Inactivation of Pkd1 in principal cells causes a more severe cystic kidney disease than in intercalated cells
2009; Elsevier BV; Volume: 75; Issue: 6 Linguagem: Inglês
10.1038/ki.2008.659
ISSN1523-1755
AutoresKalani L. Raphael, Kevin A. Strait, Peter K. Stricklett, R. Lance Miller, Raoul D. Nelson, Klaus Piontek, Gregory G. Germino, Donald E. Kohan,
Tópico(s)Urological Disorders and Treatments
ResumoRenal cysts in autosomal dominant polycystic kidney disease arise from cells throughout the nephron, but there is an uncertainty as to whether both the intercalated cells (ICs) and principal cells (PCs) within the collecting duct give rise to cysts. To determine this, we crossed mice containing loxP sites within introns 1 and 4 of the Pkd1 gene with transgenic mice expressing Cre recombinase under control of the aquaporin-2 promoter or the B1 subunit of the proton ATPase promoter, thereby generating PC- or IC-specific knockout of Pkd1, respectively. Mice, that had Pkd1 deleted in the PCs, developed progressive cystic kidney disease evident during the first postnatal week and had an average lifespan of 8.2 weeks. There was no change in the cellular cAMP content or membrane aquaporin-2 expression in their kidneys. Cysts were present in the cortex and outer medulla but were absent in the papilla. Mice in which PKd1 was knocked out in the ICs had a very mild cystic phenotype as late as 13 weeks of age, limited to 1–2 cysts and confined to the outer rim of the kidney cortex. These mice lived to at least 1.5 years of age without evidence of early mortality. Our findings suggest that PCs are more important than ICs for cyst formation in polycystic kidney disease. Renal cysts in autosomal dominant polycystic kidney disease arise from cells throughout the nephron, but there is an uncertainty as to whether both the intercalated cells (ICs) and principal cells (PCs) within the collecting duct give rise to cysts. To determine this, we crossed mice containing loxP sites within introns 1 and 4 of the Pkd1 gene with transgenic mice expressing Cre recombinase under control of the aquaporin-2 promoter or the B1 subunit of the proton ATPase promoter, thereby generating PC- or IC-specific knockout of Pkd1, respectively. Mice, that had Pkd1 deleted in the PCs, developed progressive cystic kidney disease evident during the first postnatal week and had an average lifespan of 8.2 weeks. There was no change in the cellular cAMP content or membrane aquaporin-2 expression in their kidneys. Cysts were present in the cortex and outer medulla but were absent in the papilla. Mice in which PKd1 was knocked out in the ICs had a very mild cystic phenotype as late as 13 weeks of age, limited to 1–2 cysts and confined to the outer rim of the kidney cortex. These mice lived to at least 1.5 years of age without evidence of early mortality. Our findings suggest that PCs are more important than ICs for cyst formation in polycystic kidney disease. Renal cysts in autosomal dominant polycystic kidney disease (ADPKD) arise from cells throughout the nephron. Analysis of human ADPKD cyst fluid reveals two major cyst types; one with sodium and chloride hypotonic to plasma (derived presumably from distal nephron) and the other with sodium and chloride isotonic to plasma (thought to derive from proximal nephron).1.Huseman R. Grady A. Welling D. et al.Macropuncture study of polycystic disease in adult human kidneys.Kidney Int. 1980; 18: 375-385Abstract Full Text PDF PubMed Scopus (71) Google Scholar Immunohistochemical studies, using stains for aquaporins, lectins, and other markers have yielded variable results, but suggest that a substantial percentage of cysts in ADPKD arise from distal nephron, including the collecting duct.2.Bachinsky D.R. Sabolic I. Emmanouel D.S. et al.Water channel expression in human ADPKD kidneys.Am J Physiol. 1995; 268: F398-F403PubMed Google Scholar, 3.Hayashi M. Yamaji Y. Monkawa T. et al.Expression and localization of the water channels in human autosomal dominant polycystic kidney disease.Nephron. 1997; 75: 321-326Crossref PubMed Scopus (18) Google Scholar, 4.Kovacs J. Zilahy M. Gomba S. Morphology of cystic renal lesions. Lectin and immuno-histochemical study.Acta Chir Hung. 1997; 36: 176-178PubMed Google Scholar, 5.Yoder B.K. Mulroy S. Eustace H. et al.Molecular pathogenesis of autosomal dominant polycystic kidney disease.Expert Rev Mol Med. 2006; 8: 1-22Crossref PubMed Scopus (45) Google Scholar Within the collecting duct, there is an uncertainty as to whether both cell types, namely intercalated (IC) and principal (PC) cells, give rise to cysts. No studies have determined the collecting duct cellular pattern of expression of the proteins responsible for ADPKD, polycystin-1, and -2;6.Consortium T.E.P.K.D. The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16.Cell. 1994; 77: 881-894Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 7.Mochizuki T. Wu G. Hayashi T. et al.PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1103) Google Scholar this relates to the lack of antibodies with adequate sensitivity and specificity. A second key point relates to primary cilia expression by these cell types. Numerous studies have implicated the primary cilium as a key factor in cystogenesis, as mutations of multiple different ciliary proteins lead to renal cyst formation;5.Yoder B.K. Mulroy S. Eustace H. et al.Molecular pathogenesis of autosomal dominant polycystic kidney disease.Expert Rev Mol Med. 2006; 8: 1-22Crossref PubMed Scopus (45) Google Scholar both polycystin-1 and -2 are associated spatially with the primary cilium.8.Yoder B.K. Hou X. Guay-Woodford L.M. The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia.J Am Soc Nephrol. 2002; 13: 2508-2516Crossref PubMed Scopus (689) Google Scholar Ultra-structural studies done over 45 years ago detected primary cilia on rat PCs, but not ICs.9.Latta H. Maunsbach A.B. Madden S.C. Cilia in different segments of the rat nephron.J Biophys Biochem Cytol. 1961; 11: 248-252Crossref PubMed Scopus (70) Google Scholar This conclusion was supported by one subsequent study (in 1989) focused on rat inner medullary collecting duct (which has very few ICs).10.Clapp W.L. Madsen K.M. Verlander J.W. et al.Morphologic heterogeneity along the rat inner medullary collecting duct.Lab Invest. 1989; 60: 219-230PubMed Google Scholar In contrast, modern technology has shown clearly that in the mouse, both PCs and ICs contain primary cilia. A recent report found that mouse ICs have a primary cilium by staining for acetylated α-tubulin (a marker of the ciliary axoneme) as well as by scanning electron microscopy.11.Shibazaki S. Yu Z. Nishio S. et al.Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1.Hum Mol Genet. 2008; 17: 1505-1516Crossref PubMed Scopus (168) Google Scholar Thus, based on the above considerations, there is no compelling reason a priori to suspect that ICs are unable to give rise to cysts in ADPKD. An animal model of ADPKD would be ideal to evaluate the roles of IC and PC in cyst formation. Several models of autosomal recessive polycystic kidney disease exist, including congenital polycystic kidney (cpk) mice, oak ridge polycystic kidney (Tg737) mice, and polycystic kidney (pck) rats.12.Guay-Woodford L.M. Murine models of polycystic kidney disease: molecular and therapeutic insights.Am J Physiol Renal Physiol. 2003; 285: F1034-F1049Crossref PubMed Scopus (163) Google Scholar However, these involve mutations in proteins other than polycystin-1 and -2. A number of animal models of ADPKD exist; however, most of these have proven to be problematic. Mice with homozygous mutations of the mouse orthologs of the human genes encoding polycystin-1 and -2 (Pkd1 and Pkd2) die in utero because of multiple organ involvement, some of which is not typical for ADPKD.13.Boulter C. Mulroy S. Webb S. et al.Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene.Proc Natl Acad Sci USA. 2001; 98: 12174-12179Crossref PubMed Scopus (234) Google Scholar, 14.Kim K. Drummond I. Ibraghimov-Beskrovnaya O. et al.Polycystin 1 is required for the structural integrity of blood vessels.Proc Natl Acad Sci USA. 2000; 97: 1731-1736Crossref PubMed Scopus (255) Google Scholar, 15.Lu W. Peissel B. Babakhanlou H. et al.Perinatal lethality with kidney and pancreas defects in mice with a targeted Pkd1 mutation.Nat Genet. 1997; 17: 179-181Crossref PubMed Scopus (358) Google Scholar, 16.Lu W. Shen X. Pavlova A. et al.Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1 causes cystogenesis and bone defects.Hum Mol Genet. 2001; 10: 2385-2396Crossref PubMed Scopus (148) Google Scholar, 17.Muto S. Aiba A. Saito Y. et al.Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant.Hum Mol Genet. 2002; 11: 1731-1742Crossref PubMed Scopus (125) Google Scholar Heterozygous Pkd1 and Pkd2 knockout (KO) mice develop limited renal cysts late in life and have not, therefore, been highly useful for analysis.13.Boulter C. Mulroy S. Webb S. et al.Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene.Proc Natl Acad Sci USA. 2001; 98: 12174-12179Crossref PubMed Scopus (234) Google Scholar, 16.Lu W. Shen X. Pavlova A. et al.Comparison of Pkd1-targeted mutants reveals that loss of polycystin-1 causes cystogenesis and bone defects.Hum Mol Genet. 2001; 10: 2385-2396Crossref PubMed Scopus (148) Google Scholar The Pkd2WS25 model is perhaps most similar to human ADPKD.18.Wu G. D’Agati V. Cai Y. et al.Somatic inactivation of Pkd2 results in polycystic kidney disease.Cell. 1998; 93: 177-188Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar In this spontaneous loss of heterozygosity model, recombination of the Pkd2WS25 allele leads to renal cysts if the animal contains a Pkd2 null allele. However, as PKD1 mutations account for the large majority of cases of ADPKD, continued efforts have focused on targeting this allele. These approaches have utilized largely the renal-specific KO of Pkd1 in mice. One model used mice expressing the γ-glutamyl transpeptidase promoter coupled to Cre recombinase and loxP-flanked (floxed) Pkd1 exons 2–6.19.Starremans P.G. Li X. Finnerty P.E. et al.A mouse model for polycystic kidney disease through a somatic in-frame deletion in the 5′ end of Pkd1.Kidney Int. 2008; 73: 1394-1405Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar These animals developed proximal and distal nephron cysts and survived about 4 weeks––long enough to examine potential signaling pathways, but limiting more long-term analysis. A similar strategy was employed in which, Ksp-Cre transgenic mice were bred with mice containing floxed exons 2–4 in Pkd1.11.Shibazaki S. Yu Z. Nishio S. et al.Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1.Hum Mol Genet. 2008; 17: 1505-1516Crossref PubMed Scopus (168) Google Scholar This resulted in Cre expression selectively in thick ascending limb through the collecting duct; however, the animals survived only 3 weeks, perhaps because of early (E11.5) Cre expression. To overcome early lethality, mice were generated with tamoxifen-inducible Pkd1 deletion using a Ksp-CreERT2 transgene and floxed Pkd1 exons 2–11.20.Lantinga-van Leeuwen I.S. Leonhard W.N. van der Wal A. et al.Kidney-specific inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice.Hum Mol Genet. 2007; 16: 3188-3196Crossref PubMed Scopus (136) Google Scholar When Pkd1 is disrupted at 3–6 months, a mild cystic phenotype is obtained, but when Pkd1 is disrupted at 4 days of age, severe cyst formation occurs. This latter model may hold the greatest promise for studying cystogenesis in ADPKD; however, it is still not possible, because of the issues described above, to determine the collecting duct cell type of origin of the cysts. To address this, this study employed a Cre/loxP strategy to selectively mutatePkd1 in PC and IC. We report a marked difference in the severity of cystic kidney disease in these two new mouse models of ADPKD. A total of 81 out of 389 pups (20.8%) were PC-Pkd1 KO (homozygous for the Pkd1cond allele and heterozygous for the AQP2-Cre transgene). Here, 105 animals (27.0%) were homozygous for the Pkd1cond allele, but did not have the AQP2-Cre transgene. Ninety-four animals (24.2%) were heterozygous for the Pkd1cond allele and contained the AQP2-Cre transgene. However, 109 animals (28.0%) were heterozygous for the Pkd1cond allele, but did not have the AQP2-Cre transgene. These genotype frequencies were not significantly different (P=0.18). A total of 57 PC-Pkd1 KO animals were kept with littermates until natural death. The mean survival of the PC-Pkd1 KO animals was 8.2 weeks (range 2.1–21.6 weeks, s.e.±0.7) (Figure 1). Five animals, each of the three other genotypes, were kept alive for comparison and all remained alive at 78 weeks (1.5 years). Thus, the heterozygous PC-Pkd1 KO mice had a normal life span. At birth, there were no macroscopic or microscopic renal cysts. Bilateral renal cysts were grossly apparent in all the PC-Pkd1 KO mice at week 1, with progressive cyst formation and enlargement through week 4 (Figure 2a–d and 3a). Total cysts per kidney exceeded 100 starting on week 1 (Figure 4). Cysts were most apparent in the cortex and outer medulla, whereas cysts were not observed in the papilla. Real-time PCR for polycystin-1 mRNA in the papillary collecting duct, from wild-type mice, showed this region contains 1.46 times the amount of polycystin-1 mRNA (normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) as compared with outer medulla. Hence, the absence of papillary cysts is not because of the failure of papillary CD to express polycystin-1 mRNA. No cysts were apparent macroscopically or microscopically in any control animals killed at any time point from week 1 through 39 weeks of age. No gross abnormalities were present in non-renal organs in the PC-Pkd1 KO mice.Figure 3Representative magnetic resonance imaging of kidneys obtained from (a) principal cell polycystin-1 knockout (PC)-Pkd1 KO and (b) intercalated cell polycystin-1 knockout (IC-Pkd1 KO). The magnetic resonance imaging is from PC-Pkd1 KO mice at 2 weeks of age and IC-Pkd1 KO mice at 13 weeks of age.View Large Image Figure ViewerDownload (PPT)Figure 4Number of cysts at varying ages in the principal cell polycystin-1 knockout (PC-Pkd1 KO) and the intercalated cell polycystin-1 knockout (IC-Pkd1 KO) mice. PC-Pkd1 KO bars are at maximal height––these kidneys had >100 cysts. N=10 kidneys each data point.View Large Image Figure ViewerDownload (PPT) Staining for Dolichos biflorus agglutinin confirmed that cysts are of the collecting duct origin (Figure 5a and b). AQP2 expression of cystic epithelium was variable––some cysts displayed circumferential staining, some with no staining, and others with partial staining (images not shown). The presence of renal cysts at week 1 and thereafter was associated with a higher kidney weight (Figure 6a). Body weight was less than control animals at 4 weeks and thereafter (Figure 6b). Renal insufficiency was evident as early as week 2: blood urea nitrogen (BUN) in PC-Pkd1 KO was 41.2 mg/dl (n=4, s.e.±8.4) versus 19.2 mg/dl (n=5, s.e.±1.2) in control animals (P=0.0224). Severe renal insufficiency was evident at 4 and 8 weeks of age (Figure 6c).Figure 6Kidney weight, body weight and BUN in principal (PC) and intercalated cell (IC) polycystin-1 (Pkd1) knockout mice. (a) Kidney weight of the principal cell polycystin-1 knockout (PC-Pkd1 KO) and the intercalated cell polycystin-1 knockout (IC-Pkd1 KO) expressed as mean and s.e. *P<0.0001 for the comparison of PC-Pkd1 KO and PC controls at weeks 1, 2, 4, and 8. ⁁P=0.0001 at 4 weeks and #P=0.0026 at 8 weeks for the comparison of PC-Pkd1 KO and IC-Pkd1 KO. Kidney weight comparison of IC-Pkd1 KO and IC controls was NS. (b) Body weight of PC-Pkd1 KO and IC-Pkd1 KO expressed as mean and s.e. *P<0.0001 for the comparison of PC-Pkd1 KO and PC controls at 4, 8, and 13 weeks. Body weight comparison was NS at D7 and D14. ⁁P=0.024 at 4 weeks and #P=0.0026 at 8 weeks for the comparison of PC-Pkd1 KO and IC-Pkd1 KO. Body weight comparison IC-Pkd1 KO and IC controls was NS. (c) Blood urea nitrogen (BUN) of PC-Pkd1 KO and IC-Pkd1 KO expressed as mean and s.e. *P<0.0001 for the comparisons of PC-Pkd1 KO versus PC controls and PC-Pkd1 KO versus IC-Pkd1 KO at 4 and 13 weeks. #P=0.0054 for the comparison of IC-Pkd1 KO and IC controls at 4 weeks. The difference between IC-Pkd1 KO and IC controls at 13 weeks was NS.View Large Image Figure ViewerDownload (PPT) In order to determine whether there were alterations in cyclic AMP (cAMP) levels or expression of the target protein, AQP2, total kidney cAMP content, and membrane-associated AQP2 protein levels were ascertained. There was no difference in cAMP content between the 4-week-old PC-Pkd1 KO mice (105.1±14.9 pmol cAMP/mg total cell protein, n=5) and controls (125.0±17.1 pmol cAMP/mg total cell protein, n=5). Similarly, there were no differences in glycosylated or unglycosylated AQP2 levels between the PC-Pkd1 KO and the control mice (Figure 7). A total of 26 out of 86 mice (30.2%) were IC-Pkd1 KO: homozygous for the Pkd1cond allele and contained the B1-Cre transgene. Seventeen animals (19.8%) were homozygous for the Pkd1cond allele, but did not have the B1-Cre transgene. Twenty animals (23.3%) were heterozygous for the Pkd1cond allele and contained the B1-Cre transgene. However, 23 animals (26.7%) were heterozygous for the Pkd1cond allele, but did not have the B1-Cre transgene. These genotype frequencies were not significantly different (P=0.55). In comparison with PC-Pkd1 KO, IC-Pkd1 KO animals had a very mild cystic phenotype (Figure 2e–g, 3 and 4). Two of the five IC-Pkd1 KO animals killed after 4 weeks had no cysts macroscopically or microscopically. One animal had one macroscopic cyst in one kidney. Another animal had two macroscopic cysts in one kidney and three in the other. The fifth had one macroscopic cyst in one kidney and an additional microscopic cyst in the contralateral kidney. All cysts at 4 weeks of age were limited to the cortex. At 8–9 weeks of age, one of the five IC-Pkd1 KO mice had no renal cysts. The remaining four had 1–2 macroscopically apparent cysts in each kidney. As in the 4-week-old animals, cysts were limited to the cortex. At 17 weeks, all the five animals had cysts in both kidneys (Figure 3 and 4). These averaged two cysts per kidney. Most cysts in animals at this age were relatively small (as compared with cysts in the PC-Pkd1 KO mice). ICs represent about 30% of cortical and outer medullary collecting duct cells (Figure 8); hence, the extremely low number of cysts in the IC-Pkd1 KO mice, as compared with the PC-Pkd1 KO mice, is not because of the number of ICs. Body weight and kidney weight were not different between IC-Pkd1 KO and littermate controls, and were not different than PC-Pkd1 controls (Figure 6a and b). BUN at 4 weeks was higher in IC-Pkd1 KO than in littermate controls, but not likely meaningful as the BUN at 17 weeks was similar. (Figure 6c). BUN in IC-Pkd1 KO was not different from the BUN in PC-Pkd1 controls (Figure 6c). No abnormalities were grossly apparent in any non-renal organs in the IC-Pkd1 KO animals, and no cysts were observed in any control animal through 39 weeks of age. None of the IC-Pkd1 KO animals died during 78 weeks (1.5 years) of follow-up (N=10). In this study, two new mouse models of ADPKD were created. The PC-Pkd1 KO mice were generated using the AQP2-Cre mice which express Cre selectively in renal PCs and in the male reproductive tract.21.Ahn D. Ge Y. Stricklett P.K. et al.Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention.J Clin Invest. 2004; 114: 504-511Crossref PubMed Scopus (217) Google Scholar, 22.Ge Y. Bagnall A. Stricklett P.K. et al.Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention.Am J Physiol Renal Physiol. 2006; 291: F1274-F1280Crossref PubMed Scopus (171) Google Scholar, 23.Ge Y. Stricklett P.K. Hughes A.K. et al.Collecting duct-specific knockout of the endothelin A receptor alters renal vasopressin responsiveness, but not sodium excretion or blood pressure.Am J Physiol Renal Physiol. 2005; 289: F692-F698Crossref PubMed Scopus (81) Google Scholar, 24.Nelson R. Stricklett P. Ausiello D. et al.Expression of a Cre recombinase transgene by the aquaporin-2 promoter in kidney and male reproductive system of transgenic mice.Am J Physiol. 1998; 275: C216-C226PubMed Google Scholar The PC-Pkd1 KO mice have a severe, yet viable, kidney-limited cystic phenotype. Animals develop cysts at 1 week of age, renal insufficiency at 2 weeks, and progressive kidney enlargement and cyst growth. These mice have an average life span of 8.2 weeks with a wide range in survival. Thus, PC polycystin-1 dysfunction can, in of itself, cause a profound renal cystic disease. The IC-Pkd1 KO model was generated using the B1-Cre mice which express Cre selectively in ICs within the kidney; these mice have low-level Cre expression in the brain, intestine, and uterus.25.Miller R. Lucero O. Riemondy K. V-ATPase B1-subunit promoter drives expression of Cre-Recombinase in intercalated cells of the kidney.Kidney Int. 2008Google Scholar The IC-Pkd1 KO mice had very few cysts with minimal to no impairment in renal function. Cysts in the IC-Pkd1 KO mice were confined largely to the cortex, and particularly near the capsule. This very mild phenotype and renal cyst localization is not because of an incomplete IC Cre expression; B1-Cre mice confer virtually 100% IC-specific target gene recombination throughout the cortex and outer medulla as evidenced by crossing B1-Cre mice with ROSA26-YFP (yellow fluorescent protein) reporter animals.25.Miller R. Lucero O. Riemondy K. V-ATPase B1-subunit promoter drives expression of Cre-Recombinase in intercalated cells of the kidney.Kidney Int. 2008Google Scholar Furthermore, we found that IC constitute 30% of the collecting duct cell types in the cortex and 28% in the outer medulla (another study found that IC make up 40% of the cortical and outer medullary collecting duct cells26.Teng-umnuay P. Verlander J.W. Yuan W. et al.Identification of distinct subpopulations of intercalated cells in the mouse collecting duct.J Am Soc Nephrol. 1996; 7: 260-274PubMed Google Scholar); hence, the low number of cysts and their absence in outer medulla is not because of the limited numbers of IC. As it is currently not possible to localize polycystin-1 protein or mRNA to specific collecting duct cell types, the reasons for the limited number of cysts and the failure to form outer medullary cysts are speculative. Mouse IC contain a primary cilium;11.Shibazaki S. Yu Z. Nishio S. et al.Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1.Hum Mol Genet. 2008; 17: 1505-1516Crossref PubMed Scopus (168) Google Scholar hence, failure to form cysts is not because of the lack of this structure. It may be that IC lacks expression of, and/or functionally important, polycystin-1. In this regard, it is possible that cysts in the IC-Pkd1 KO mice arise solely from the connecting segment. B1-Cre-mediated recombination (using the ROSA26-YFP reporter mice) occurs in the connecting segment cells that co-express AQP2;25.Miller R. Lucero O. Riemondy K. V-ATPase B1-subunit promoter drives expression of Cre-Recombinase in intercalated cells of the kidney.Kidney Int. 2008Google Scholar hence, it is conceivable that these ‘hybrid’ cells, which have features of both IC and PC, express the V-ATPase characteristic of ICs as well as the functionally important polycystin-1. Confirmation of this is problematic as there is no way to specifically target the connecting segment. Furthermore, segmental nephron markers are lost largely in cystic epithelium and do not identify accurately the cell type of origin.27.Jiang S.T. Chiou Y.Y. Wang E. et al.Defining a link with autosomal-dominant polycystic kidney disease in mice with congenitally low expression of Pkd1.Am J Pathol. 2006; 168: 205-220Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Thus, although the current data suggest that the collecting duct and the connecting segment cysts in ADPKD arise only from cells with PC characteristics, confirmation is required. The PC-Pkd1 KO mice do not express cysts in the renal papilla, yet mouse papillary collecting ducts possess a primary cilia11.Shibazaki S. Yu Z. Nishio S. et al.Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1.Hum Mol Genet. 2008; 17: 1505-1516Crossref PubMed Scopus (168) Google Scholar and polycystin-1 mRNA levels that exceed those in the outer medulla. Failure to express Cre recombinase in the papillary collecting duct is not the reason for cyst absence, as AQP2-Cre mice express active Cre in virtually every PC in this nephron segment.21.Ahn D. Ge Y. Stricklett P.K. et al.Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention.J Clin Invest. 2004; 114: 504-511Crossref PubMed Scopus (217) Google Scholar, 22.Ge Y. Bagnall A. Stricklett P.K. et al.Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention.Am J Physiol Renal Physiol. 2006; 291: F1274-F1280Crossref PubMed Scopus (171) Google Scholar, 23.Ge Y. Stricklett P.K. Hughes A.K. et al.Collecting duct-specific knockout of the endothelin A receptor alters renal vasopressin responsiveness, but not sodium excretion or blood pressure.Am J Physiol Renal Physiol. 2005; 289: F692-F698Crossref PubMed Scopus (81) Google Scholar, 24.Nelson R. Stricklett P. Ausiello D. et al.Expression of a Cre recombinase transgene by the aquaporin-2 promoter in kidney and male reproductive system of transgenic mice.Am J Physiol. 1998; 275: C216-C226PubMed Google Scholar, 28.Zharkikh L. Zhu X. Stricklett P.K. et al.Renal principal cell-specific expression of green fluorescent protein in transgenic mice.Am J Physiol Renal Physiol. 2002; 283: F1351-F1364Crossref PubMed Scopus (19) Google Scholar In contrast, in the Ksp-Cre Pkd1 model, tubule ectasia is apparent in the renal papilla.11.Shibazaki S. Yu Z. Nishio S. et al.Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1.Hum Mol Genet. 2008; 17: 1505-1516Crossref PubMed Scopus (168) Google Scholar The reason for the different papillary phenotypes is uncertain, but may relate to differences in the timing of Cre expression: Cre is active much earlier in renal development in the Ksp-Cre Pkd1 mice (E11.5) as compared with the PC-Pkd1 KO mice (E18.5). The mechanisms responsible for massive cyst formation in the PC-Pkd1 KO mice remain conjectural. Although cystogenesis is associated strongly with defective cilia, there is no evidence to suggest that deficiency of polycystin-1 in PCs leads to structural abnormalities in cilia. Studies in Pkd1 KO endothelial cells,29.Nauli S.M. Kawanabe Y. Kaminski J.J. et al.Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.Circulation. 2008; 117: 1161-1171Crossref PubMed Scopus (309) Google Scholar epithelial cells lining kidney cysts from patients with ADPKD30.Natoli T.A. Gareski T.C. Dackowski W.R. et al.Pkd1 and Nek8 mutations affect cell-cell adhesion and cilia in cysts formed in kidney organ cultures.Am J Physiol Renal Physiol. 2008; 294: F73-F83Crossref PubMed Scopus (36) Google Scholar, 31.Nauli S.M. Rossetti S. Kolb R.J. et al.Loss of polycystin-1 in human cyst-lining epithelia leads to ciliary dysfunction.J Am Soc Nephrol. 2006; 17: 1015-1025Crossref PubMed Scopus (135) Google Scholar, 32.Xu C. Rossetti S. Jiang L. et al.Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+ signaling.Am J Physiol Renal Physiol. 2007; 292: F930-F945Crossref PubMed Scopus (105) Google Scholar and in Caenorhabditis elegans with disruption of a Pkd1 homolog33.Barr M.M. Sternberg P.W. A polycystic kidney-disease gene homologue required for male mating behaviour in C elegans.Nature. 1999; 401: 386-389Crossref PubMed Scopus (392) Google Scholar all indicate that primary cilia are present and are grossly intact. More specifically, collecting duct cells from mice with Pkd1 disruption have grossly intact primary cilia.11.Shibazaki S. Yu Z. Nishio S. et al.Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1.Hum Mol Genet. 2008; 17: 1505-1516Crossref PubMed Scopus (168) Google Scholar, 34.Nauli S. Alenghat F. Luo Y. et al.Polycystins 1 and 2 mediate mechanosensation in the primar cilium of kidney cells.Nat Genet. 2003; 33: 129-137Crossref PubMed Scopus (1514) Google Scholar In addition, these studies found no evidence that AQP2 levels or cellular cAMP content were altered in the PC Pkd1 KO mice. These observations are of interest in that vasopressin can directly regulate cyst growth in a model of ADPKD,35.Wang X. Wu Y. Ward C.J. et al.Vasopressin directly regulates cyst growth in polycystic kidney disease.J Am Soc Nephrol. 2008; 19: 102-108Crossref PubMed Scopus (195) Google Scholar cAMP stimulates human PKD epithelial cell growth and fluid secretion,36.Belibi F.A. Reif G. Wallace D.P. et al.Cyclic AMP promotes growth and secretion in human polycystic kidney epithelial cells.Kidney Int. 200
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