The disease is one of the major causes of chronic renal failure

Summary and Discussion

Samenvatting

7

Summary and Discussion

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common inherited disorder that predominantly manifests with progressive development of ß uid- Þ lled cysts in both kidneys. Cyst formation ultimately results in chronic renal failure. The disease is one of the major causes of chronic renal failure. Cysts arise from a defect in renal tubule epithelium function (Chapter 1, Figure 4).

Due to an as yet unknown mechanism, a subset of epithelial cells undergoes a signiÞ cant change that enables them to escape the regulatory mechanisms that normally regulate their function. These cells expand and grow out to form an isolated cyst. Cyst enlargement then occurs via ß uid accumulation in the cyst lumen due to mislocalization of ion channels. Progressive development of cysts and accompanying Þ brosis of the surrounding tissue disrupts renal function and ultimately results in chronic renal failure. In the majority of patients, the disease can be accounted for by a mutation in the either the PKD1 gene (1,2) or the PKD2 gene (3-5). The precise function of polycystin-1 and polycystin- 2, the proteins encoded by the PKD1 and PKD2 gene respectively, remains to be elucidated. Therefore, it is still unclear how a mutation in polycystin-1 or polycystin-2 results in cyst formation. Data suggest that polycystins can exert multiple functions depending on the environment it is expressed in (cell type, diff erentiation status, etc.). Polycystin-1 and polycystin-2 have been shown to play a role in the cellular response upon extra-cellular stimuli, whether these are chemical (growth factors, stresses) or mechanical stimuli (ß uid ß ow). Both polycystin-1 and polycystin-2 can regulate the cellular response by modulating signalling cascades, cell adhesion and/or cystoskeletal integrity, all of which are tightly coupled. Depending on the stimulus received, cell type, diff erentiation status, and other cellular and environmental factors, diff erent functional aspects may be required to facilitate proper cellular responses. The molecular basis of polycystin-1 and polycystin-2 function in these signal transduction routes has not been elucidated yet. Polycystins may eff ect these signaling events via their ion channel activity or by modulating activity of interaction partners. It is clear however, that in the kidney, polycystins are required to maintain proper renal architecture and especially tubular integrity, since a defect in polycystin inevitably leads to polycystic kidney disease. Our work has focused on the renal pathology, because polycystic kidney disease and concomitt ant chronic renal failure is the major clinical threat for ADPKD patients. We set out to investigate the functions of polycystin-1 and polycystin-2 in order to gain further insight into the molecular and cellular processes that are disrupted in ADPKD cyst formation. Potential signaling pathways modulated by polycystin-1 were identiÞ ed using a widely implemented approach based on reporter constructs. A C-terminal polycystin-1 construct was co-expressed in cells with luciferase reporter constructs to detect Wnt/β-catenin signaling or AP-1 activation (Chapter 2). In our hands, polycystin- 1 did not modulate Wnt/β-catenin, whereas AP-1 activity was regulated by polycystin-1. Results were conÞ rmed using immunoß uorescence microscopy and western blot analysis to detect activated signaling components in normal and cystic cells and tissue. Using this approach we excluded Wnt/β-catenin signaling Chapter 7

and identiÞ ed AP-1 activation as a potential player in ADPKD cyst formation.

The role of AP-1 in cyst formation was further analyzed in ADPKD cystic tissue using immunohistochemical and western blot analysis (Chapter 3). Since AP-1 is regulated by MAPK’s, we explored the potential role of MAPK signaling by analyzing cystic epithelial cells derived from human ADPKD patients and from mouse renal cysts (Chapter 4). We report that ERK signalling activity is signiÞ cantly impaired in human and mouse renal cystic epithelial cells and that polycystin-1 and polycystin-2 can modulate ERK activity. Our data clearly demonstrate that polycystins modulate intracellular signalling pathways, including MAPKs and AP-1 transcription factors. Moreover, both MAPK signalling and AP-1 activity are defective in cystic cells and tissue, indicating that these signalling events contribute to cyst formation or progression and may be crucial for maintaining renal epithelial cellular integrity and function. Direct immunoprecipitation studies will provide further insight into the mechanism by which polycystin exert their eff ect on MAPKs and AP-1. Other groups have reported that polycystins also modulate other intracellular signalling pathways including, NFAT and JAK-STAT transcription factors, and the mTOR pathway (7-9). These observations show that polycystins do not exclusively modulate MAPK signalling and AP-1 activity, but rather regulate a broad range of signalling routes within the cell. Since it is less likely that polycystins exert these eff ects via direct interaction with all of the corresponding intermediates, we hypothesize that polycystins may have selective molecular eff ects via which intracellular signalling events are achieved. One of the most likely candidates to relay the signal from polycystins is Ca²⁺ that is increased in the cell aft er activation of the polycystin channel. Other possible candidates are molecules up-stream of the signalling pathways and can be identiÞ ed by direct immunoprecipitation experiments. Also, sub-cellular localization of polycystins can provide a relay mechanism: depending on the sub-cellular localization of the polycystin complex, in the plasma membrane at adhesion junctions, in the primary cilium, or in the cytosol, diff erent cellular responses are achieved. Based on this, polycystins have been proposed to function as multi-purpose cellular tools that can act as a “master switch” between diff erent signalling routes.

In Chapter 5, we describe down-regulation of PKD1 and PKD2 aft er cellular stress as a potential factor in cyst formation. These data suggest that stress induced down-regulation of PKD1 and PKD2 during life may be an additional non- genetic modifying factor in cyst formation. Sub-lethal DNA damage is normally accumulated during life. In the case of ADPKD, germ-line inactivation of one allele in combination with down-regulation of PKD1 and PKD2, may just be suffi cient to initiate cyst formation and progression. In this respect, it would be interesting to asses the eff ect of cellular damage on mouse models for Pkd1 and Pkd2. To test this hypothesis, we propose to induce cellular damage to heterozygous knock-out mice or to cross heterozygous mice with DNA-repair deÞ cient mice. Based on our current data, we expect more severe and progressive polycystic kidney disease.

Chapter 6 reviews potential therapeutic intervention strategies for cyst formation in ADPKD. Data on EGF/EGFR and AVP/cAMP signalling as potential targets for therapeutic intervention in ADPKD are outlined and discussed. Some of these reports present conß icting data. For instance, inhibition of EGFR activity

by pharmaceutical compounds such as EKI-785 and EKI-569, have been reported to improve disease progression in mouse models for polycystic kidney disease (10,11). In contrast, EGF supplemental treatment for polycystic kidney disease has been reported (12,13). Our data (Chapter 4) provide direct molecular evidence for impaired ERK signaling activity in human and mouse renal cystic cells at early and end-stage of disease and thereby establish a rationale for the EGF supplemental therapy proposed by Gatt one II et al. and Ricker et al. (12,13).

In conclusion, much eff ort has been undertaken to unravel the molecular defects in polycystic kidneys disease. Our data contribute to this endeavour and identify MAPK signalling and AP-1 activity defects in both early and late polycystic kidneys disease. These results can be further conÞ rmed by crossing mouse models for polycystic kidney disease with mouse models for these signalling components, for instance ERK knock-out mice. Comparison of single and compound

heterozygous mice will provide further insight whether MAPK signalling and AP-1 activity are indeed mediators in polycystic kidney disease. These insights are crucial to develop directed therapeutic strategies for ADPKD.

References

1. The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. The European Polycystic Kidney Disease Consortium. Cell 1994;78:725.

2. Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. The International Polycystic Kidney Disease Consortium. Cell 1995;81:289-298.

3. Kimberling WJ, Kumar S, Gabow PA, et al. Autosomal dominant polycystic kidney disease: localization of the second gene to chromosome 4q13-q23. Genomics 1993;18:467-472.

4. Peters DJ, Spruit L, Saris JJ, et al. Chromosome 4 localization of a second gene for autosomal dominant polycystic kidney disease. Nat Genet 1993;5:359-362.

5. Mochizuki T, Wu G, Hayashi T, et al. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 1996;272:1339-1342.

6. Puri S, Magenheimer BS, Maser RL, et al. Polycystin-1 activates the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway. J Biol Chem 2004;279:55455-55464.

7. Bhunia AK, Piontek K, Bolett a A, et al. PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2. Cell 2002;109:157-168.

8. Shillingford JM, Murcia NS, Larson CH, et al. The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease. Proc Natl Acad Sci U S A 2006;103:5466-5471.

9. Torres VE, Sweeney WE, Jr., Wang X, et al. EGF receptor tyrosine kinase inhibition att enuates the development of PKD in Han:SPRD rats. Kidney Int 2003;64:1573-1579.

10. Sweeney WE, Futey L, Frost P, et al. In vitro modulation of cyst formation by a novel tyrosine kinase inhibitor. Kidney Int 1999;56:406- 413.

11. Gatt one VH, 2nd, Lowden DA, Cowley BD, Jr. Epidermal growth factor ameliorates autosomal recessive polycystic kidney disease in mice. Dev Biol 1995;169:504-510.

12. Ricker JL, Gatt one VH, 2nd, Calvet JP, et al. Development of autosomal recessive polycystic kidney disease in BALB/c-cpk/cpk mice. J Am Soc Nephrol 2000;11:1837-1847.

Chapter 7