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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 . 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 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 , 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 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. Hum Mol Genet 12: 2703–2710, 2003 nisms by which cells along the renal tubule communicate to 6. Masyuk TV, Huang BQ, Ward CJ, Masyuk AI, Yuan D, Splinter PL, elicit coordinated behaviors and maintain a constant tubu- Punyashthiti R, Ritman EL, Torres VE, Harris PC, LaRusso NF: Defects lar diameter are not well understood. It would be interesting in cholangiocyte fibrocystin expression and ciliary structure in the PCK to determine whether urine flow-mediated dispersal of fi- rat. Gastroenterology 125: 1303–1310, 2003 7. Harris, PC: 2008 Homer W. Smith Award: Insights into the pathogen- brocystin-containing ELVs represents one such mechanism esis of polycystic kidney disease from gene discovery. JAmSoc of long-range communication between cells of the renal tu- Nephrol 20: 1188–1198, 2009 bule. In tissues such as the Drosophila melanogaster wings 8. Hildebrandt F, Attanasio M, Otto E: Nephronophthisis: Disease mech- and eyes, where PCP is also observed, asymmetric expres- anisms of a . J Am Soc Nephrol 20: 23–35, 2009 sion of core PCP produce a gradient along the 9. Menezes LF, Cai Y, Nagasawa Y, Silva AM, Watkins ML, Da Silva AM, proximal–distal plane of the tissue.20 This is thought to re- Somlo S, Guay-Woodford LM, Germino GG, Onuchic LF: Polyductin, sult in the establishment of PCP. Unequal distribution of the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and . Kidney Int 66: 1345–1355, fibrocystin-containing ELVs along the renal tubule may 2004 serve a similar function in the mammalian kidney. 10. Bakeberg JL, Tammachote R, Woollard JR, Hogan MC, Tuan H-F, Li The mouse expressing tagged-endogenous fibrocystin is a M, van Deursen JM, Wu Y, Huang BQ, Torres VE, Harris PC, Ward valuable reagent with which some of the other questions raised CJ: Epitope-tagged Pkhd1 tracks the processing, secretion, by the study could be addressed. For example, what are the and localization of fibrocystin. J Am Soc Nephrol 22: 2266–2277, spatial and temporal expression patterns of fibrocystin and 2011 11. Woollard JR, Punyashtiti R, Richardson S, Masyuk TV, Whelan S, what are the consequences of fibrocystin ELVs binding on pri- Huang BQ, Lager DJ, vanDeursen J, Torres VE, Gattone VH, LaRusso mary cilia? The ELVs were found to contain proteins that could NF, Harris PC, Ward CJ: A mouse model of autosomal recessive potentially modulate cilia-regulated and polycystic kidney dis- polycystic kidney disease with biliary duct and proximal tubule dilata- ease (PKD) relevant signaling pathways.12 Whether the con- tion. Kidney Int 72: 328–336, 2007 tents of ELVs affect ciliary signaling in the kidney and impact 12. Hogan MC, Manganelli L, Woollard JR, Masyuk AI, Masyuk TV, Tam- the pathogenesis of PKD remains to be seen. Finally, what are machote R, Huang BQ, Leontovich AA, Beito TG, Madden BJ, the other contents of fibrocystin ELVs? In addition to proteins, Charlesworth MC, Torres VE, LaRusso NF, Harris PC, Ward CJ: Char- acterization of PKD protein-positive exosome-like vesicles. JAmSoc if these ELVs are also found to contain mRNAs, miRNAs, or Nephrol 20: 278–288, 2009 other noncoding RNAs, a novel mechanism of intercellular 13. Hiesberger T, Gourley E, Erickson A, Koulen P, Ward CJ, Masyuk communication in kidney will be uncovered—does the pro- TV, Larusso NF, Harris PC, Igarashi P: Proteolytic cleavage and teomic makeup of fibrocystin ELVs change with kidney devel- nuclear translocation of fibrocystin is regulated by intracellular opment, after kidney injury, during kidney regeneration or in Ca2ϩ and activation of protein kinase C. J Biol Chem 281: 34357– Pkhd1 null mice? 34364, 2006 14. Kaimori JY, Nagasawa Y, Menezes LF, Garcia-Gonzalez MA, Deng J, Imai E, Onuchic LF, Guay-Woodford LM, Germino GG: Polyductin undergoes notch-like processing and regulated release from primary DISCLOSURES cilia. Hum Mol Genet 16: 942–956, 2007 VP is supported by NIH grant (K08 DK084311-01). 15. Fischer E, Legue E, Doyen A, Nato F, Nicolas JF, Torres V, Yaniv M,

J Am Soc Nephrol 22: 2139–2155, 2011 Editorials 2149 EDITORIALS www.jasn.org

Pontoglio M: Defective planar cell polarity in polycystic kidney dis- Hispanics, have higher ESRD rates.6 The incident rates of ease. Nat Genet 38: 21–23, 2006 ESRD for African Americans and Hispanics are 3.5 and 1.5 16. Nishio S, Tian X, Gallagher AR, Yu Z, Patel V, Igarashi P, Somlo S: Loss 4 of oriented cell division does not initiate cyst formation. J Am Soc times greater than non-Hispanic whites, respectively. An- Nephrol 21: 295–302, 2010 other unique feature is that although dialysis therapy is ex- 17. Karner C, Wharton KA Jr, Carroll TJ: Planar cell polarity and vertebrate pected to be lifesaving, some 20% of dialysis patients die organogenesis. Semin Cell Dev Biol 17: 194–203, 2006 each year, resulting in a low 5-year survival (Ͻ35%), worse 18. Karner CM, Chirumamilla R, Aoki S, Igarashi P, Wallingford JB, Carroll than many fatal cancers. Over half of the CKD deaths are TJ: Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis. Nat Genet 41: 793–799, 2009 attributable to cardiovascular or infectious deaths. The eti- 19 McNeill H: Planar cell polarity and the kidney. J Am Soc Nephrol 20: ology of the poor ESRD survival is unknown. A 4-decade 2104–2111, 2009 focus on treating conventional cardiovascular risk factors in 20. McNeill H: Planar cell polarity: Keeping hairs straight is not so simple. dialysis patients, including hyperlipidemia and hyperten- Cold Spring Harb Perspect Biol 2:a003376, 2010 sion, has changed the mortality only marginally, evidenced by slight improvement over the last few years, which is pos- sibly linked to increased use of cardiovascular and renopro- See related article, “Epitope-Tagged Pkhd1 Tracks the Processing, Secretion, 7 and Localization of Fibrocystin,” on pages 2266–2277. tective agents. Strangely enough, there is a unique connection between the two aforementioned distinctive features of the ESRD patients in that, for reasons that have remained unex- Is the Malnutrition-Inflammation plained, dialysis patients from minority groups have greater Complex the Secret behind longevity than non-Hispanic whites. The ESRD racial sur- vival disparities of dialysis patients can even be referred to as Greater Survival of African- a survival paradox for African Americans, in whom the American Dialysis Patients? lower dialysis mortality contrasts sharply with the general population in which African Americans have a shorter life ʈ expectancy than whites.4 Kamyar Kalantar-Zadeh*†‡§ and Keith C. Norris‡ Although a recent study found that the survival advantage *Harold Simmons Center for Chronic Disease Research and Epide- miology and †Division of Nephrology and Hypertension, Los Ange- of African-American dialysis patients exists mainly among les Biomedical Research Institute at Harbor-UCLA, Torrance, Califor- those older than 50 years of age,5 the study’s reference group ‡ § nia; David Geffen School of Medicine and Department of comprised all white patients including Hispanic whites, who Epidemiology School of Public Health, University of California–Los ʈ Angeles, Los Angeles, California; and Charles R. Drew University of are also known to have greater survival than non-Hispanic Medicine and Science, Los Angeles, California whites.8 Indeed, the greater survival of African Americans and Hispanics persists despite adjustments for demographics, res- J Am Soc Nephrol 22: 2150–2152, 2011. doi: 10.1681/ASN.2011101002 idency, dialysis modality or technique, and causes of death, among others.6 Examining these unusual disparities and paradoxes may Chronic dialysis therapy legislated in the United States be the key to discovering factors that can improve longevity through the ESRD program was started in the early 1970s as in all CKD patients and probably in other populations with an early prototype of universal health care coverage for a chronic disease and will be a major step to improving out- chronic disease. Since then, several unique features of dial- comes for all patients. In line with ongoing efforts to dis- ysis patients have stood out without any clear explanation, cover the roots of the racial survival disparities in CKD, including racial and ethnic disparities, cardiovascular dis- several candidate factors have been suggested4: racial/ethnic 1 ease, and extremely high mortality. Racial and ethnic dis- differences in nutritional and inflammatory profile and di- crepancies in ESRD patients have their roots in the earlier et; differences in mineral-bone disorders, including higher stages of chronic kidney disease (CKD), among others, be- parathyroid hormone levels in African-American patients 2 cause of access to care and the higher likelihood of hyper- leading to higher likelihood of receiving active vitamin D 1,3 tension and diabetes in African Americans. Across virtu- agents; differences in psychosocial status and coping mech- ally all age groups, one-third of dialysis patients in the anisms, including perception of quality of life; differences in United States are African American as compared with 14% dialysis treatment and techniques; and genetic or other in- 4,5 of the general population. Other minorities, including herent differences related to CKD and cardiovascular dis- ease progression. Published online ahead of print. Publication date available at www.jasn.org. Emerging data indicate that the nutritional-inflammatory

Correspondence: Dr. Keith C. Norris, Charles R. Drew University of Medicine axis is an important and biologically plausible mechanism in and Science, 1731 East 120th Street, Los Angeles, CA 90059. Phone: 323-323- engendering survival differentials across race.6 At least two- 5702; Fax: 323-357-3486; E-mail: [email protected] thirds of all dialysis patients show evidence of muscle and fat Copyright © 2011 by the American Society of Nephrology wasting and increased levels of inflammatory markers, in-

2150 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 2139–2155, 2011