quantification of the fluorescent staining intensity of glomerular Angptl4. *P<0.01 vs. normal controls; #P<0.01 vs. PHN rats. (D) Quantitative real-time PCR of Angptl4 from kidney tissue. *P<0.01 vs. normal controls; #P<0.05 vs. PHN rats. (E) Western blot analysis of glomerular Angptl4 expression in PHN rats. (F) Quantification of the western blot analysis of glomerular Angptl4 expression. *P<0.01 vs. normal controls; #P<0.05 vs. PHN rats. Glomerular Angptl4 expression was upregulated in PHN rats and tacrolimus notably diminished glomerular Angptl4. (G) Western blot analysis of urine Angptl4 excretion in PHN rats. (H) Quantification of the western blot analysis of urine Angptl4 excretion in PHN rats. *P<0.01 vs. normal controls; #P<0.05 vs. PHN rats. Urine Angptl4 excretion was upregulated in PHN rats and tacrolimus notably diminished urine Angptl4. (n = 10 for every group at each time point).

Figure 4. Angptl4 in podocytes gradually migrated to the GBM in passive Heymann nephritis (PHN) rats (magnification, x400).

(A) Immunofluorescence of glomerular Angptl4 with RECA-1, an endothelial marker, in PHN rats on day 7. (B) Immunofluorescence of glomerular Angptl4 with OX-7, a mesangial cell marker, in PHN rats on day 7. (C–E) Immunofluorescence of glomerular Angptl4 with synaptopodin, a podocyte marker, in PHN rats on days 7, 14, and 21. The overlap rates of Angptl4 and synaptopodin on days 7, 14, and 21 were 71.16%, 71.58%, and 56.23%, respectively. (F–H) Immunofluorescence of glomerular Angptl4 with laminin, a GBM marker, in PHN rats on days 7, 14, and 21. The overlap rates of Angptl4 and laminin on days 7, 14, and 21 were 17.69%, 35.37%, and 29.75%, respectively.

Figure 5. Tacrolimus reduced the expression of glomerular desmin, a biomarker of podocyte injury, and the relationship between Angptl4, desmin, and urine protein.

(A and B) Immunofluorescence of glomerular desmin in normal rats and in tacrolimus-treated and untreated PHN rats (magnification, x400). (C) Quantification of the fluorescent staining intensity of glomerular desmin. *P<0.01 vs. normal controls; #P<0.05 vs. PHN. Tacrolimus reduced the expression of glomerular desmin. (D) Relationship between Angptl4 and desmin in PHN rats. The expression of Angptl4 was upregulated to its peak prior to desmin expression. (E) Relationship between Angptl4 and urine protein in PHN rats. The expression of Angptl4 was upregulated to its peak prior to proteinuria. (F) Relationship between desmin and urine protein in PHN rats. Podocyte injury and proteinuria had the same trends.

Figure 6. Expression of Angptl4 in human glomerulonephritis and its relationship with desmin and urine protein.

(A) Immunofluorescence of glomerular Angptl4 with synaptopodin in MN patients (magnification, x400). The overlap rate of Angptl4 and synaptopodin was 58.72%. (B) Immunofluorescence of glomerular Angptl4 and desmin in MN and MsPGN patients with similar nephrotic-range proteinuria (magnification, x400). Angptl4 and desmin expression levels were markedly upregulated in the MN patient compared with the MsPGN patient. (C and D) Quantification of the immunofluorescence intensities of glomerular Angptl4 and desmin in MN and MCD and non-podocytopathy (MsPGN). *P<0.01 vs. MsPGN. The expression levels of Angptl4 and desmin were notably increased in MN and MCD patients relative to MsPGN patients. (E) Scatter diagram of Angptl4 and desmin in 20 patients. A correlative analysis revealed positive correlations between glomerular Angptl4 and desmin expression. (F) Scatter diagram of desmin and urine protein in podocytopathy patients (13 patients). Correlative analysis revealed positive correlations between desmin and proteinuria. (G and H) The scatter diagrams reveal the relationships between Angptl4, cholesterol, and triglycerides in 20 patients. There were no correlations between glomerular Angptl4 expression and cholesterol or between Angptl4 and triglycerides. (I) Western blot analysis of urine Angptl4 excretion in MN and MsPGN patients with similar nephrotic-range proteinuria. (J) Quantification of the western blot analysis of urine Angptl4 excretion in MN and MsPGN patients. *P<0.05 vs. MsPGN. Urine Angptl4 excretion was more obvious in MN patients than in MsPGN patients.