EDITORIALS www.jasn.org Tagged Fibrocystin Sheds Its cystin are not present in the mouse kidney. To prove that splicing does not occur, the authors performed long-range Secrets overlapping RT-PCRs using primers that amplified exons 1–21, 19–34, 32–52, 48–52, and 48–67. These reactions Vishal Patel generated PCR products of predicted size, and their se- Department of Internal Medicine and Division of Nephrology, Uni- quences aligned perfectly with the full-length cDNA encod- versity of Texas Southwestern Medical Center at Dallas, Dallas, Texas ing Pkhd1. If splicing had occurred, due to the exclusion of exons from the transcribed mRNA, PCR products of vary- J Am Soc Nephrol 22: 2148–2150, 2011. ing lengths would have been produced. doi: 10.1681/ASN.2011101005 To determine whether alternative forms of fibrocystin are made, the authors utilized an elegant in vivo approach. They Autosomal recessive polycystic kidney disease (ARPKD), a rare generated mice that harbored a loxP flanked transcriptional monogenic disorder with an incidence of 1:20,000 live births, is STOP cassette in intron–2 and coding-sequences of two characterized by massive bilateral kidney enlargement due to SV5–Pk tags in exon–3 of the Pkhd1 gene. As a consequence, 1 cystic dilation of the renal tubules. In addition, liver cysts and Pkhd1 transcription is disrupted and mutant mice develop hepatic fibrosis are also observed in patients with ARPKD. In liver cysts and fibrosis. Consistent with a previous report, the neonates and infants, ARPKD manifests as severe kidney fail- mutant mice do not develop kidney cysts.11 Only proximal ure in utero, oligohydramnios, and pulmonary hypoplasia, tubule dilation was observed in female mice at older ages. Next, causing respiratory failure, a major cause of perinatal mortal- using a cre/loxP recombination strategy, the transcriptional ity. ARPKD can also manifest in older children and adoles- STOP cassette was removed. This resulted in the re-expression cents. The clinical features include kidney cysts, progressive of the Pkhd1 gene. Since the coding sequences of SV5-Pk tags renal failure, systemic hypertension, and portal hypertension were present in exon-3 of the Pkhd1 gene, fibrocystin was due to hepatic fibrosis. Over 50% of these patients develop end-stage renal disease and require kidney transplantation be- tagged with SV5-Pk epitope at its N-terminus. Characteriza- fore the age of 20.1,2 tion of these mice demonstrates normal histology of the kidney ARPKD is caused by mutations in PKHD1 (polycystic kid- and liver, indicating that epitope-tagging of fibrocystin does ney and hepatic disease 1), which encodes a large protein called not disrupt its function in vivo. Using antibodies that easily fibrocystin or polyductin.3,4 Fibrocystin is predicted to be a detect the epitope, the authors show that fibrocystin is primar- type I membrane protein with a large extracellular N-terminal ily made as a 500-kD protein. domain, a single transmembrane segment, and a short cyto- While the results provide strong evidence against extensive plasmic C-terminus.3,4 In addition to other places in the cell, PKHD1 splicing and the presence of alternative forms of fibro- fibrocystin is located on the primary cilium, a hair-like organ- cystin, they do not conclusively resolve the issue. It is possible elle present on the surface of most cells in the body.5 Lack of that some of the alternative transcripts are expressed in a spa- fibrocystin results in stunted primary cilia.6 These findings in- tiotemporal manner; therefore, these transcripts will only be dicate that abnormalities of primary cilia may underlie the detected at specific stages of development or in specific cell pathogenesis of ARPKD.7,8 types. Another possibility is that if some of these transcripts are The molecular mechanism by which mutations in PKHD1 not abundantly expressed, they may elude detection. If tran- produce ARPKD is not known. It has been proposed that scripts that exclude exon-3 are translated, the encoded protein mouse Pkhd1 and the human PKHD1 genes undergo extensive would not contain the epitope tag and therefore would not be differential splicing.3,9 Moreover, at least three of these tran- detected. Finally, there are considerable differences in the phe- scripts are translated into smaller alternative forms of fibrocys- notypes of patients with ARPKD and Pkhd1 null mice.2,11 In tin, while numerous other alternative forms remain undiscov- contrast to ARPKD patients who develop kidney cysts and re- 3,9 ered. Therefore, determining the functional consequences of nal failure in utero or during childhood, the Pkhd1 null mice the pathogenic mutations poses a significant challenge. display normal kidneys at birth and develop only mild cystic 10 In this issue of JASN, Bakeberg et al. have revisited the kidney disease at older ages. This raises the possibility that the importance of Pkhd1 splicing and fibrocystin processing. mouse Pkhd1 and the human PKHD1 genes are processed and Surprisingly, the authors show that splicing is not a major function slightly differently. feature of mouse Pkhd1 and that alternative forms of fibro- The study by Bakeberg et al. provides important insights and has raised several unanswered questions. The authors Published online ahead of print. Publication date available at www.jasn.org. have previously shown that the mature cleaved form of fi- brocystin is found in exosome-like vesicles (ELVs) isolated Correspondence: Dr. Vishal Patel, Department of Internal Medicine/Nephrol- 12 ogy, 5323 Harry Hines Boulevard, F5206, Dallas, TX 75390. Phone: 214-648- from the mouse urine. Exosomes are small extracellular 2754; Fax: 214-648-2071; E-mail: [email protected] vesicles that are derived from multivesicular bodies.12 Tak- Copyright © 2011 by the American Society of Nephrology ing advantage of the easily-detectable tagged-endogenous 2148 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 2139–2155, 2011 www.jasn.org EDITORIALS fibrocystin, the authors confirm their previous findings in REFERENCES the present study. So what is the functional significance of fibrocystin-containing ELVs? Posttranslational processing 1. Igarashi P, Somlo S: Genetics and pathogenesis of polycystic kidney produces multiple smaller products of the full-length fibro- disease. J Am Soc Nephrol 13: 2384–2398, 2002 cystin inside the cell and the N-terminal ectodomain is shed 2. Williams SS, Cobo-Stark P, James LR, Somlo S, Igarashi P: Kidney cysts, pancreatic cysts, and biliary disease in a mouse model of auto- 13,14 out of the cell. Exosomes represent a mechanism by somal recessive polycystic kidney disease. Pediatr Nephrol 23: 733– which the processed intracellular fibrocystin is transported 741, 2008 out of the cell. In the extracellular space, fibrocystin-con- 3. Onuchic LF, Furu L, Nagasawa Y, Hou X, Eggermann T, Ren Z, Berg- taining ELVs rapidly associate with primary cilia.12 There- mann C, Senderek J, Esquivel E, Zeltner R, Rudnik-Schoneborn S, fore, ELVs may be the means by which cells communicate Mrug M, Sweeney W, Avner ED, Zerres K, Guay-Woodford LM, Somlo with each other. S, Germino GG: PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglob- A novel function ascribed to fibrocystin is maintenance ulin-like plexin-transcription-factor domains and parallel beta-helix 1 15,16 of planar cell polarity (PCP) in the kidney. In addition repeats. Am J Hum Genet 70: 1305–1317, 2002 to apical-basal polarity, cells in multicellular organisms are 4. Ward CJ, Hogan MC, Rossetti S, Walker D, Sneddon T, Wang X, Kubly also polarized along the plane of the tissue, which is referred V, Cunningham JM, Bacallao R, Ishibashi M, Milliner DS, Torres VE, to as PCP.17 Coordinated cellular behaviors, convergent ex- Harris PC: The gene mutated in autosomal recessive polycystic kidney tension, and oriented cell division—processes regulated by disease encodes a large, receptor-like protein. Nat Genet 30: 259– PCP signaling—result in the establishment and mainte- 269, 2002 5. Ward CJ, Yuan D, Masyuk TV, Wang X, Punyashthiti R, Whelan S, 18,19 nance of PCP in the kidney. PCP signaling ensures that Bacallao R, Torra R, LaRusso NF, Torres VE, Harris PC: Cellular and renal tubule diameter remains relatively unchanged during subcellular localization of the ARPKD protein; fibrocystin is expressed tubule elongation or regeneration after injury. The mecha- on primary cilia. 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