Dados do Trabalho

Título

WHAT MAKES THE DEPOSITS DENSE IN DENSE DEPOSIT DISEASE

Introdução

C3 glomerulopathy (C3G) is a disease resulting from dysregulation of the alternative pathway (AP) of complement. C3G includes C3 Glomerulonephritis (C3GN) and Dense Deposito Disease (DDD); both are characterized by bright glomerular C3 staining. However, on electronic microscopy, DDD is characterized by dense osmiophilic mesangial and intramembranous deposits, while the deposits of C3GN are not dense. To date, it has not been established what makes the deposits dense in DDD.

Material e Método

We performed laser microdissection of glomeruli followed by mass spectrometry (MS/MS) in 15 cases of DDD and 29 cases of C3GN to determine the proteomic profile and differences between C3GN and DDD. Kidney biopsy tissue from formalin fixed paraffin embedded material (FFPE) was used for MS/MS studies. 6μm-thick sections of FFPE tissue were mounted on Director slides, deparaffinized and stained with hematoxylin and eosin. Nonsclerotic glomeruli were identified using polarized light and resected with laser microdissection. FFPE fragments from each microdissection were collected in a cap containing a cell lysis buffer and analyzed individually. Proteins were extracted from the FFPE fragments using heat and denatured via sonication. Extracted proteins were digested overnight using trypsin and the resulting peptide mixture was analyzed on a mass spectrometer coupled to a nano-flow high performance liquid chromatography system. The resulting MS/MS spectra were analyzed using Mascot and X!Tandem.

Resultados

As expected, there was overlap in the proteomic profile of C3GN and DDD (Figure 1). Both disease entities showed high total spectral counts (TSC) of C3, CFHR5, CFHR1, CFHR2 and CFH. Although high TSC of terminal complement proteins (C5-C9) were present in C3GN and DDD, there was a 6-9-fold increase of C5-9 in DDD compared to C3GN. An unexpected finding was the 7-9-fold increase of apoliproteins (APO): APOE, APOA5, APOA2 and APOA4, in DDD compared to C3GN (Figure 2). Control cases showed no accumulation of APO. We also detected increased accumulation of HTRA1, C4b binding protein and SAP proteins in DDD. Immunohistochemistry is being performed to confirm and localize APO in dense deposits of DDD compared to deposits in C3GN.

Discussão e Conclusões

There is a higher burden of terminal complement pathway proteins in DDD compared to C3GN. In addition, extensive deposition of apolipoproteins APOE and APOA5 likely cause the deposits to appear dense in DDD.

Palavras Chave

C3 glomerulopathy, dense deposit disease, mass spectrometry, proteomics, complement