Kidney Week

Abstract: FR-PO684

A Missense Mutation in Exon 10 of WT1 Might Lead to Focal Segmental Glomerulosclerosis Due to Its Mislocalization and Downstream Dysregulation

Session Information

• Pediatric Nephrology - 1
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Pediatric Nephrology

• 1900 Pediatric Nephrology

Authors

• Kanamori, Toru, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo-ku, Tokyo, Japan

Toru Kanamori, MS

• Udagawa, Tomohiro, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan

Tomohiro Udagawa, MD, PhD

• Okutsu, Mika, Tokyo Ika Shika Daigaku Daigakin Ishigaku Sogo Kenkyuka, Bunkyo-ku, Tokyo, Japan

Mika Okutsu

• Mori, Takayasu, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan

Takayasu Mori, MD, PhD

• Sohara, Eisei, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan

Eisei Sohara, MD, PhD

• Uchida, Shinichi, Tokyo Ika Shika Daigaku, Bunkyo-ku, Tokyo, Japan

Shinichi Uchida, MD, PhD

Background

Wilms’ tumor suppressor gene 1 (WT1) is associated with the development of the urogenital system and regulates genes involved in sex differentiation. While mutation of exon 8 or 9, and intron 9 of WT1 are known as a cause of focal segmental glomerulosclerosis (FSGS) and sexual disorders, the pathological role of the mutation in exon 10 is largely unknown in FSGS. We found a missense Arg495Gly in a Japanese boy with FSGS connecting to the end stage kidney disease. His renal podocytes histopathology showed that WT1 is localized in not only the cytoplasm but also the nucleus. Additionally, his kidney sample’s expressions of NPHS1 were patchy pattern rather than normal smooth and linear pattern.

Methods

To analyze the pathogenic mechanism of the mutation in FSGS, we evaluated WT1 localization in cultured HEK293T cell transfected with expression constructs encoding the wildtype or WT1^R495Q protein by immunofluorescence staining and western blotting with nuclear/cytoplasm fractionations. Then, to examine the effect of WT1^R495Q on the regulation of key podocyte genes, we quantitatively assessed mRNA expression levels isolated from wild type or mutation-expressing cells by qPCR.

Results

The WT1 localization was different between cells transfected WT1wildtype/WT1^R495Q under phosphorylation-accelerated conditions induced by Forskolin. WT1^R495Q is not affected by Forskolin stimulation, while WT1wildtype affected by Forskolin stimulation and expression of WT1 in the nuclear is decreased and in the cytoplasm is increased. Compared to wild types, the expression of the NPHS1 gene is decreased in the cells transfected with WT1^R495Q. When wild type and mutant WT1 were co-transfected with the same dose, the expression of NPHS1 was the same as when only the mutant was transfected and its expression was reduced in a mutant dose-dependent manner.

Conclusion

We demonstrated WT1^R495Q mutation could affect on WT1 protein localization and dysregulates NPHS1 expression in podocytes. This type of WT1 mutation on exon10 could be loss of function mutation and have a dominant negative effect on expression of podocyte genes. These may have influence podocyte recovery systems using phosphorylation which is essential for regulation of podocyte-specific protein.