Sgk-1 is a positive regulator of constitutive albumin uptake in renal proximal tubule cells.
OBJECTIVE
Receptor-mediated endocytosis of albumin by the renal proximal tubule requires a number of proteins including megalin/cubilin, sodium/hydrogen exchanger isoform 3 (NHE3) and ClC-5, as well as the PSD-95/Dlg/Zo-1 (PDZ) scaffold sodium/hydrogen exchanger regulatory factor 2 (NHERF2). Despite members from the AGC kinase family, v-Akt Murine Thymoma Viral Oncogene (Akt or Protein Kinase B) and Serum/Glucocorticoid regulated Kinase 1 (Sgk-1) regulating a number of essential proteins in the albumin handling pathway, their role in uptake is largely unknown.


METHODS
Opossum kidney (OK) cells were exposed to Texas-Red albumin, in the presence of silencing constructs against Sgk-1, Akt and NHERF2, in addition to the NHE3 inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and NHE3 activator dexamethasone. Target protein was also measured by Western blot analysis in OK cells following exposure to dexamethasone and albumin.


RESULTS
Silencing Sgk-1 or overexpression of a dominant negative mutant (DN-Sgk-1) led to a significant reduction of albumin endocytosis compared to control. Conversely, over-expression of wildtype (WT) or constitutively active (CA) Sgk-1 significantly increased uptake. Previous reports have shown Sgk-1 can activate NHE3 through an interaction mediated by NHERF2. We found that silencing both Sgk-1 and NHERF2 demonstrated no additive effect on uptake, suggesting signaling via similar endpoints. Treatment with dexamethasone increased Sgk-1 protein levels and increased albumin endocytosis in OK cells. Interestingly, silencing Akt also lead to a reduction in albumin endocytosis, however in cells silenced for both Sgk-1 and Akt, the additive change in albumin uptake demonstrated that these proteins may act via separate pathways.


CONCLUSIONS
We have characterized a Sgk-dependent pathway that regulates albumin uptake in the proximal tubule which also includes NHE3 and NHERF2. These data provide further insights into this essential tubular process.
Biomedical and Lifestyle Diseases Unit, School of Biomedical and Health Sciences, Victoria University, St Albans, Melbourne, VIC, Australia. deanne.skelly@unimelb.edu.au
Deanne H
DH
Hryciw
Wade A
WA
Kruger
Jessica F
JF
Briffa
Craig
C
Slattery
Adelyn
A
Bolithon
Aven
A
Lee
Philip
P
Poronnik
D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism*; D009893:Opossums; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012709:Serum Albumin / Q000032:analysis / Q000378:metabolism*; D014018:Tissue Distribution; D014966:Xanthenes / Q000032:analysis / Q000493:pharmacokinetics; D000099184:Serum-Glucocorticoid Regulated Kinases
D017874:Immediate-Early Proteins; D017346:Protein Serine-Threonine Kinases; D012709:Serum Albumin; D014966:Xanthenes; D000099184:Serum-Glucocorticoid Regulated Kinases; C034657:Texas red