BASIC RESEARCH www.jasn.org Disruption of IFT Complex A Causes Cystic Kidneys without Mitotic Spindle Misorientation † ‡ † Julie A. Jonassen,* Jovenal SanAgustin, Stephen P. Baker, and Gregory J. Pazour *Department of Microbiology and Physiological Systems, †Program in Molecular Medicine, and ‡Department of Information Sciences, University of Massachusetts Medical School, Worcester, Massachusetts ABSTRACT Intraflagellar transport (IFT) complexes A and B build and maintain primary cilia. In the mouse, kidney- specific or hypomorphic mutant alleles of IFT complex B genes cause polycystic kidneys, but the influence of IFT complex A proteins on renal development is not well understood. In the present study, we found that HoxB7-Cre–driven deletion of the complex A gene Ift140 from collecting ducts disrupted, but did not completely prevent, cilia assembly. Mutant kidneys developed collecting duct cysts by postnatal day 5, with rapid cystic expansion and renal dysfunction by day 15 and little remaining parenchymal tissue by day 20. In contrast to many models of polycystic kidney disease, precystic Ift140-deleted collecting ducts showed normal centrosomal positioning and no misorientation of the mitotic spindle axis, suggesting that disruption of oriented cell division is not a prerequisite to cyst formation in these kidneys. Precystic collecting ducts had an increased mitotic index, suggesting that cell proliferation may drive cyst expansion even with normal orientation of the mitotic spindle. In addition, we observed significant increases in expression of canonical Wnt pathway genes and mediators of Hedgehog and tissue fibrosis in highly cystic, but not precystic, kidneys. Taken together, these studies indicate that loss of Ift140 causes pro- nounced renal cystic disease and suggest that abnormalities in several different pathways may influence cyst progression. J Am Soc Nephrol 23: 641–651, 2012. doi: 10.1681/ASN.2011080829 Genesis of primary cilia—specialized microtubular- by kinesin-2 and cytoplasmic dynein-2.7–10 IFT based sensory structures that project from most complex B comprises at least 13 proteins11 and is cells—requires intraflagellar transport (IFT), a required for ciliary assembly.12–16 In the mouse, bidirectional process that builds, maintains, strong alleles of IFT complex B genes typically pro- and disassembles these organelles. IFT also sup- duce midgestational lethality14,15 before renal de- ports diverse signaling roles played by primary velopment. Hypomorphic mutations in Ift88 or cilia that influence development, differentiation, kidney-specific deletion of Ift20, two IFT complex and cell cycle regulation.1–3 In the kidney, primary B genes, cause renal cyst formation.13,16 Similarly, cilia play vital roles in promoting tubular develop- strong alleles of the kinesin-2 or the dynein-2 IFT ment and maintaining normal renal morphology motors produce midgestational lethality,15,17,18 and function. Mutations that produce structural or functional defects in renal cell primary cilia cause abnormal proliferation of tubular epithelia, in- creased fluid secretion, and polycystic kidney Received August 19, 2011. Accepted November 23, 2011. 4–6 disease. Discerning processes controlling IFT- Published online ahead of print. Publication date available at mediated ciliary assembly and function is essential www.jasn.org. for understanding the pathogenic mechanisms un- Correspondence: Dr.GregoryJ.Pazour,PrograminMolecular derlying cystic renal diseases and other ciliopa- Medicine, University of Massachusetts Medical School, Biotech II, thies. Suite 213, 373 Plantation Street, Worcester, MA 01605. Email: The IFTsystem consists of two large protein com- [email protected] plexes, IFT complexes A and B, that are transported Copyright © 2012 by the American Society of Nephrology J Am Soc Nephrol 23: 641–651, 2012 ISSN : 1046-6673/2304-641 641 BASIC RESEARCH www.jasn.org whereas kidney-specific deletion of kinesin-2 results in cystic localize to the spindle pole during mitosis.39–42 In contrast, disease.19 IFT140 does not seem to be associated with the spindle pole In contrast, very little is known about what, if any, role bodies during mitosis (Figure 1D). In control postnatal (p)5 is played by IFT complex A proteins on mammalian renal de- kidneys, IFT140 labels the base of the cilium (Figure 1E) just velopment and renal physiology. In invertebrate organisms, adjacent to the centrosome (Figure 1F). Staining of experimen- mutation or RNA interference depletion of individual IFT tal kidneys indicates that, at most, very short cilia remain at p5 complex A proteins produces cilia that are shortened and often and no IFT140 staining is observed. These results indicate that fl dilated and accumulate ciliary proteins.12,20–26 Similarly, RNA the conversion of the Ift140 ox allele to the Ift140null2 allele is interference knockdown of complex A proteins in mamma- efficient and that IFT140 is required for ciliary assembly. lian cells generated shortened cilia that accumulated IFT-B HoxB7-Cre expression begins with mesonephric duct de- proteins.27 In mice, null alleles of individual IFT complex A velopment 6–9daysbeforebirth43 before formation of the proteins, including IFT139,28 IFT122,29,30 and IFT121,31 pro- ureteric bud, the progenitor of adult collecting ducts. Collect- duced defects in skeletal, craniofacial, and nervous systems ing duct deletion of Ift140 led to pronounced postnatal renal that caused embryonic or neonatal death. Because of the cyst formation (Figure 2, A and B). At p0, there are modest early lethality of IFT complex A mutants, there is no informa- medullary collecting duct dilations but no renal cysts. By p5, tion as to whether IFT complex A defects would disrupt renal extensive medullary cysts are evident, with minimal cortical developmentorfunctioninmice.InIft122 zebrafish mor- cysts. By p10, cysts are present in medullary and cortical re- phants, pronephric cysts were observed32;incontrast,Ift140 gions, and by p20, extensive cysts are found throughout the morphants showed no apparent ciliary or sensory neuron de- kidney, with little remaining parenchymal tissue (Figure 2B). fects.33 Missense mutations in IFT-A proteins have been Kidney weights increased progressively (Figure 2C), and blood described in patients with cranioectodermal dysplasia/Sensen- urea nitrogen levels were elevated in mutants at p15 and p20 brenner’ssyndrome,31,32,34,35 a ciliopathy associated with (Figure 2D), consistent with renal failure during the third extensive craniofacial, skeletal, heart, liver, and ectodermal ab- week of life. normalities. Some cranioectodermal dysplasia patients exhibit Control collecting ducts were highly ciliated at birth, renal disease characterized by extensive glomerular sclerosis, whereas experimental p0 collecting ducts had no or very short renal cysts, interstitial fibrosis with focal inflammatory cell cilia (Figure 3A). There was no ciliary loss in noncollecting infiltration, scattered tubular atrophy, and chronic renal fail- duct cells (Figure 3A), supporting the specificity of HoxB7-Cre ure.31,32,36 Nonetheless, our understanding of how IFT com- for the collecting duct system.43 Although there may be a pro- plex A proteins influence renal development and cystic disease gressive loss of these shortened cilia over time, many collecting is extremely limited, and the present studies addressed this duct cells still carried stumpy cilia at p10 and p20 (Figure 3A). question by characterizing IFT140 function in mouse kidney. These shortened cilia show increased staining with IFT88 compared with controls (Figure 3B). Similarly, Ift140-deleted cultured cells showed increased ciliary staining with IFT88 RESULTS and IFT27 antibodies (Figure 3C). During normal tubular development, the mitotic spindle of To understand the role of IFT140 in cystic kidney disease, dividing cells orients in parallel to the longitudinal axis of the we used Knockout Mouse Project (KOMP) embryonic stem (ES) nephron.44 In many different polycystic kidney disease (PKD) cells37,38 to create flox and null1 alleles (Figure 1A). Animals models,16,45,46 randomization of the mitotic spindle axis may fl homozygous for Ift140 ox are viable with no detectable pheno- be a critical part of the cystogenic program. At p5, many ex- types, whereas animals homozygous for Ift140null1 die at mid- perimental collecting ducts were already dilated; these ducts gestation and will be described in a separate publication. In this were excluded from analysis to focus on events occurring be- work, we used HoxB7-Cre to delete Ift140 in the collecting ducts. fore the duct lost its normal architecture. In control collecting fl Control animals have the genotype HoxB7-Cre, Ift140 ox/+,and ducts, mitotic spindles aligned with the long axis of the tubule. fl experimental animals are HoxB7-Cre, Ift140 ox/null1.Theflox al- In contrast to what we observed in Ift20-deleted kidneys,16 loss lele is converted to the null2 allele by Cre, and therefore, exper- of Ift140 did not alter mitotic spindle orientation in precystic imental animals are Ift140null2/null1 in the collecting ducts. An collecting ducts (Figure 4, A and B). Whereas deletion of Ift20 antibody generated against mouse IFT140 (Figure 1B) does from the collecting ducts caused mislocalization of the cen- not detect any IFT140 in extracts made from cell lines derived trosome from the center of the apical end of the cell,16 deletion from experimental collecting ducts (Figure 1B), indicating that of Ift140 did not produce this phenotype,