Kidney Week

Abstract: SA-PO488

Proximal Tubular Intracellular Pathways Leading to Cystogenesis and Tubular Damage in Tuberous Sclerosis

Session Information

• Cystic Kidney Diseases: Basic/Translational
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1001 Genetic Diseases of the Kidneys: Cystic

Authors

• Nechama, Morris, Hadassah Hebrew University Medical Center, Jerusalem, Israel

Morris Nechama,

• Volovelsky, Oded, Hadassah Hebrew University Medical Center, Jerusalem, Israel

Oded Volovelsky,

Background

Tuberous sclerosis (TS) is a genetic disorder caused by inactivating mutations in either the Tsc1 or Tsc2 genes. These mutations induce mTOR activation resulting in cell growth and tumorigenesis. The renal presentation of TS includes angiomyolipoma and cystic disease and is associated with high morbidity and mortality. The exact molecular mechanisms leading to the tubular cell damage and cyst formation remain poorly understood.

Methods

Tsc1 was deleted in nephron progenitor cells (NPCs) by mating Six2 Cre ^tg/+ /Tsc^-/+ males with Tsc1^f/f females. Tubular damage and cyst formation were examined by H&E staining of kidney sections and IHC for the proximal tubule marker LTL and the proliferation marker KI-67. c-Myc expression was examined by IHC and western blots. c-Myc - hamartin protein interaction was demonstrated by Co-Immunoprecipitation. Rapamycin (IP, 1 mg/kg every other day) was injected to pregnant females between gestation days E12.5-E16.5. For isolation of proximal tubule cells, kidneys from embryos at gestation date E18.5, were digested using Collagenase/Dispase and cells stained for Prominin1 (a marker for proximal tubule cells) and sorted by flow cytometry.

Results

Tsc1 knockout in NPCs led to a lethal phenotype of severe renal cystic disease. The tubular damage was demonstrated already at E15.5 with a modified ciliary structure in the cyst lying epithelial cells. The tubular damage and cyst formation were associated with an increased proliferation as measured by Ki-67 staining, as well as c-Myc overexpression. Moreover, c-Myc interacted with hamartin which directly affects c-Myc expression. Rapamycin injection throughout pregnancy prevented tubular damage and cyst formation and prolonged offspring life span from P2 to P14 days. Rapamycin also prevented the increase in Ki67 and c-Myc overexpression. To uncover the molecular pathways involved in this process, we isolated embryonic proximal tubular cells from control and Tsc1 KO mice without or with mTOR inhibition by rapamycin Expression profiling confirmed the increase in c-Myc expression, as above. In addition, it identified specific target genes that can contribute to the tubular cell damage and cytogenesis.

Conclusion

Tubular damage and cyst formation in TS correlate with increased cell proliferation and c-Myc expression, as well as modifications of specific molecular and biochemical pathways.

Funding

• Government Support - Non-U.S.