The C-Terminal Fragment of Agrin (CAF), a Novel Marker of Renal Function, Is Filtered by the Kidney and Reabsorbed by the Proximal Tubule

PLoS One. 2016 Jul 5;11(7):e0157905. doi: 10.1371/journal.pone.0157905. eCollection 2016.

Abstract

Agrin, a multidomain proteoglycan and neurotrypsin, a neuronal serine protease, are important for forming (neuromuscular) synapses. Proteolytical activity of neurotrypsin produces a C-terminal fragment of agrin, termed CAF, of approximately 22 kDA molecular size which also circulates in blood. The presence of CAF in urine suggests either glomerular filtration or secretion into urine. Blood levels of CAF have been identified as a potential novel marker of kidney function. Here we describe that several nephron segments in the mouse kidney express agrin and neutrotrypsin in addition to the localization of both protein in the glomerulum. Agrin mRNA and protein was detected in almost all nephron segments and mRNA abundance was highest in the inner medullary collecting duct. Neurotrypsin mRNA was mostly detected in the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the inner medullary collecting duct. Moreover, we show that the proximal tubule absorbs injected recombinant CAF by a process shared with receptor-mediated and fluid phase endocytosis. Co-injection of CAF with recombinant human transferrin, a substrate of the receptor-mediated endocytic pathway as well as with FITC-labelled dextran (10 kDa), a marker of fluid phase endocytosis, showed partial colocalization of CAF with both markers. Further colocalization of CAF with the lysosomal marker cathepsin B suggested degradation of CAF by the lysosome in the proximal tubule. Thus, the murine kidney expresses agrin and neurotrypsin in nephron segments beyond the glomerulum. CAF is filtered by the glomerulum and is reabsorbed by endocytosis by the proximal tubule. Thus, impaired kidney function could impair glomerular clearance of CAF and thereby increase circulating CAF levels.

MeSH terms

  • Agrin / genetics
  • Agrin / metabolism*
  • Animals
  • Biomarkers / blood
  • Biomarkers / metabolism*
  • Biomarkers / urine
  • Endocytosis
  • Gene Expression Profiling / methods
  • Glomerular Filtration Rate
  • Humans
  • Immunoblotting
  • Kidney / metabolism
  • Kidney / physiology*
  • Kidney Tubules, Proximal / metabolism
  • Kidney Tubules, Proximal / physiology*
  • Loop of Henle / metabolism
  • Loop of Henle / physiology
  • Male
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microscopy, Confocal
  • Nephrons / metabolism
  • Nephrons / physiology
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Proteoglycans / genetics
  • Proteoglycans / metabolism
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacokinetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism

Substances

  • Agrin
  • Biomarkers
  • C-terminal agrin fragment
  • Peptide Fragments
  • Proteoglycans
  • Recombinant Proteins
  • Serine Endopeptidases
  • neurotrypsin

Grant support

This study has been supported by an EU FP7 EUROSTAR grant ("Early Diagnosis and Monitoring of Renal Diseases, E! 6038 KIDNEY") to D. Steubl, M. Roos, A. Mäder, S. Hettwer, and C.A. Wagner, and a grant from the Swiss Commission for Technology and Innovation ("CTI no. 15622.1 PFLS-LS") to A. Mäder, S. Hettwer, and C.A. Wagner.