Abstract

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    & Nephronophthisis-associated ciliopathies. J. Am. Soc. Nephrol. 18, 1855–1871 (2007).

  2. 2.

    & Clinical spectrum and pathogenesis of nephronophthisis. Curr. Opin. Nephrol. Hypertens. 21, 272–278 (2012).

  3. 3.

    , & Ciliopathies. N. Engl. J. Med. 364, 1533–1543 (2011).

  4. 4.

    et al. Identification of 99 novel mutations in a worldwide cohort of 1,056 patients with a nephronophthisis-related ciliopathy. Hum. Genet. published online; doi: (5 April 2013).

  5. 5.

    et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways. Cell 145, 513–528 (2011).

  6. 6.

    et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J. Cell Biol. 192, 1023–1041 (2011).

  7. 7.

    , , , & Inv acts as a molecular anchor for Nphp3 and Nek8 in the proximal segment of primary cilia. Cytoskeleton (Hoboken) 67, 112–119 (2010).

  8. 8.

    , , , & The ciliary protein Nek8/Nphp9 acts downstream of Inv/Nphp2 during pronephros morphogenesis and left-right establishment in zebrafish. FEBS Lett. 586, 2273–2279 (2012).

  9. 9.

    et al. NEK8 mutations affect ciliary and centrosomal localization and may cause nephronophthisis. J. Am. Soc. Nephrol. 19, 587–592 (2008).

  10. 10.

    , , , & A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics 7, 4228–4234 (2007).

  11. 11.

    et al. Missense mutation in sterile α motif of novel protein SamCystin is associated with polycystic kidney disease in (cy/+) rat. J. Am. Soc. Nephrol. 16, 3517–3526 (2005).

  12. 12.

    et al. Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region. J. Cell Sci. 122, 44–54 (2009).

  13. 13.

    et al. A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish. Development 129, 5839–5846 (2002).

  14. 14.

    , , & Nephrocystin-3 is required for ciliary function in zebrafish embryos. Am. J. Physiol. Renal Physiol. 299, F55–F62 (2010).

  15. 15.

    , , & Xenopus Bicaudal-C is required for the differentiation of the amphibian pronephros. Dev. Biol. 307, 152–164 (2007).

  16. 16.

    , & The role of Xenopus frizzled-8 in pronephric development. Biochem. Biophys. Res. Commun. 321, 487–494 (2004).

  17. 17.

    et al. Inversin relays Frizzled-8 signals to promote proximal pronephros development. Proc. Natl. Acad. Sci. USA 107, 20388–20393 (2010).

  18. 18.

    et al. Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia. Am. J. Hum. Genet. 82, 959–970 (2008).

  19. 19.

    et al. Mutations of NPHP2 and NPHP3 in infantile nephronophthisis. Kidney Int. 75, 839–847 (2009).

  20. 20.

    et al. Genotype-phenotype correlation in 440 patients with NPHP-related ciliopathies. Kidney Int. 80, 1239–1245 (2011).

  21. 21.

    et al. Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy. J. Clin. Invest. 120, 791–802 (2010).

  22. 22.

    et al. Factor inhibiting HIF (FIH) recognizes distinct molecular features within hypoxia-inducible factor-α (HIF-α) versus ankyrin repeat substrates. J. Biol. Chem. 287, 8769–8781 (2012).

  23. 23.

    et al. Factor inhibiting HIF limits the expression of hypoxia-inducible genes in podocytes and distal tubular cells. Kidney Int. 78, 857–867 (2010).

  24. 24.

    et al. Involvement of hypoxia-inducible transcription factors in polycystic kidney disease. Am. J. Pathol. 170, 830–842 (2007).

  25. 25.

    et al. VEGF receptor inhibition slows the progression of polycystic kidney disease. Kidney Int. 72, 1358–1366 (2007).

  26. 26.

    , & The Wilms tumor genes wt1a and wt1b control different steps during formation of the zebrafish pronephros. Dev. Biol. 309, 87–96 (2007).

  27. 27.

    et al. The Rac1 regulator ELMO1 controls vascular morphogenesis in zebrafish. Circ. Res. 107, 45–55 (2010).

  28. 28.

    et al. Regulation of ciliary polarity by the APC/C. Proc. Natl. Acad. Sci. USA 106, 17799–17804 (2009).

  29. 29.

    , , , & Tandem affinity purification of ciliopathy-associated protein complexes. Methods Cell Biol. 91, 143–160 (2009).

  30. 30.

    et al. Experimental protein mixture for validating tandem mass spectral analysis. OMICS 6, 207–212 (2002).

  31. 31.

    , , & A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 (2003).

Download references

Acknowledgements

We are grateful to all patients and family members for their participation. We thank A. Sammarco, C. Engel, B. Müller, L. Schomas, S. Bräg and M. Klein for excellent technical assistance, the staff of the Life Imaging Center (LIC) in the Center for Systems Biology, Albert-Ludwigs-University Freiburg for excellent confocal microscopy resources and the support in image recording and analysis, and U. Lanner and E. Haaf of the proteomics core facility. We thank N. Katsanis and J. Willer (Duke University) for providing us with expression constructs for NEK7 and INVS. We thank K. Coene for her help in generating affinity proteomics data for NEK8. We thank E. Jones for making the 3G8 and 4A6 antibodies available through the European Xenopus stock centre. V.F., T.E., H.J.B. and C. Bergmann are employees of Bioscientia, a member of Sonic Healthcare. D.B. is a Higher Education Funding Council for England (HEFCE) Clinical Reader and is supported by Kids Kidney Research. A.K.-Z., G.W., E.W.K., F.G., T.B.H. and S.S.L. are supported by the Deutsche Forschungsgemeinschaft (DFG; KFO 201). E.W.K. is supported by the DFG (KU 1504). C. Boehlke is supported by the Else-Kröner-Fresenius Stiftung. G.W. and T.B.H. are supported by the Excellence Initiative of the German Federal and State Governments (EXC 294-BIOSS). M.U., R.R. and G.W. are supported by the European Community's Seventh Framework Programme (grant agreement 241955, SYSCILIA). R.R. is supported by the Netherlands Organisation for Scientific Research (NWO Vidi-91786396 and Vici-016.130.664). K.B. and M.U. are supported by the European Community's Seventh Framework Programme under grant agreement 278568, PRIMES. This study was supported in part by the Excellence Initiative of the German Federal and State Governments (GSC-4, Spemann Graduate School) and by grants from the Agence Nationale de la Recherche to S.S. (R09087KS and RPV11012KK) and the Fondation pour la Recherche Médicale (DEQ20071210558). This research was supported by grants from the US National Institutes of Health to F.H. (DK068306 and DK090917). F.H. is an Investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist and a Frederick G.L. Huetwell Professor. C. Bergmann received support from the DFG (BE 3910/4-1, ZE 205/14-1 and SFB/TRR57), the Deutsche Nierenstiftung and the PKD Foundation.

Author information

Author notes

    • Sylvia Hoff
    •  & Jan Halbritter

    These authors contributed equally to this work.

Affiliations

  1. Department of Medicine, Renal Division, University of Freiburg Medical Center, Freiburg, Germany.

    • Sylvia Hoff
    • , Daniel Epting
    • , Christopher Boehlke
    • , Christoph Schell
    • , Takayuki Yasunaga
    • , Martin Helmstädter
    • , Miriam Mergen
    • , Florian Grahammer
    • , Tobias B Huber
    • , E Wolfgang Kuehn
    • , Albrecht Kramer-Zucker
    • , Gerd Walz
    • , Carsten Bergmann
    •  & Soeren S Lienkamp
  2. Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Freiburg, Germany.

    • Sylvia Hoff
    •  & Christoph Schell
  3. Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

    • Sylvia Hoff
    • , Christoph Schell
    •  & Martin Helmstädter
  4. Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Jan Halbritter
    •  & Friedhelm Hildebrandt
  5. Center for Human Genetics, Bioscientia, Ingelheim, Germany.

    • Valeska Frank
    • , Tobias Eisenberger
    • , Hanno J Bolz
    •  & Carsten Bergmann
  6. Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences and Institute for Genetic and Metabolic Disease, Radboud University Medical Centre, Nijmegen, The Netherlands.

    • Thanh-Minh T Nguyen
    • , Jeroen van Reeuwijk
    •  & Ronald Roepman
  7. Institute National de la Santé et de la Recherche Médicale (INSERM) U983, Hôpital Necker–Enfants Malades, Paris, France.

    • Emilie Filhol
    •  & Sophie Saunier
  8. Paris Descartes–Sorbonne Paris Cité University, Imagine Institute, Paris, France.

    • Emilie Filhol
    •  & Sophie Saunier
  9. Institute for Ophthalmic Research, Division of Experimental Ophthalmology and Medical Proteome Center, University of Tuebingen, Tuebingen, Germany.

    • Karsten Boldt
    • , Nicola Horn
    •  & Marius Ueffing
  10. Research Unit Protein Science, Helmholtz Zentrum München–German Research Center for Environmental Health, Munich and Neuherberg, Germany.

    • Marius Ueffing
  11. Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA.

    • Edgar A Otto
  12. Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands.

    • Mariet W Elting
  13. Department of Pediatric Nephrology, VU University Medical Center, Amsterdam, The Netherlands.

    • Joanna A E van Wijk
  14. University College London (UCL) Institute of Child Health and Pediatric Nephrology, Great Ormond Street Hospital, London, UK.

    • Detlef Bockenhauer
  15. Department of Histopathology, Great Ormond Street Hospital, London, UK.

    • Neil J Sebire
  16. Department of Pediatrics, Aarhus University Hospital, Skejby, Aarhus, Denmark.

    • Søren Rittig
  17. Institute of Pathology, Aalborg University Hospital, Aalborg, Denmark.

    • Mogens Vyberg
  18. Department of Nephrology, Aalborg University Hospital, Aalborg, Denmark.

    • Troels Ring
  19. Department of Pediatrics and Adolescent Medicine, University of Freiburg Medical Center, Freiburg, Germany.

    • Martin Pohl
  20. Department of Pediatric Nephrology, Hannover Medical School, Hannover, Germany.

    • Lars Pape
  21. Department of Pediatrics, Children's Hospital Lucerne, Lucerne, Switzerland.

    • Thomas J Neuhaus
  22. Center of Pediatric Nephrology and Transplantation, Cairo University and Egyptian Group for Orphan Renal Diseases (EGORD), Cairo, Egypt.

    • Neveen A Soliman Elshakhs
  23. Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota, USA.

    • Sarah J Koon
    •  & Peter C Harris
  24. Center for Biological Signaling Studies (BIOSS), Freiburg, Germany.

    • Tobias B Huber
    • , E Wolfgang Kuehn
    •  & Gerd Walz
  25. Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany.

    • Hanno J Bolz
  26. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

    • Friedhelm Hildebrandt
  27. Center for Clinical Research, University of Freiburg, Freiburg, Germany.

    • Carsten Bergmann

Authors

  1. Search for Sylvia Hoff in:

  2. Search for Jan Halbritter in:

  3. Search for Daniel Epting in:

  4. Search for Valeska Frank in:

  5. Search for Thanh-Minh T Nguyen in:

  6. Search for Jeroen van Reeuwijk in:

  7. Search for Christopher Boehlke in:

  8. Search for Christoph Schell in:

  9. Search for Takayuki Yasunaga in:

  10. Search for Martin Helmstädter in:

  11. Search for Miriam Mergen in:

  12. Search for Emilie Filhol in:

  13. Search for Karsten Boldt in:

  14. Search for Nicola Horn in:

  15. Search for Marius Ueffing in:

  16. Search for Edgar A Otto in:

  17. Search for Tobias Eisenberger in:

  18. Search for Mariet W Elting in:

  19. Search for Joanna A E van Wijk in:

  20. Search for Detlef Bockenhauer in:

  21. Search for Neil J Sebire in:

  22. Search for Søren Rittig in:

  23. Search for Mogens Vyberg in:

  24. Search for Troels Ring in:

  25. Search for Martin Pohl in:

  26. Search for Lars Pape in:

  27. Search for Thomas J Neuhaus in:

  28. Search for Neveen A Soliman Elshakhs in:

  29. Search for Sarah J Koon in:

  30. Search for Peter C Harris in:

  31. Search for Florian Grahammer in:

  32. Search for Tobias B Huber in:

  33. Search for E Wolfgang Kuehn in:

  34. Search for Albrecht Kramer-Zucker in:

  35. Search for Hanno J Bolz in:

  36. Search for Ronald Roepman in:

  37. Search for Sophie Saunier in:

  38. Search for Gerd Walz in:

  39. Search for Friedhelm Hildebrandt in:

  40. Search for Carsten Bergmann in:

  41. Search for Soeren S Lienkamp in:

Contributions

S.H. performed Xenopus and biochemical experiments. D.E. performed zebrafish studies. C. Boehlke, C.S., T.Y., M.H. and M.M. analyzed cilia in various models. J.H., E.F., E.A.O., V.F., T.E., H.J.B., S.S., F.H. and C. Bergmann performed mutational analysis. J.v.R., T.-M.T.N., K.B., N.H., M.U. and R.R. performed affinity proteomic and network analyses. M.W.E., J.A.E.v.W., D.B., N.J.S., S.R., M.V., T.R., M.P., L.P., T.J.N., N.A.S.E., S.J.K. and P.C.H. recruited subjects and provided clinical information. S.H., D.E., T.Y., F.G., T.B.H., E.W.K., A.K.-Z., G.W. and S.S.L. designed experiments and analyzed data. S.H., J.H., R.R., S.S., C. Bergmann, F.H., G.W. and S.S.L. wrote the manuscript, with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Friedhelm Hildebrandt or Carsten Bergmann or Soeren S Lienkamp.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Tables 2 and 3

Excel files

  1. 1.

    Supplementary Table 1

    Supplementary Table 1

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ng.2681

Further reading

Newsletter Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing