Membranous nephropathy (MN) is a specific entity of glomerulonephritis, and its glomerular inflammation is characterized by the deposition of immune complexes in the glomerular basement membrane and proteinuria. However, the molecular mechanisms underlying MN glomerular inflammation are not fully understood. This study was designed to investigate the role of clusterin (CLU) in MN development using a mouse model of cationic bovine serum albumin-induced MN (CBS).
Materials and methods
- Animals and cells
WT B6 and CLU-KO background B6 mice (female, 7-8 weeks old) were received from breeding colonies at the animal care center at the Jack Bell Research Center (Vancouver, British Columbia, Canada).
The conditionally immortalized heat-sensitive mouse podocytes (HSMPs) were a kind gift from Dr. Stuart Shankland (School of Medicine, University of Washington, Seattle, WA, USA) .29 These cells were grown at 33 ° C in RPMI 1640 culture medium (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), 2 mmol / L glutamine (Sigma-Aldrich Canada, Oakville, ON, Canada ), 10 mmol / L of HEPES (Sigma-Aldrich Canada), 1 mmol / L of sodium pyruvate (Sigma-Aldrich Canada) and 10 U / mL of recombinant mouse interferon (IFN) -γ (Life Technologies, Inc. ). For the experiments, HSMPs were induced to inactivity and phenotype differentiated at 37 ° C in the same medium without the addition of IFN-γ.
- Induction of MN in mice by BSA and experimental groups
MN in mice (WT or CLU-KO) was induced by immunization with BSA according to the previously described protocol with minor modifications.26 In summary, BSA solution (2 mg / mL BSA in PBS, pH 7.4) (Chondrex, Inc., Redmond, WA, USA) was fully emulsified with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich Canada).
The resulting emulsion (0.1 ml/mouse, 0.1 mg BSA) was injected subcutaneously at the base of the tail. After two weeks of immunization, mice were injected with a solution of BSA intraperitoneally (IP) (0.2 ml/mouse, 0.4 mg BSA) every other day (qod) for a total of 5 times. . Two WT control groups were included: one treated with PBS vehicle (here PBS-WT) and the other treated with BSA (here BSA-WT). The experimental group was CLU-KO mice treated with BSA (herein BSA-KO).
- Urine collection and protein determination
Urine samples were collected using a metabolic cage (3600M021, Tecniplast, Toronto, ON, Canada). Briefly, mice were individually housed in the metabolic cage for 8 h during the dark phase of a 12 h light / dark cycle without adaptive training. The mice were fed ad libitum the same food and drinking water as in the “home” cages. Urine samples (1 to 1.5 ml per mouse) were collected and centrifuged at 5000 × g to pellet cellular debris, and the supernatant (urine) was stored in aliquots at -80 ° C until use. Total protein levels in urine samples were measured using the Dimension Vista® 1500 system (Siemens Healthineers Canada, Oakville, ON, Canada) at the Central Chemistry Laboratory of Vancouver General Hospital (Vancouver, BC, Canada).
- Measurement of serum creatinine (SCr) and blood urea nitrogen (BUN)
At the end of the experiments (day 50 after the initial immunization with BSA), blood samples were collected by cardiac puncture from sacrificed mice and the serum was stored at -80 ° C until use. Both SCr and BUN were measured at the Central Chemistry Laboratory of Vancouver General Hospital (Vancouver, BC, Canada).
Here, we show that after immunization with BSA, SCr and proteinuria increased in CLU-KO mice but not in WT mice. Similarly, severe glomerular atrophy and mesangial expansion along with C3 deposition were only found in the kidneys of CLU-KO mice, but not in WT mice. However, there were no differences in serum IgG and complement 3 levels between CLU-KO and WT mice. In the serum of WT mice, CLU bound to anti-BSA IgG, complements (eg, C8), proteinase/protease inhibitors, and antioxidant proteins to form a complex, and incubation with WT serum reduced podocyte lysis. complement-dependent in cultures.
Our data suggest that a CLU deficiency induces BSA-initiated glomerular inflammation of MN in a disease-resistant strain of mice, suggesting an anti-glomerular inflammatory role of CLU in resistance to MN development. This function may be due, at least in part, to the formation of the CLU-anti-BSA Igs complex that prevents glomerular inflammation or injury in disease-resistant mice.
glomerular inflammatory disease, membranous nephropathy, immune complex, glomerulonephritis, mouse model.