Quantitative assessment of glomerular basement membrane collagen IV α chains in paraffin sections from patients with focal segmental glomerulosclerosis and Alport gene variants

Pongpratch Puapatanakul, Suramath Isaranuwatchai, Ankanee Chanakul, Jerasit Surintrspanont, Kroonpong Iampenkhae, Talerngsak Kanjanabuch, Kanya Suphapeetiporn, Vivek Charu, Hani Y. Suleiman, Kearkiat Praditpornsilpa, Jeffrey H. Miner

Research output: Contribution to journalArticlepeer-review

Abstract

Focal segmental glomerulosclerosis (FSGS) lesions have been linked to variants in COL4A3/A4/A5 genes, which are also mutated in Alport syndrome. Although it could be useful for diagnosis, quantitative evaluation of glomerular basement membrane (GBM) type IV collagen (colIV) networks is not widely used to assess these patients. To do so, we developed immunofluorescence imaging for collagen α5(IV) and α1/2(IV) on kidney paraffin sections with Airyscan confocal microscopy that clearly distinguishes GBM collagen α3α4α5(IV) and α1α1α2(IV) as two distinct layers, allowing quantitative assessment of both colIV networks. The ratios of collagen α5(IV):α1/2(IV) mean fluorescence intensities (α5:α1/2 intensity ratios) and thicknesses (α5:α1/2 thickness ratios) were calculated to represent the levels of collagen α3α4α5(IV) relative to α1α1α2(IV). The α5:α1/2 intensity and thickness ratios were comparable across all 11 control samples, while both ratios were significantly and markedly decreased in all patients with pathogenic or likely pathogenic Alport COL4A variants, supporting validity of this approach. Thus, with further validation of this technique, quantitative measurement of GBM colIV subtype abundance by immunofluorescence, may potentially serve to identify the subgroup of patients with FSGS lesions likely to harbor pathogenic COL4A variants who could benefit from genetic testing.

Original languageEnglish
Pages (from-to)1049-1057
Number of pages9
JournalKidney International
Volume105
Issue number5
DOIs
StatePublished - May 2024

Keywords

  • focal segmental glomerulosclerosis
  • immunofluorescence
  • type IV collagen

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