Histopathological analysis of renal cystic epithelia in the Pkd2WS25/- mouse mod - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:48 |只看该作者 |倒序浏览 |打印

作者：R. Brent Thomson, SueAnn Mentone, Robert Kim, Karen Earle, Eric Delpire, Stefan Somlo,  Peter S. Aronson作者单位：1 Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8029; and Department of Anesthesiology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232 , C! Y4 P$ \# D7 [# B


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      【摘要】
   It has been proposed that autosomal dominant polycystic kidney disease (ADPKD)affected renal epithelial cells undergo a phenotypic transition from a highly differentiated absorptive state to a much less differentiated secretory state during cystogenesis and that this transition is accompanied by loss of epithelial cell polarity and mistargeting of specific membrane proteins. We conducted a detailed evaluation of this hypothesis in the Pkd2 WS25/ - mouse model of ADPKD. Ultrastructural analysis of Pkd2 WS25/ - cysts by electron microscopy confirmed that cystic epithelial cells progressively dedifferentiate with cyst enlargement. Immunocytochemical analysis of both early- and late-stage cysts with antibodies directed against Na -K -ATPase, Ksp-cadherin, and E-cadherin failed to detect evidence of altered cyst cell polarity. Na -K -ATPase and Ksp-cadherin were expressed exclusively on the basolateral membranes (BLM) of epithelial cells in all early cysts. Expression levels of both Na -K -ATPase and Ksp-cadherin decreased progressively with the degree of cyst cell dedifferentiation, but neither protein was ever mislocalized. Highly dedifferentiated cysts did not express immunodetectable levels of either Na -K -ATPase or Ksp-cadherin. E-cadherin was expressed prominently on the BLM of all cysts. Cysts were subsequently stained with an antibody directed against the secretory isoform of the Na -K -Cl - cotransporter NKCC1. NKCC1 expression was detected on the BLM of advanced cysts only. Our data are consistent with a model of progressive cystic epithelial cell dedifferentiation in which fluid accumulation in late-stage cysts is mediated by transepithelial secretion of chloride rather than secretion of sodium by apical Na -K -ATPase. $ W1 ?  V4 U$ {+ Q: {/ H5 L
      【关键词】 polarity Na K ATPase NKCC Kspcadherin Ecadherin
   THE PHYSIOLOGICAL MECHANISMS involved in renal cyst formation in patients affected by autosomal dominant polycystic kidney disease (ADPKD) are still largely unknown. Intensive genetic analyses of families affected by ADPKD have revealed that the majority of reported cases are directly attributable to mutations in the genes that encode either polycystin-1 or polycystin-2. The functions of polycystin-1 and polycystin-2 are not yet completely understood, but it is generally believed that their functions are related and that a disruption in the function of either protein initiates a common cystogenic pathway (for a review, see Ref. 18 ). In the currently accepted model of cystogenesis, single progenitor cells are believed to enter a dedifferentiating hyperplastic pathway that ultimately gives rise to the expansive fluid-filled cysts that are the hallmark of ADPKD. Implicit in this model is the notion that the cystic epithelium undergoes a phenotypic transition from a highly differentiated absorptive state to a much less differentiated secretory state.
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It has been suggested that alterations in epithelial cell polarity due to mistargeting of specific membrane proteins may play a significant role in this transition. Charron et al. ( 8 ) reported that cultured human ADPKD cyst cells have a disrupted cytoarchitecture and coupled with mislocalization of E-cadherin may have a generalized basolateral sorting defect. Wilson et al. ( 34, 35 ) proposed that the mistargeting of a fully functional Na -K -ATPase to the apical membrane of cystic epithelial cells could result in net basal-to-apical Na flux, which in turn could drive luminal fluid accumulation and cyst expansion. Although apical mislocalization of the Na -K -ATPase has been reported in several different models of polycystic kidney disease (for example, see Refs. 1 and 26 ), it has not been universally accepted as the principal mechanism underlying either fluid secretion or cyst expansion in ADPKD.. n* `2 A* E" X

Grantham et al. ( 13 ) proposed that cyst luminal fluid accumulation may be driven by net basal-to-apical Cl - movements in a manner similar to that observed in classical secretory epithelia such as sweat duct or airway (for a review, see Ref. 30 ) rather than by apical secretion of Na  . In support of this hypothesis, Brill et al. ( 6 ) reported that they could find no evidence of mislocalization of Na -K -ATPase in either excised human ADPKD cysts or human ADPKD cyst cells in culture. Moreover, studies by Hanaoka et al. ( 14 ) and Brill et al. ( 6 ) indicated that apical Cl - exit could be facilitated by the cystic fibrosis transmembrane regulator (CFTR) protein, and inhibitor studies by Mangoo-Karim et al. ( 23 ) suggested that the basolateral entry step may be mediated by the Na -K -2Cl - cotransporter NKCC1.
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To address this controversy and to determine if altered cell polarity plays a role in cyst development in ADPKD, we conducted an extensive ultrastructural and immunocytochemical characterization of renal cysts in the Pkd2 WS25/ - adult mouse model of ADPKD. Cyst initiation in this model is due to a somatic inactivation of the second Pkd2 allele, and cystogenesis has been shown to proceed in a manner similar to that reported for human ADPKD ( 36, 37 ). Importantly, the use of an animal model permitted us to perform rapid in situ perfusion fixation of the kidneys, eliminating the potential for ischemia-induced mistargeting of Na -K -ATPase (see Refs. 7 and 24 ) and affording optimal preservation of both cellular ultrastructure and protein localization. Ultrastructural analysis by both transmission and scanning electron microscopy confirmed that cyst epithelial cells undergo a dramatic dedifferentiation as cystogenesis proceeds. Immunocytochemical analysis of both early- and late-stage cysts indicates that neither altered cell polarity nor mislocalization of Na -K -ATPase appears to play a role in cyst expansion and fluid secretion in ADPKD. Consistent with the model of progressive cystic epithelial cell dedifferentiation in which fluid secretion is mediated by transepithelial Cl - movements rather than apical secretion of Na  , we detected expression of the Na -K -2Cl - cotransporter NKCC1 on the basolateral membranes of advanced cyst epithelial cells.

METHODS

Animals and reagents. Pkd2 WS25/ - animals were generated by crossing Pkd2 / - and Pkd2 WS25/ mice as described by Wu et al. ( 36 ). To facilitate tissue preparation for microscopy, all animals used in this study were 1 to 2 mo of age. Kidneys from animals of this age contained large numbers of cysts in all stages of development but retained enough noncystic tissue to permit adequate perfusion fixation of both the cysts and the unaffected renal parenchyma.  E0 Q" [0 J; a# G% u5 k+ j
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Two different mouse monoclonal antibodies directed against the 1 -subunit of Na -K -ATPase were used in this study: antibody C464.6 (Upstate Biotechnology, Lake Placid, NY) was used at a dilution of 1:50 and antibody Alpha-5 (Developmental Studies Hybridoma Bank, Iowa City, IA; Ref. 22 ) was used at a dilution of 1:100. The anti-E-cadherin mouse monoclonal antibody Clone 36 (Transduction Laboratories, Lexington, KY) was used at a dilution of 1:100.) @9 G+ |- h8 |# v
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The anti-Ksp-cadherin antibody is an affinity-purified rabbit polyclonal antibody that we generated against the human isoform of Ksp-cadherin. Rabbits were immunized with a maltose-binding protein (MBP; New England Biolabs, Beverly, MA) fusion protein containing the COOH-terminal 267 amino acids (KspFP563-829) of the human isoform of Ksp-cadherin. Sera was negatively purified against bacterial lysates containing only the MBP and then positively purified on a column containing the MBP-KspFP563-829 fusion protein. Specific reactivity was confirmed by Western blot analysis of Cos-7 cells transiently transfected with full-length rabbit Ksp-cadherin. Specificity was further verified by Western blot analysis of human, mouse, rabbit, and dog kidney microsomes and immunocytochemical analysis of human and mouse kidney cryosections. The purified serum was used for immunocytochemistry in the present study at a dilution of 1:100.

The anti-NKCC1 antibody ( 19 ) is an affinity-purified rabbit polyclonal antibody directed against the mouse isoform of NKCC1 and was used at a dilution of 1:200. A rabbit antimouse Tamm-Horsfall antibody (THP; a generous gift of Dr. J. Hoyer) was used at a dilution of 1:200 for identification of cysts of cortical thick ascending limb origin ( 16 ). A rabbit anti-thiazide-sensitive Na-Cl cotransporter (NCC, TSC) antibody (a generous gift of Dr. S. Hebert) was used at a dilution of 1:200 for identification of cysts of distal convoluted tubule origin ( 28 ). The fluorescein-conjugated lectin Lotus tetragonolobus (Vector Laboratories, Burlingame, CA) was used at a dilution of 1:10 for identification of cysts of proximal tubule origin ( 20 ). Rhodamine-conjugated Dolichos biflorus agglutinin (Vector Laboratories) was used at a dilution of 1:100 for identification of cysts of collecting duct origin ( 20 ). Alexa Fluor anti-mouse IgG (488 and 594) and anti-rabbit IgG (488) secondary antibodies were used at a dilution of 1:200 (Molecular Probes, Eugene, OR).6 K3 n; P* y3 b+ V# ~  a' N5 ~# Q! u
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Electron microscopy. Mice were anesthetized by intraperitoneal injection of pentobarbital sodium. The kidneys were cleared (PBS) and fixed (3% paraformaldehyde/3% glutaraldehyde) by cardiac perfusion. The kidneys were then removed, cut into 2- to 4-mm-thick sagittal sections, and postfixed in the same fixative for 1 h at 4°C. Tissue destined for transmission electron microscopy (TEM) was further cut into 2- to 4-mm blocks for subsequent processing. Cortical tissue only was used for the TEM study. The tissue sections were washed in 0.1 M cocodylate buffer containing 7.5% sucrose (pH 7.4, 3 changes; 1 h; 4°C) and then incubated in veronal acetate-buffered 1% osmium tetroxide for 2 h at 4°C (1 h for TEM sections). Tissue used for scanning electron microscopy (SEM) was again washed in cocodylate buffer (3 changes; 1 h; 4°C), dehydrated through a graded ethanol series, critical point dried, mounted on stubs, sputter coated with PdAu, and then visualized on an International Scientific Instruments SS-40 scanning electron microscope. Tissue used for TEM was washed and incubated in a Kellenberger veronalacetate buffer containing 0.5% uranyl acetate for 2 h at room temperature, rinsed in H 2 O, dehydrated through a graded ethanol series, and embedded in Epon 812. Ultrathin 80-nm sections were cut on a Reichert Ultracut E ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM910 electron microscope.
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Immunocytochemistry. Kidneys from six nonsibling mice were perfused and cleared in the same manner as above, except that PLP (2% paraformaldehyde, 750 mM lysine, and 10 mM sodium periodate in a phosphate buffer, pH 7.4) was used as the fixative. The cortices were separated from the kidneys, cut into 2- to 4-mm blocks, and postfixed in the same fixative for an additional 4 h. To maintain the structural integrity of the fragile cyst walls, all tissue blocks were embedded in Epon 812 as above except that the tissue blocks were not exposed to osmium tetroxide or uranyl acetate. One-micron sections were cut on the Reichert Ultracut E ultramicrotome and mounted on Superfrost Plus glass slides (Electron Microscopy Sciences, Fort Washington, PA). Sections were etched for 5 min in a solution containing 2 g KOH, 10 ml 100% methanol, and 5 ml propylene oxide, washed for 5 min in 100% methanol (2 changes), rinsed in TBS (50 mM Tris · HCl, 100 mM NaCl, pH 7.4), and then subjected to antigen retrieval as outlined by Biemesderfer et al. ( 5 ). Briefly, sections were immersed in a boiling 10 mM sodium citrate buffer (pH 6) and then heated for an additional 20 min at the 40% power setting of a 700-W microwave oven, allowed to cool, washed in TBS, quenched in 0.5 M ammonium chloride with 0.1% BSA for 15 min, rinsed in TBS, incubated in 1% SDS in TBS for 5 min, and finally washed in TBS. Sections to be stained with mouse primary antibodies were incubated with unconjugated anti-mouse IgG diluted 1:5 in TBS/0.1% BSA/10% goat serum for 1 h at room temperature. All other sections were blocked with 0.1% BSA and 10% goat serum in TBS for 1 h at room temperature. All sections were washed with TBS and then incubated with the respective primary antibodies diluted in TBS/0.1% BSA/10% goat serum overnight at 4°C. The next morning sections were washed in high-salt TBS (527 mM NaCl) with 0.1% BSA ( x 5; 5 min each wash) and then incubated with the appropriate fluoro-chrome-labeled secondary antibody diluted in TBS/0.1% BSA/10% goat serum for 1 h at room temperature. Sections were washed for 5 min in high-salt TBS, 20 min in standard TBS (4 changes), mounted in VectaShield (Vector Laboratories), and then visualized on a Zeiss Axiophot phase-contrast microscope. Antibodies and lectins used for double-labeling experiments were tested in control pilot studies to verify lack of cross-reactivity between reagents. Sequential serial sections were labeled with specific cortical tubule segment markers to determine cyst tubule segments of origin.

Cyst staging. The criteria used to developmentally stage cysts were based on cyst size, preliminary TEM ultrastructural surveys of PKD2 WS25/ - ADPKD cysts, and recommendations by Devuyst et al. ( 10 ). Generally, cysts were considered early stage if on cross section there were less than 50 cyst-lining epithelial cells, intermediate stage if there were 51-200 cyst-lining epithelial cells, and advanced stage if there were greater than 200 cyst-lining epithelial cells. The relative position of a section within a cyst was determined by examination of adjacent serial sections spanning 40 µm in each direction.
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Ultrastructural analysis of Pkd2 WS25/ - ADPKD cysts. In small cysts that are presumably in the early stages of cyst expansion, the ultrastructure of the cyst cells is highly reminiscent of unaffected tubular epithelial cells ( Fig. 1 A ). The cells are cuboidal to columnar and have approximately normal numbers of mitochondria, well-developed lateral junctions, well-organized basement membranes, and prominent cilia. None of the early cyst cells that we examined retained sufficient "normal" ultrastructural details to unequivocally assess the tubule segment of origin. As cyst expansion proceeds, the cells take on a more squamoid appearance ( Fig. 1 B ) and lose all resemblance to unaffected tubular epithelial cells. The mitochondria become much less prominent, but the cells continue to retain well-defined lateral junctions and a well-organized basement membrane. Significant deposits of collagen begin to appear in the region just below the basement membrane. In very large cysts that presumably represent the final stages of cyst expansion, the cyst cells are highly attenuated and acquire a pronounced endothelial-like appearance ( Fig. 1 C ). Mitochondria are extremely rare, but again the cells continue to retain well-defined lateral junctions ( Fig. 1 C, inset ). The basement membrane loses much of its organization and becomes intimately associated with large deposits of striated collagen. We did not observe thickening or lamination of the basement membrane per se at any stage of cyst progression.

Fig. 1. Representative transmission electron micrographs of early- through late-stage Pkd2 WS25/ - autosomal dominant polycystic kidney disease (ADPKD) cysts. A : early cyst (magnification x 8,595). B : late intermediate cyst (magnification x 6,440). C : extremely advanced cyst (magnification x 14,291). C, inset : lateral junction of cystic epithelial cells in extremely advanced cyst (magnification x 54,351). A total of 48 cysts were analyzed by transmission electron microscopy; 29 were considered to be early stage, 13 were considered to be intermediate stage, and 6 were considered to be advanced. Arrows indicate basement membranes; * indicate collagen deposits.8 S& }1 q! N3 r) W1 @
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SEM of the lumens of similarly staged ADPKD cysts provides further support for the cellular dedifferentiation hypothesis. In the initial stages of cyst growth, the cyst epithelial cells appear to be highly proliferative and form elaborate chords, ridges, and labyrinths within the cyst lumens ( Fig. 2 A ). Early cyst cells maintain regular well-defined lateral borders and have prominent cilia ( Fig. 2 B ). The degree of secondary cell-surface ultrastructure (e.g., microvilli) varies from cyst to cyst and presumably reflects the cyst tubule segment of origin. As cyst size increases, the cell borders become less regular and the cilia less prominent ( Fig. 2 C ). In many late-intermediate and early-advanced stage cysts, we observed more than one cell phenotype (for example, see Fig. 2 C arrow). These atypical cyst cells were not present in large numbers (1-5% of the total cyst cell population) and appeared to be randomly interspersed throughout the cyst walls. It is unclear if these cells are differentially staged progeny of the original cyst progenitor cell or if they are derived from outlying cell populations. Cellular heterogeneity was never observed in extremely advanced cysts ( Fig. 2 D ). The cells in advanced cysts appeared to be much larger than those seen in early cysts, and they have irregular cell borders and inconspicuous cilia. The cyst walls are very uniform and show little evidence of the cellular proliferation that was evident in the earlier stages of cyst growth.# z' Q4 k  ]6 Q6 y8 @

Fig. 2. Representative scanning electron micrographs of Pkd2 WS25/ - ADPKD cysts. A : luminal surface of a comparatively small 123-µm-diameter advanced stage cyst illustrating the extent to which cyst cells are capable of proliferating (magnification x 544). B : high magnification of an early cyst cell. Note the regular cell borders and the prominent cilia (magnification x 2,367). C : luminal surface of a late intermediate cyst. The arrow indicates an atypical cyst cell of the type frequently observed in cysts of this size class (magnification x 1,878). D : luminal surface of an extremely advanced 3.2-mm-diameter cyst (magnification x 1,801). Total number of cysts analyzed = 64.2 ?- Z9 z; ?/ Q

Immunocytochemical analysis of PKD2 WS25/ - ADPKD cysts. To determine the localization of Na -K -ATPase in PKD2 WS25/ - cysts and to facilitate comparison with previously published reports, both Alpha-5 and C464.6 anti-Na -K -ATPase antibodies were used in this study. With the use of antibody Alpha-5, Wilson et al. ( 34 ) reported that the 1 -subunit of Na -K -ATPase was localized to the apical membrane of ADPKD cyst cells in immersion-fixed human nephrectomy samples and human ADPKD cyst cells in culture. With the use of antibody 6H (clone C464.6), Brill et al. ( 6 ) reported that the 1 -subunit of Na -K -ATPase was localized to the basolateral membrane of immersion-fixed excised human ADPKD cysts and human ADPKD cyst cells in culture. Under the conditions used in our study, the staining patterns for the two antibodies were identical ( Fig. 3 ). They each labeled the basolateral membranes of both normal and cystic epithelia. Sixty cysts from six mice were positively labeled with Na -K -ATPase antibodies (see Table 1 ) and in no instance was apical localization ever detected.

Fig. 3. Immunolocalization of Na -K -ATPase in Pkd2 WS25/ - ADPKD cystic epithelia. A : overview of antibody C464.6 staining depicting basolateral labeling in both normal and cystic epithelia. Cysts 1 and 2 were Dolichos biflorus agglutinin (DBA) positive indicating collecting duct origin, and cyst 3 was NCC (TSC) positive indicating a distal convoluted tubule origin (magnification x 98). B : higher magnification of cyst 3 labeled with antibody C464.6 (magnification x 258). C : basolateral immunostaining of both normal and cystic epithelia with anti-Na -K -ATPase antibody Alpha-5. The cyst was positively labeled with the anti-Tamm-Horsfall antibody (THP) indicating a thick ascending limb origin (magnification x 707).+ t; b& X8 j' i* K8 v, C. R, Q$ j
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Table 1. Nephron segment of origin of Na - -K -ATPase-, Ksp-cadherin-, E-cadherin-, and NKCC1-positive cysts
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In addition to assessing cyst Na -K -ATPase distribution, we also performed double-labeling experiments with an antibody directed against Ksp-cadherin. Ksp-cadherin is a kidney-specific member of the cadherin superfamily of cell adhesion molecules and is expressed exclusively on the basolateral membrane of all tubular segments of the mammalian kidney ( 33 ). If cystic epithelial cells truly have a generalized basolateral sorting defect, one would predict that other basolateral proteins, such as Ksp-cadherin, would also be missorted. This proved not to be the case. As with Na -K -ATPase, apical localization of Ksp-cadherin in ADPKD cysts was never detected (see Fig. 4 and Table 1 ).
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Fig. 4. Immunolocalization of Ksp-cadherin in Pkd2 WS25/ - ADPKD cystic epithelia. A : overview of basolateral Ksp-cadherin staining in both normal and cystic epithelia. The immunolabeled section is the anti-Ksp-cadherin portion of a double-labeling experiment done with the anti-Na -K -ATPase antibody C464.6. The anti-Na -K -ATPase portion of the experiment was presented in Fig. 3, A and B (magnification x 98). B : high magnification of the cyst labeled with * in A (magnification x 258).

Given our inability to detect evidence of a generalized basolateral sorting defect or of altered cyst cell polarity, we suspected that E-cadherin may actually not be mislocalized in native ADPKD cyst cells. With the use of the anti-E-cadherin antibody Clone 36, Charron et al. ( 8 ) reported that E-cadherin was not expressed on the lateral membranes of ADPKD cells in culture but rather that it was sequestered in perinuclear vesicular structures. With the use of the same antibody in the Pkd2 WS25/ - ADPKD native cysts, we found no evidence of cystic epithelial intracellular sequestration of E-cadherin. Rather, we observed prominent E-cadherin staining on the lateral membranes of all cystic epithelial cells (see Fig. 5 and Table 1 ).% Q0 e  n% H$ A3 B

Fig. 5. Immunolocalization of E-cadherin in Pkd2 WS25/ - ADPKD cystic epithelia. The anti-E-cadherin antibody clone 36 clearly labels the basolateral membranes of cystic epithelia. The cyst was DBA positive, indicating a collecting tubule origin (magnification x 354).# I" t2 D5 v, X" h2 d$ G/ ?

Although neither Na -K -ATPase nor Ksp-cadherin was ever detected on the apical membrane of Pkd2 WS25/ - native cysts, we did observe a progressive decrease in the level of expression of both proteins that appeared to be concomitant with the extent of cyst expansion and the degree of cyst cell dedifferentiation from a cuboidal to a squamoid phenotype. Na -K -ATPase immunostaining was never observed in cysts with either a highly attenuated squamoid epithelium or extensive surrounding fibrotic tissue ( Fig. 6 A ). Ksp-cadherin immunostaining was also typically absent in advanced cysts but was occasionally detected in squamoid epithelial cysts that were not surrounded by extensive fibrotic tissue. In Fig. 6 B, for example, both cysts have a highly attenuated squamoid epithelium, but only the upper cyst was totally surrounded by fibrotic tissue. In the region below the lower cyst (not included in the field shown in Fig. 6 B ), normal appearing tubules were in direct apposition with the cyst wall and there was no evidence of surrounding fibrotic tissue. We interpret this to indicate that the lower cyst is less advanced than the upper cyst. The decrease in Na -K -ATPase and Ksp-cadherin expression levels could not be correlated with the cyst tubule segment of origin because advanced cysts also tended to lose expression of the tubule origin markers used in this study (see Table 1 ). In the field shown in Fig. 6, A and B, the lower cyst was THP positive, whereas the upper cyst was negative for all tubule segment markers. This observation is consistent with the notion that the upper cyst is more highly advanced and dedifferentiated than the lower cyst. E-cadherin immunostaining, on the other hand, was detected at high levels on the lateral membranes of all cysts regardless of the degree of cyst expansion or dedifferentiation. In Fig. 6 C, for example, the anti-E-cadherin antibody is shown labeling cyst epithelial cells that were negative for Na -K -ATPase, Ksp-cadherin, and all tubule segment markers.( C$ _/ m5 n% v' B& ~

Fig. 6. Immunolocalization of Na -K -ATPase, Ksp-cadherin, and E-cadherin in advanced Pkd2 WS25/ - ADPKD cysts. A : tissue section was labeled with the anti-Na -K -ATPase antibody C464.6. Positive immunostaining was not detected in either the highly dedifferentiated cystic epithelium or the surrounding fibrotic tissue. Areas of the section outside the field showed strong basolateral staining in both unaffected tubule segments and early cysts. The lower cyst was THP positive, indicating a thick ascending limb origin, but the upper cyst was not positively labeled by any of the tubule segment markers used in this study (magnification x 327). B : double labeling of the same tissue section depicted in A with the anti-Ksp-cadherin antibody. Note the prominent basal immunostaining in the lower THP-positive cyst (magnification x 327). C : prominent lateral E-cadherin immunostaining of cystic epithelial cells that were not positively labeled by anti-Na -K -ATPase or anti-Ksp-cadherin antibodies or tubule segment-specific markers (magnification x 211).$ L& D2 a; `! `1 w

If Na -K -ATPase is not mislocalized to the apical membrane of ADPKD cysts, primary active luminal Na extrusion is unlikely to account for cyst fluid secretion. Sullivan et al. ( 32 ) suggest that ADPKD cyst fluid accumulation is not driven by apical sodium transport but rather by net basal-to-apical chloride flux. They propose that the cystic epithelium undergoes a transition to a secretory phenotype similar to that observed in airway epithelia and that the transepithelial chloride flux is mediated by the basolateral bumetanide-sensitive Na -K -2Cl - cotransporter NKCC1 and the apical chloride channel CFTR. Hanaoka et al. ( 14 ) and Brill et al. ( 6 ) provided compelling evidence for CFTR involvement in the apical exit step, but to date there has not been a clear demonstration of NKCC1 localization on the basolateral membranes of native ADPKD cyst cells.
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In normal kidneys, NKCC1 expression is confined to the basolateral membrane of the inner medullary collecting duct, the glomerular and extraglomerular mesangium, the glomerular afferent arterioles, and the papillary epithelium ( 19 ). Consistent with these findings, NKCC1 expression in the noncystic regions of the cortices of Pkd2 WS25/ - ADPKD cystic kidneys was restricted to structures associated with glomeruli ( Fig. 7 A ). The vast majority of the cortical cysts that we observed did not express immunodetectable levels of NKCC1 (see Table 1 ). In particular, anti-NKCC1 antibody labeling was never observed in cyst cells that expressed immunodetectable levels of Na -K -ATPase. For example, the tissue section illustrated in Fig. 7 A depicting NKCC1-negative cysts is a serial section subsequent to that labeled with the anti-Na -K -ATPase antibody shown in Fig. 3 A. Moderate levels of NKCC1 expression were detected on the basolateral membrane of squamoid cell cysts whose epithelia had typically dedifferentiated to the point of no longer expressing detectable levels of the tubule segment-specific markers (see Fig. 7, B and C, and Table 1 ). NKCC1 expression was occasionally detected in squamoid cell cysts that were positive for segment-specific marker labeling, but in the two observed cases the segment-specific marker labeling was weak and inconsistent. Coexpression of NKCC1 with Ksp-cadherin was rare and occurred in less than 10% of the cysts that expressed NKCC1.

Fig. 7. Immunolocalization of NKCC1 in Pkd2 WS25/ - ADPKD cysts. A : immunolabeling of the cystic renal cortex with the anti-NKCC1 antibody. Note the positive staining in the glomerular-associated structures indicated by arrowheads and the lack of staining in the early cysts indicated by *. See Figs. 3 A and 4 A for sequential serial-section labeling with anti-Na -K -ATPase and anti-Ksp-cadherin antibodies (magnification x 92). B : anti-NKCC1 immunolabeling of an advanced cyst that does not express detectable levels of Na -K -ATPase, Ksp-cadherin, or tubule segment-specific markers. Arrowhead indicates glomerular mesangial staining (magnification x 278). C : heterogeneous anti-NKCC1 cyst staining. Cyst 1 is a DBA/Ksp-cadherin-positive, Na -K -ATPase-negative cyst (data not shown) that stains moderately with the anti-NKCC1 antibody. Cyst 2 is a NKCC1-negative glomerular cyst that is also negative for all tubule segment markers, Na -K -ATPase and Ksp-cadherin. Note the lack of anti-NKCC1 mesangial staining in the glomerular rudiment indicated by *. Cyst 3 expressed high levels of basolateral NKCC1 but was negative for all tubule segment markers, Na -K -ATPase and Ksp-cadherin (magnification x 740).9 z6 [) j) |% H3 ]: D3 A0 i

DISCUSSION
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The ultrastructural morphology of the mouse Pkd2 WS25/ - ADPKD cysts is virtually indistinguishable from that of human ADPKD cysts (for example, see Refs. 7, 9, 11, 12 ). As reported for human ADPKD cystic kidneys, we observed two predominant cyst types: those with highly differentiated cuboidal or columnar epithelial cells and those with attenuated dedifferentiated squamoid epithelial cells. Our data suggest that the two cyst cell populations are related and that they represent different stages of cyst development. Small early cysts were predominantly lined by cuboidal or columnar epithelial cells, and large advanced cysts were predominantly lined by dedifferentiated squamoid epithelial cells. Intermediate cysts had less clearly demarcated cell types and frequently appeared to have both cuboidal and squamoid cells in the same cyst. Carone et al. ( 7 ) observed a similar cellular phenotypic heterogeneity in a subpopulation of human ADPKD cysts. The cyst cell phenotype appears to directly reflect the degree of cyst progression. In the early stages of cystogenesis, the cyst cells are well differentiated and resemble normal tubular epithelial cells. The tissue surrounding the cyst at this stage shows little or no evidence of fibrosis or abnormal collagen deposition. As cyst expansion proceeds, the cyst cells gradually dedifferentiate to the attenuated squamoid phenotype seen in advanced macrocysts. The surrounding tissue becomes extremely fibrotic, and large deposits of collagen become intimately associated with the highly disorganized cyst basement membrane.
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Early studies of human ADPKD cystic kidneys also identified two cyst types, but it was largely believed that the two cyst types were unrelated and that the phenotypic differences reflected the respective cyst tubule segments of origin (for a review, see Ref. 32 ). Cysts lined by dedifferentiated squamoid epithelial cells were reported to contain a fluid with a high-sodium content that was similar to that observed in serum and proximal nephron segments. These cysts were therefore believed to be derived from proximal tubules and were referred to as "proximal" or "nongradient" cysts. Cysts lined with cuboidal or columnar cells had sodium concentrations that were low relative to serum levels and very similar to that observed in distal nephron segments. These cysts were considered to be derived from the distal nephron and were referred to as "distal" or "gradient" cysts. The cyst tubule segment of origin was determined solely on the basis of luminal sodium concentrations and an ability to maintain a transepithelial sodium gradient. The identification of the tubule segment of origin was rarely corroborated by cyst cell ultrastructure because the vast majority of the sampled cysts had typically dedifferentiated to the point of no longer expressing recognizable segment-specific characteristics (for example, see Ref. 12 ). The ultrastructure of the "distal" cyst cells was reminiscent of cells observed in distal segments of unaffected nephrons, but the "proximal" cyst cells had no resemblance to normal proximal tubule cells. Statistical analyses of human ADPKD cyst fluid compositions suggested that roughly 67% of the sampled cysts were of proximal origin and that the remaining 33% were of distal origin ( 11, 17 ). The ratio of "proximal" cysts to "distal" cysts closely reflected the expected ratio of proximal nephron mass to distal nephron mass and was therefore taken as additional supporting evidence that the two cyst types reflected the nephron segment of origin. This conclusion implies that cystogenesis is a totally random event without a clear segment-specific predilection for the site of cyst formation.$ m# W5 @, h( f* \0 u# n. N4 P0 I
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Our data (see Table 1 ) and that of Wu et al. ( 36 ) argue strongly against a simple stochastic model for the site of cyst formation in ADPKD-affected kidneys. Seventy to eighty percent of the cysts that were observed in the Pkd2 WS25/ - mouse renal cortex could be unequivocally identified as being of either distal tubule or collecting duct origin. It is possible that this correlation is specific to the Pkd2 WS25/ - model, but recent immunocytochemical studies on human ADPKD cystic kidneys suggest that this is not the case. In two studies examining immunolocalization of aquaporin-1 ( 10 ) and of gp330 ( 2 ), only 30-44% of over 1,000 cysts were considered to be of proximal origin. Similar studies of aquaporin-2 immunolocalization ( 10, 15 ) suggested that 30-33% of the cysts were of collecting duct origin. Presumably the bulk of the remaining 33% of the cysts is derived from other distal segments arguing that 66 and not 33% of the cysts are of distal origin, and 33 and not 67% of the cysts are of proximal origin. If this is true, the cyst fluid sodium concentration and hence the cyst cell ultrastructure would be unlikely to correlate with the cyst segment of origin in the studies of human ADPKD kidneys described above." x5 w2 |8 L7 A. L
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Similar to Grantham et al. ( 12 ), we observed cellular phenotypic heterogeneity in a significant subpopulation of ADPKD cysts. The source of the secondary cyst cell types is unclear. Data presented by Grantham et al. ( 12 ) suggest that the cysts in question in their study had a collecting tubule origin and that the secondary cells were most likely intercalated cells. The same may be true to some extent in our study, but the ultrastructural evidence for a collecting tubule cyst origin is less compelling. Regardless of the cyst tubule segment of origin, it is difficult to reconcile the cyst cellular heterogeneity with the hypothesis of Qian et al. ( 29 ) for cyst cell monoclonality. With the use of a PCR-based cell clonality assay with human ADPKD cysts, Qian et al. demonstrated that at least 82% of the cysts (and quite probably more) were derived from single cyst progenitor cells. If only collecting tubule-derived cysts showed cellular heterogeneity, one could postulate that both the intercalated- and principal-like cells were derived from a single cyst progenitor cell. The exact origin of intercalated cells in vivo is unclear, but it appears that both they and principal cells may be derived from a common ureteric bud cell lineage during nephrogenesis ( 31 ). Perhaps ADPKD-induced cellular dedifferentiation places collecting tubule-derived cyst progenitor cells into a limited pleuripotent state that allows the subsequent transition to either an intercalated- or principal-like cyst cell. A greater difficulty comes with cysts that do not appear to be of collecting duct origin. In these instances, one could postulate a polyclonal cyst origin or the recruitment of cyst cells from an outlying cell population. The strongest argument against either of those possibilities in our study is that the secondary cells were always isolated and randomly dispersed throughout the cyst wall. If there truly was an independently derived mitotically active subpopulation of cells within a cyst, one would expect to see clustering of phenotypically identical cells. Moreover, the cellular phenotypic heterogeneity was not preserved in all cyst stages. Heterogeneity was only observed in intermediate or early advanced cysts coincident with the general timing of the transition from a cuboidal to a squamoid cyst cell phenotype. This suggests that the apparent "atypical" cells may actually be normal cyst cells in a slightly delayed state of dedifferentiation and that they would eventually be phenotypically indistinguishable from their neighbors upon completion of the transition to the squamoid dedifferentiated phenotype.* A2 M$ s4 P( E, {3 D  o- A( w, |) C

The role of the primary cilium in the etiology of polycystic kidney disease is currently receiving considerable attention. Studies in mouse models of PKD suggest that defects in primary cilia may directly induce formation of renal cysts (for a review, see Ref. 18 ). In orpk mice, for example, a mutation in the polaris gene has been shown to lead to severe stunting of the primary cilia in the collecting tubule and concomitant cyst formation ( 27 ). In the Pkd2 WS25/ - model of ADPKD, all cells in both early- and intermediate-stage cysts, regardless of the cyst tubule segment of origin, have well-developed normal appearing cilia. Cells in advanced cysts also have primary cilia, but they appear to be much less prominent. Grantham et al. ( 12 ) reported similar findings in human ADPKD renal cysts. These observations suggest that if a ciliary defect is involved in cyst formation in ADPKD, it likely does not have an ultrastructural component. This is not wholly unexpected given that mutations in the C. elegans orthologs of the PKD1 and PKD2 genes induced functional disruptions of the sensory cilia without producing grossly obvious ciliary ultrastructural defects ( 3, 4 ). Nauli et al. ( 25 ) recently proposed that polycystin-1 and polycystin-2 form heterodimeric complexes on the surface of the primary cilium and that these complexes act as mechanotransductive elements to convey information on luminal fluid flow and lumen diameter to the morphoregulatory centers of renal tubular epithelial cells. They suggest that a functional disruption of this complex could lead to cellular dedifferentiation and tissue remodeling and that cyst formation due to mutations in PKD1 or PKD2 is likely due to a dysfunction in this signaling pathway rather than an overt alteration of ciliary ultrastructure.( D3 T' K4 N; q/ B6 J' B' ~
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The only ultrastructural detail that appears to be significantly different between Pkd2 WS25/ - ADPKD cysts and human ADPKD cysts is the degree of basement membrane perturbation. Cuppage et al. ( 9 ) reported that there were abnormalities in human ADPKD cyst basement membranes but that there was not a consistent pattern in the defect. Carone et al. ( 7 ), however, reported that all of the human ADPKD cysts observed in their study had thickened reticulated basement membranes. We did not observe either thickening or reticulation of cyst basement membranes at any stage of cyst growth. The basement membranes of all early and intermediate cysts were not noticeably different from those of unaffected tubule segments. It was not until the cyst epithelial cells were highly dedifferentiated that disorganization of the cyst basement membrane became apparent. Consistent with the human ADPKD studies, we observed a significant increase in interstitial fibrosis and collagen deposition in regions in direct apposition with advanced cysts. The factors responsible for the fibrosis and the collagen deposition in both the Pkd2 WS25/ - and human ADPKD cystic kidneys remain uncertain.  ]' |  U, n% \
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It is clear that cyst cells undergo a dramatic dedifferentiation as cyst expansion proceeds. However, from an ultrastructural perspective, we could detect no evidence to suggest that the cystic epithelium ever dedifferentiates to the point of losing cellular polarity. Well-defined lateral junctions were preserved in even the most advanced cysts. Consistent with this observation, Na -K -ATPase, Ksp-cadherin, and E-cadherin were all correctly targeted in cysts derived from every segment of the tubular nephron and the collecting system. Moreover, we detected significant levels of anti-E-cadherin immunostaining on the lateral membranes of all cyst cells regardless of the stage of cyst expansion or cyst cell dedifferentiation. It is possible that some proteins may truly be mislocalized in ADPKD cystic epithelia, but our observations in the Pkd2 WS25/ - mice suggest that this would be an uncommon event and that it would likely be due to a perturbation in a processing pathway that is specific for that particular protein and not due to a generalized basolateral sorting defect.

Our immunolocalization data do not support the model of apical Na -K -ATPase-mediated, sodium-driven cyst fluid secretion. In advanced cysts at least, the data are consistent with the model of chloride-driven cyst fluid accumulation proposed by Sullivan et al. ( 32 ) in which chloride uptake is mediated by the basolateral Na -K -2Cl - cotransporter NKCC1. In a preliminary study of NKCC1 expression in human ADPKD cystic kidneys, Lebeau et al. ( 21 ) report that 36% of all cysts examined expressed immunodetectable levels of NKCC1 on their basolateral membranes. This number compares favorably with our observations in Pkd2 WS25/ - cystic mouse kidneys (see Table 1 ), but it is unclear if they observed a similar correlation of NKCC1 expression with cyst expansion and dedifferentiation. Because we specifically focused on cortical cysts in our study, the NKCC1 expression that we observed must have been a direct result of an ADPKD-induced phenotypic transformation rather than the consequence of cystic expansion of the inner medullary nephron segments that normally express NKCC1. If advanced ADPKD cyst cells are using basolateral NKCC1 to drive luminal fluid secretion in a manner similar to that proposed for airway epithelial cells, one would presume that Na -K -ATPase activity would also be required on the basolateral membrane of the same fluid-secreting cells. Grantham et al. ( 13 ) clearly demonstrated that basolateral ouabain inhibited chloride-dependent cyst fluid secretion in excised human ADPKD cysts. In our study, Na -K -ATPase was never detected in the advanced cyst cells that expressed NKCC1. We suspect that Na -K -ATPase may have been present on the basolateral membrane but that it was expressed at such low levels that it was below the level of detection with the antibodies that were used in our study. Given the extraordinarily low rate of fluid secretion that must occur in cysts in vivo, it is possible that very few copies of Na -K -ATPase are actually required to maintain the necessary ionic gradients. The disparity of NKCC1 immunostaining between early- and late-stage cysts suggests that the mechanism of cyst fluid accumulation evolves as cystogenesis proceeds. It is likely that the earliest cysts retain connections with the nephron segment of origin and that the cyst fluid is initially glomerular filtrate. As the cyst expands and the connection with the parent nephron is lost, fluid accumulation likely occurs by NKCC1-mediated NaCl and fluid secretion.' _; S- w4 Y! M" E3 M
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In summary, we confirmed that ADPKD cyst cells undergo a dramatic dedifferentiation as cystogenesis proceeds. We could detect no evidence of a generalized basolateral sorting defect or of mislocalization of either Na -K -ATPase, E-cadherin, or Ksp-cadherin, and we therefore conclude that altered cyst cell polarity does not play a role in either cyst expansion or cyst fluid secretion in ADPKD. We directly demonstrated the expression of the Na -K -2Cl - cotransporter isoform NKCC1 on the basolateral membrane of late cysts, supporting the model of chloride and fluid secretion proposed by Grantham and colleagues ( 13, 32 ).

DISCLOSURES
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57328 (Yale PKD Center).; Y+ t- a: F' u) [' R7 u
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ACKNOWLEDGMENTS
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We thank B. Piekos and T. Ardito for expert technical assistance with the scanning and transmission electron microscopy.
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