Abstract: SA-PO1035

A Transcriptional Network Controlling Epithelial Barrier Function in the Collecting Duct Is Necessary to Maintain Renal Medullary Osmolality

Session Information

Category: Fluid, Electrolytes, and Acid-Base

  • 702 Water/Urea/Vasopressin, Organic Solutes

Authors

  • Hinze, Christian, Charité, Berlin, Germany
  • Ruffert, Janett, Max Delbrueck Center for Molecular Medicine, Berlin, Germany
  • Walentin, Katharina, Max Delbrueck Center for Molecular Medicine, Berlin, Germany
  • Mutig, Kerim, Charité, Berlin, Germany
  • Bachmann, Sebastian, Charité, Berlin, Germany
  • Schumann, Michael, Charité, Berlin, Germany
  • Himmerkus, Nina, Institute of Physiology, CAU, Kiel, Germany
  • Bleich, Markus, Institute of Physiology, CAU, Kiel, Germany
  • Schmidt-Ott, Kai M., Charité, Berlin, Germany
Background

Osmolytes are accumulated in the renal medulla generating a gradient between the hypertonic interstitial medullary tissue and the urine to facilitate urinary concentration. The transcription factor grainyhead-like 2 (GRHL2) is highly expressed in renal collecting ducts. We previously showed that mice with collecting duct-specific GRHL2 deficiency show a decreased urine concentrating ability and a predisposition for prerenal acute kidney injury. We also demonstrated a loss of barrier function in GRHL2-deficient collecting ducts, as shown by a reduced transepithelial resistance in freshly isolated collecting ducts from the inner stripe of the outer medulla. However, the functional consequences of the epithelial barrier loss remained unclear.

Methods

We generated collecting duct-specific GRHL2 knockout mice (Hoxb7/Cre; Grhl2flox/-, further referred to as GRHL2CD-/-) and measured tissue osmolalities in the cortex, the inner stripe of the outer medulla (ISOM) and the inner medulla (IM) in control and GRHL2CD-/- mice. We used inner medullary collecting duct (IMCD-3) cells with a CRISPR/Cas9-induced Grhl2 knockout and wildtype control cells for functional analyses. GRHL2 target genes were identified by microarray gene expression analyses from control and GRHL2CD-/- kidneys and by Grhl2 chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) from wildtype kidneys.

Results

Our data show a significantly decreased tissue osmolality in ISOM and IM of GRHL2CD-/- kidneys compared to control kidneys. GRHL2 knockout IMCD-3 cells when compared with wildtype cells showed a significantly reduced transepithelial resistance and an increased paracellular flux of sodium and chloride. Integration of microarray and ChIP-seq data indicated that Grhl2 target genes were involved in tight junction assembly.

Conclusion

These data functionally link collecting duct epithelial barrier function with urinary concentrating ability for the first time. We identify a transcriptional network regulated by the transcription factor GRHL2, which is necessary to maintain tight epithelial barriers across the collecting duct epithelium, thereby preventing leakage of sodium and chloride into the urine and preserving a high medullary osmolarity.