Renal Cysts of inv/inv Mice Resemble Early Infantile Nephronophthisis
Carrie L. Phillips*,,
Karen J. Miller,
Adele J. Filson*,
Jens Nürnberger*,
Jeffrey L. Clendenon*,
Gregory W. Cook*,
Kenneth W. Dunn*,
Paul A. Overbeek,
Vincent H. Gattone, II and
Robert L. Bacallao*
Department of Medicine, *Division of Nephrology and Department of Pathology, and Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana; and Department of Molecular and Cellular Biology, Baylor College of Medicine, Texas Medical Center, Houston, Texas.
Correspondence to Dr. Carrie L. Phillips, Indiana University School of Medicine, Department of Medicine, Division of Nephrology, 950 West Walnut, R2-202, Indianapolis, IN 46202-5188. Phone: 317-274-1266; Fax: 317-274-8575; E-mail: cphilli3{at}iupui.edu
ABSTRACT. Cystic kidney disease has been linked to mutationsin the Invs gene in mice with inversion of embryonic turning(inv/inv) and the INVS (NPHP2) gene in infants with nephronophthisistype 2 (NPHP2). The inv mouse model features multiorgan defectsincluding renal cysts, altered left-right laterality, and hepatobiliaryduct malformations transmitted in an autosomal recessive manner.Affected mice usually die of renal and liver failure by postnatalday 7. Although cardiopulmonary and liver anomalies have beencarefully detailed, renal cysts have yet to be fully characterizedin inv/inv. By use of three-dimensional visualization by two-photonmicroscopy, this study provides the first comprehensive analysisof in situ cyst formation and progression in inv/inv kidneys.At embryonic day 15, there is dilatation of Bowman’s capsulefollowed temporally by corticomedullary cysts involving collectingducts, proximal tubules, and thick ascending limbs. Collectingducts of newborn inv/inv mice are uniformly and diffusely cysticfrom medulla to cortex, with normal diameters found only attheir most proximal tips. Proximal tubules form fusiform cyststhat alternate with segments of normal or narrowed caliber alongtorturous convolutions. Because defective cilia have been linkedto situs inversus and cystogenesis, we examined inv/inv ciliaby scanning and transmission electron microscopy. The formerdetected monocilia of expected length in cystic collecting ductsand proximal tubules; the latter demonstrated the usual 9 +2 pattern in respiratory cilia. The inv mutant mouse has renalcysts resembling infantile NPHP2 and will provide broader insightinto the role cilia play in renal cystogenesis.
Inherited cystic kidney diseases comprise a large category ofdisorders characterized by cystic kidneys and multiorgan pathology.Both autosomal recessive and autosomal dominant patterns oftransmission have been described in humans and murine models(1). Regardless of the gene defect in humans, cystic expansionof epithelial-lined renal tubules leads to progressive lossof renal function and eventual dependence on dialysis or transplantation.Autosomal dominant polycystic kidney disease (ADPKD) is symptomaticin adulthood and attributed to mutations in the PKD1 gene encodingpolycystin-1, and PKD2 encoding polycystin-2 (2). A number ofgene mutations have been recently described in infants and childrenwith renal cysts (3) including PKHD1 underlying autosomal recessivepolycystic kidney disease (ARPKD) (4,5), NPHP1 in juvenile NPHP1(6), INVS in infantile nephronophthisis type 2 (NPHP2) (7),NPHP3 in adolescent NPHP3 (8), and NPHP4 in a second juvenileform (9). Although a complex cascade of proteins is known toinfluence tubulogenesis (10) and maintain normal renal function,less is understood about how certain gene mutations initiatecystogenesis.
Renal cysts arise from unchecked proliferation of epithelialcells, loss of epithelial cell polarity, and defects in epithelialcommunication with an altered extracellular matrix (11–13).These perturbations may be explained, in part, by disruptionof calcium homeostasis that is normally maintained when polycystin-1and -2 interact to regulate cation channels (2). The interactionof polycystins with NPHP gene products is unknown but the answermay be found in cilia and junctional complexes. Flow generatedby motile cilia in the embryonic node appears to establish theleft-right vertebrate axis (14), whereas nonmotile, primarycilia on the apical surface of renal tubular cells are postulatedto act as mechanosensory receptors (15). Primary cilia may detectchanges in urine flow or content and transmit signals to junctionalcomplexes to regulate epithelial cell differentiation. Inversin,the product of Invs, has recently been found in cilia (16,17)as have other cyst-associated proteins including cystin (18),polaris (19), polycystin-1 and -2 (20,21), fibrocystin (4),and nephrocystin (7), adding further evidence to the postulatedrole of defective cilia assembly as a proximate cause of renalcysts. A relationship between cilia and junctional complexesis suggested by colocalization of polycystin-1 with E-cadherinand the catenins (22), inversin with N-cadherin and the catenins(23), and nephrocystin with inversin (7).
Development of animal models that reliably mimic human diseasewill facilitate the study of cyst-associated proteins in organellessuch as cilia. Invs is one of several murine genes associatedwith renal cysts, but inv/inv mice were also reported to havealterations in left-right asymmetry, cardiovascular defects,an anomalous hepatobiliary system, and premature death (24–29).Although INVS has been sequenced and mapped in humans (as NPHP2)(7,30) and mice (as Invs), the function of inversin is unknown.
Initial reports of the inv mutant model noted renal cysts intwo-dimensional (2-D) histologic sections of newborn kidneysshown at relatively low magnifications. Kidneys of affectednewborn mice were described as having "severely dilated collectingducts," whereas heterozygotes had no phenotypic abnormalities(24,27). Morgan et al. (17) reported normal appearing monociliain kidneys but did not designate the tubule segment. We carefullystudied inv mutants as a model of early infantile nephronophthisisand characterized renal changes from embryonic through postpartumdevelopment by use of bright-field and electron microscopy andtwo-photon fluorescence microscopy with three-dimensional (3-D)analysis.
To understand the genesis of renal cysts, we used two-photonmicroscopy and digital image processing to generate 3-D imagesof in situ cysts. Compared with confocal microscopy, two-photonmicroscopy more efficiently collects scattered fluorescencewithout significant photobleaching of fluorescently labeled,thick biologic specimens (31). Because fluorescence probes maybe targeted to specific renal molecules, two-photon technologyis ideal for determining the relationship of renal cysts tocontiguous and adjacent nephron segments and surrounding matrix.3-D images produced by volume-rendering software that uses two-photondata are less labor intensive in acquisition and offer moreinformation than microdissection or conventional 2-D techniques(32–34).
Animals
Handling of mice conformed to institutional animal care guidelinesestablished by the National Institutes of Health. After halothaneinhalation and nuchal dislocation, a laparotomy was performedon pregnant mice, and embryos were removed at days E15 or E17and fixed in 10% buffered formalin for histology. Kidneys harvestedfrom newborn mice at postpartum day 1 (P1), day 5 (P5), andday 11 (P11) were fixed in 10% buffered formalin (Fisher Scientific,Fair Lawn, NJ) for bright-field microscopy. Day P2 to P5 kidneyswere immersion-fixed in 4% paraformaldehyde (Fisher Scientific)for two-photon microscopy (31). Kidneys and trachea were fixedin 3% glutaraldehyde-formaldehyde in 0.1 M sodium cacodylate(Tousimis, Rockville, MD) for scanning electron microscopy (SEM)or transmission electron microscopy (TEM). Lack of pigmentationin albino control mice (+/+) allowed for phenotypic distinctionfrom homozygotes (inv/inv) with situs inversus, or heterozygotes(inv/+) with intact left-right asymmetry (24,27). Body and kidneyweights were collected on a range of newborn mice availableat time of study.
Bright-field Microscopy
All formalin-fixed kidneys were embedded in paraffin, sectioned3 to 4 µm in thickness, and stained with periodic acid–Schiff.2-D sections were collected with a Leica DMLB microscope (Bannockburn,IL) equipped with a Polaroid DMC digital camera (Bedford, MA).Two kidneys from each animal (inv/inv, inv/+ and +/+) were examinedat E15 (n = 2), E17 (n = 4), P1 (n = 6), and P11 (n = 1). Atubule was deemed cystic if the diameter was twice that of +/+littermates.
Electron Microscopy
SEM of kidney was performed on an Amray 1000A (KLA Tencor, Bedford,MA). For TEM, thin sections of trachea were examined on a PhilipsCM10 TE (FEI, Mahwah, NJ). Two inv/inv and +/+ animals wereexamined by each method as described (35).
Immunohistochemistry
Paraformaldehyde-fixed kidneys were Vibratome sectioned up to200 µm thick (Technical Products International, Inc.,St. Louis, MO), washed in 1 x PBS, and incubated overnight (4°C)with one or more of the following: (1) rhodamine- or fluorescein-labeledlectins (Vector Labs, Burlingame, CA), (2) rhodamine-phalloidin(Molecular Probes, Eugene, OR), which labels filamentous (F)actin and/or (3) Tamm-Horsfall protein antibody (BiomedicalTechnologies, Stoughton, MA), to label thick ascending limbof Henle. Fluorescein-goat anti-rabbit (Jackson Immunoresearch,West Grove, PA) was used as secondary antibody for Tamm-Horsfallantibody. Peanut agglutinin (PNA) and Dolichos biflorus (DBA)lectins label collecting ducts. PNA and Lotus tetragonolobus(LTG) label proximal tubules. Lectins, phalloidin, and antibodieswere diluted 1:200 in 2% BSA, 0.1% Triton X-100, and 1x PBS(Fisher Scientific). After a 4-h wash in 1x PBS, sections wereplaced on glass coverslips mounted to No. 1.5 culture dishes(MatTek, Ashland, MA). Formalin-fixed, paraffin-embedded, 5-µm-thicksections of newborn kidney (day P1) were deparaffinized througha xylene-ethanol gradient, labeled with DBA lectin followedby anti-DBA antibody, and developed with diaminobenzidine aschromagen (Vector Labs).
Two-Photon Fluorescence Microscopy
Optical sections were collected on a Bio-Rad MRC1024 confocal/2photon system (Bio-Rad, Hercules, CA) fitted to a Nikon Eclipseinverted microscope with x20, x40, or x60 water-immersion objectives(Nikon, Melville, NY) at the Indiana Center for Biological Microscopy(http://nephrology.iupui.edu/imaging). Illumination was providedby a Spectra-Physics (Mountain View, CA) Tsunami Lite Titanium-Sapphirelaser tuned to a wavelength of 800 nm. The objective collarwas matched to the coverslip thickness. Data sets were collectedas Z-series spaced from 0.4 µm (x60 objective) to 1.0µm (x20 objective).
Image Analysis
3-D images were produced by Voxx (36), a volume rendering program(available free from http://nephrology.iupui.edu/imaging/voxx).Segmentation and surface rendering of tubule luminal space wasperformed with Amira (TGS, San Diego, CA). Adobe Photoshop (SanJose, CA) was used to assemble and label final figures. To generatecomposites of Vibratome kidney sections, neighboring Z-serieswere collected via two-photon microscopy, rendered in Voxx,saved as TIFF files, and assembled in mosaics by Adobe Photoshop.
Gross Development inv/inv exhibit situs inversus (Figure 1) and usually die withinthe first week of life, although one pup survived to P11. +/–mice followed up to 1 yr do not exhibit significant gross abnormalities(data not shown). Compared with age-matched +/+ littermates,inv/inv mice showed poor body growth, jaundice and developedprogressively enlarging renal cysts (Figure 2). By day P5, inv/invmice were azotemic with twice the concentration of blood ureanitrogen as compared with unaffected (+/+ and +/inv) littermates(data not shown).
Figure 1. (top) Day P5 wild-type (+/+) and mutant (inv/inv) mice. The mutant mouse had decreased body size, yellow discoloration of skin, and cystic kidneys. Spleen (short arrows) and the apex of the heart (long arrows) are on the right side of inv/inv and the left side of +/+ littermate. The bottom panel shows larger cystic kidneys of an inv/inv as compared with normal-sized kidneys from a +/+ littermate.
Figure 2. Graphs showing kidney (A) and body (B) mass of inv/inv () mice compared with +/+ control littermates () during the first week of life. (A) Cystic kidneys of P7 inv/inv mice weighed more than twice as much as +/+ kidneys. (B) Body weight of +/+ mice progressively increased whereas little weight gain was noted in inv/inv mice (n = number of mice per time point available).
Light Microscopy
We inspected 2-D histologic sections of formalin-fixed, paraffin-embeddedkidneys from embryonic (days E15, E17) and postpartum (daysP1, P11) mice with bright-field microscopy. Relatively rapiddevelopment and progression of epithelial-lined cysts were foundin nephrons of inv/inv mice (Figure 3). Expansion of Bowman’sspace surrounding deep cortical glomeruli was the earliest defectobserved in E15 inv/inv (Figure 3B), and this feature persistedthrough day P11 (Figure 3F). By day E17, cysts began to appearin collecting ducts and proximal tubules (data not shown) andthese progressed in size and number in the cortex and medullaof newborn day P1 mice (Figure 3, C and D), whereas kidneysof age-matched +/inv and +/+ littermates developed normally(data not shown). Day P11 inv/inv kidneys showed diffuse andelaborate cystic expansion of proximal tubules, collecting ducts,and Bowman’s space (Figure 3F) that was similar but moreextensive than was seen at day P1. A few inv/inv tubular lumina(<5%) contained dystrophic calcifications (data not shown).Kidneys from +/+ and +/inv kidneys showed densely packed tubuleswith normal nephron development at day P11 (Figure 3E).
Figure 3. Two-dimensional, bright-field microscopic images of periodic acid–Schiff–stained kidneys. (A) +/+, day E15 (original magnification, x200). (B) inv/inv, day E15 (original magnification, x200). Expansion of Bowman’s space surrounding deep cortical glomeruli (open arrows) was noted in day E15 inv/inv kidneys (B) but not +/+ (A). (C) inv/inv, day P1 (original magnification, x40). (D) inv/inv, day P1 (original magnification, x200). Cysts involving collecting ducts, proximal tubules and Bowman’s spaces (*) are seen in P1 inv/inv. (E) +/+, day P11 (original magnification, x40). (F) inv/inv, day P11 (original magnification, x40). When compared with the dense parenchyma of +/+ kidney at day P11 (E), inv/inv shows diffuse cortical and medullary cysts (F).
Two-Photon Fluorescence Microscopy and 3-D Rendering.
Vibratome sections of paraformaldehyde-fixed newborn mouse kidneys(P2 to P5) were labeled with fluorescently tagged lectins, phalloidin,and/or anti–Tamm-Horsfall. Z-series of optical slicesfrom each tissue section were acquired on a two-photon microscopeand explored in near–real time by Voxx (Figures 4 through 9).
Figure 4. Single-channel, two-photon microscopy and three-dimensional rendering of day P5 proximal tubules (PT), thin descending loops of Henle (TL), and collecting ducts (CD). (A) +/+, PNA lectin, six adjacent Z-series assembled into one image. (B) inv/inv, PNA lectin, six adjacent Z-series with longitudinal orientation of tubules. Manual rotation of individual volumes in Voxx distinguished PT and CD that falsely appear connected in nonrotated state (*). Dilated S3 segments of PT (**) narrow distally into TL. Scale bar for A and B = 100 µm. (C) +/+, PNA lectin, 205 µm (x-axis) x 205 µm (y-axis) x 77 µm (z-axis). (D) inv/inv, PNA lectin, 205 x 205 x 79 µm. (E) +/+, LTG lectin, 205 x 205 x 42 µm. (F) inv/inv, LTG lectin, 205 x 205 x 88 µm. (G and H) inv/inv, LTG lectin, 180 x 180 x 66 µm. Surface of PT is rendered opaque in G and transparent in H to visualize attenuated lumen (arrow).
Figure 5. Two-photon microscopy, three-dimensional rendering and segmentation of proximal tubules. (A) +/+, day P5, 205 (x-axis) x 205 (y-axis) x 31 µm (z-axis); (B) inv/inv, day P5, 205 x 205 x 29 µm; (C) inv/inv, day P5, 205 x 205 x 60 µm. Cystic proximal tubules (PNA-lectin, tan in B and C) were surrounded by relatively abundant F-actin in the interstitium (phalloidin, green in A through C). (E, F) Segmentation with Amira revealed in situ fusiform cysts, hairpin turns and narrowed segments in a single day P4 convoluted proximal tubule (tan in D, original volume, 512 x 512 x 91 µm, with DBA-labeled collecting ducts [green]; E = translucent, front view; F = opaque, back view). (G–J) Optical two-dimensional (2-D) slices at 10-µm intervals, taken from the volume rendered in (D), show cysts with narrowed segments (J, arrow). The numbers 1 to 9 in panels D through J allow comparison of 2-D and three-dimensional renderings.
Figure 6. Dual-color, two-photon microscopy and three-dimensional rendering of day P2 +/+ (A) and inv/inv (B) show collecting ducts (DBA lectin, green) in relationship to proximal tubules (LTG lectin, tan). Arrows indicate thin descending loops of Henle that in inv/inv narrowed at their transitions from S3 segments. Some of these thin loops have diverticuli or flaring at their distal ends (*). A was assembled from 3 adjacent Z-series and B from 12. Scale bar = 100 µm.
Figure 7. Single-channel, two-photon microscopy with three-dimensional rendering shows longitudinal view of day P5, DBA-positive collecting ducts (B, inv/inv, 14 adjacent Z-series) that were diffusely dilated throughout their entire lengths except at proximal tips (arrows). Compare with normal caliber collecting ducts of +/+ (A, eight adjacent Z-series). Scale bar = 100 µm. Inset: immunoperoxidase staining with DBA shows cross sections of dilated collecting ducts (*) in two dimensions (day P1).
Figure 8. Dual-channel, two-photon microscopy and three-dimensional renderings of collecting ducts (DBA lectin, green). (A) Day P5 +/+ with phalloidin (tan); image assembled from 2 adjacent Z-series; arrows point to proximal tubules; G = glomerulus. (B) Day P5 inv/inv with phalloidin (tan); collecting duct cysts with focal absence of DBA labeling at proximal ends are indicated with an asterix; P = proximal tubules with visible apical brush borders; image assembled from 28 adjacent Z-series. Scale bar for A and B = 100 µm. (C) Day P2 inv/inv collecting ducts with LTG-lectin checkerboard labeling of intercalated cells (tan) against DBA-positive principal cells (green); assembled from 4 adjacent Z-series. (D, E) Day P5 inv/inv with phalloidin (tan), 205 x 205 x 50 µm volume is presented in different rotations to emphasize heterogeneous labeling of epithelial cells within single cysts.
Figure 9. Dual-color, two-photon microscopy and three-dimensional rendering of day P5 +/+ (A) and inv/inv (B) outer medulla. Thick ascending limbs labeled with anti–Tamm-Horsfall (green) run parallel to DBA-positive collecting ducts (brown). Panel A was collected over a depth of 30 µm (single Z-series). Panel B was collected over 64 µm (two adjacent Z-series). Scale bar for A and B = 100 µm.
Single-Channel, Two-Photon Microscopy of Proximal Tubules.
In kidneys of P5 +/+ mice, proximal tubules of uniform caliberwere found among compact arrays of cortical nephrons labeledwith PNA or LTG (Figure 4, A, C and E). These lectins labeledapical brush borders, intracellular vesicles, and basal aspectsof epithelial cells in proximal tubules of unaffected mice.A considerably different morphology was found in lectin-labeledcystic kidneys of day P3 and P5 inv/inv, which showed fusiform,not sacular, dilatation of convoluted and straight proximaltubules (Figure 4, B, D, and F through H) that were bridgedby segments of normal or narrowed calibers (apparent in Figure 4, B and G).Within progressively expanding cysts, the apicalbrush border microvilli appeared to be either intact or focallythinned. Intact brush borders suggested that epithelial polarityis at least structurally intact in inv/inv proximal tubules.Rarified microvilli appeared similar to loose, ill-fitting piecesof jigsaw puzzles (Figure 4D). Fusiform dilatation of collectingducts was seen with PNA labeling (Figure 4, B and D).
Multicolor, Two-Photon Microscopy, and Segmentation of Proximal Tubules.
To analyze proximal tubules in situ, Vibratome sections werelabeled with two fluorescence markers (Figures 5 and 6). PNA-labeledproximal tubules from inv/inv mice showed variable luminal wideningand cysts (Figure 5, B through D). The interstitium appearedrelatively disorganized and more abundant (Figure 5, B and C)when sections were dual-labeled with phalloidin. Similarly labeled+/+ kidneys had evenly dispersed glomeruli and uniform tubuleswith little intervening interstitium (Figure 5A).
We used Amira software to determine the luminal pathway withintortuous convoluted proximal tubules (Figure 5, E and F), atask that proved difficult and time-consuming by 2-D (Figure 5, G through J).The lumen was analyzed, rather than the basalsurface, because this space represents the route of urine flowthat may be functionally obstructed. We identified normal, cystic,and narrowed regions within the lumen of a single, serpentinetubule. Although disconnected, blind pouches were not identified,there were cystic segments next to markedly narrowed segmentsthat may function like blind pouches (between segments 2 and4, 5 and 6, or 6 and 7 in Figure 5, D through J). Similar narrowingwas detected where S3 segments transitioned to descending thinloops of Henle in the outer medulla, where occasional diverticuliwere noted (Figure 6). Rare, small outpocketings were seen alongproximal tubules (segment 4, Figure 5D).
Single-Channel, Two-Photon Microscopy of Collecting Ducts.
Collecting ducts from inv/inv mice were labeled with DBA lectinand imaged by two-photon microscopy (Figure 7). Composites ofmicroscopy volumes showed that inv/inv collecting ducts werediffusely expanded up to 20 times the diameter of +/+. The caliberof inv/inv collecting ducts approached that of +/+ only at theirimmature proximal ends in the outermost cortex (Figure 7B).Like +/+, the inv/inv collecting ducts maintained their parallelalignment from medulla to cortex. DBA-positive cysts were easilyidentified by immunoperoxidase (Figure 7, inset); however, the2-D nature of this technique gives the false impression thatcystic collecting ducts are not parallel.
Multicolor, Two-Photon Microscopy Shows Heterogeneous Labeling of Collecting Ducts. Figure 8 shows heterogeneous labeling of cysts arising in inv/invcollecting ducts that were dual-labeled with DBA and phalloidin,or DBA and LTG lectin. In collecting duct cysts in the outercortex, there was loss of DBA labeling at many of the proximaltips (Figure 8, B, D, and E). This consistently occurred atthe entrance and incorporation of distal tubule cells into thecyst wall, indicating that individual fusiform cysts can affecta region of tubule regardless of tubule segment boundaries.Further heterogeneity was demonstrated when both DBA-positiveprincipal cells and LTG-positive intercalated cells were foundin the same collecting duct cysts (Figures 6B and 8C).
Multicolor, Two-Photon Microscopy of Thick Ascending Limbs.
In the medulla, anti–Tamm-Horsfall antibody labeled thickascending limbs that were of normal caliber in +/+ kidneys (Figure 9A)but appeared dilated in inv/inv kidneys (Figure 9B).
Scanning and Transmission Electron Microscopy
We used SEM to examine monocilia in renal tubules (Figure 10).In day P5 inv/inv kidneys, normal-appearing monocilia were detectedat apical surfaces of cystic collecting ducts (Figure 10C) andproximal tubules (Figure 10D). By use of high-resolution TEM,we examined respiratory cilia from trachea of inv/inv mice (Figure 10E).Within the axoneme membrane, we were able to identifythe normal arrangement of nine peripheral doublet microtubulesaround a central pair (9 + 2). Neighboring cilia showed parallelalignment of ciliary axes (data not shown) (37).
The study presented here provides the first comprehensive analysisof cyst development and progression in kidneys of inv/inv mice.We observed a steady progression of corticomedullary cysts ininv/inv kidneys from embryonic day 15 to postpartum day 11 ina pattern resembling NPHP in humans. NPHP is characterized bycorticomedullary cysts, thickening and attenuation of tubularbasement membranes, tubular atrophy, periglomerular and interstitialfibrosis, and round cell infiltrates in the interstitium (7,38).In familial juvenile NPHP1, Sherman et al. (39) described cystsin collecting ducts and distal convoluted tubules, and diverticulainvolving loops of Henle and the distal nephron. Gagnadoux etal. (40) proposed an infantile variant of NPHP when describingdiffuse chronic tubulointerstitial nephritis characterized bymicrocystic dilatation of proximal tubules and Bowman’sspace in seven infants who progressed to end-stage renal disease(ESRD) before age 2 yr. The suggestion that infantile NPHP maybe associated with INVS was found in a Bedouin family with amutation on chromosome 9q22–31, the synthenic region formurine Invs (41). This family was studied by Otto et al.(7),who reported Invs in mice is orthologous to human NPHP2, havingidentified nine distinct recessive mutations in seven familieswith NPHP2. All patients developed ESRD by 5 yr of age; threefamilies had documented renal cysts and one patient had situsinversus.
Early reports of inv/inv mice described cysts only in collectingducts of newborns (24,27). We show cysts in collecting ducts,proximal tubules, thick ascending limbs, and Bowman’scapsules that steadily progress from embryonic through newborndevelopment. Normal and narrowed segments are identified withinindividual proximal tubules. Descending thin loops of Henleshow narrowing and focal diverticula. Newborn inv/inv mice developsevere azotemia and most succumb to renal failure within 1 wk,consistent with the rapid disease progression reported in infantileNPHP2. However, we did not observe tubular atrophy and interstitialfibrosis described in NPHP2 because this chronicity is not likelyto manifest during the short life span of inv/inv mice.
At E15 the most mature, deep cortical glomeruli show dilationof Bowman’s capsule. By E17, proximal tubule cysts developin association with the more numerous collecting duct cysts.Although collecting ducts may appear haphazardly arranged in2-D, our 3-D imaging shows inv/inv collecting ducts run parallelalong their expected linear path from medulla to cortex. Duringmaturation these ducts uniformly dilate throughout their lengthsexcept at their proximal immature tips in the superficial cortex.Cysts in inv/inv collecting ducts and proximal tubules are predominantlyfusiform with rare lateral out-pocketing of renal tubule epithelialcells. Although isolated blind pouches were not identified,our segmentation of proximal tubule lumina did identify cystsbridged by narrowed segments that may function as blind pouches.Proximal tubules and collecting ducts from cpk mice also exhibitfusiform dilation while undergoing cystogenesis (32,33). Out-pocketingof renal tubular epithelial cells in the pathogenesis of renalcysts is associated with human ADPKD (34) and is thought tobe the result of loss of heterozygosity in the clonal cell thatgives rise to a cyst. However, loss of heterozygosity in individualsomatic cells is not required for cyst formation in ARPKD orNPHP in humans or mice, including inv/inv, because all cellsin these cystic kidneys inherit two copies of the mutated gene.
inv/inv kidneys showed complexity in shape and cellular compositionof cysts. First, in proximal tubules, fusiform cysts are contiguouswith either normal or narrow segments similar to linked sausages.Normal caliber tubule segments are found between the fusiformcysts in the cpk model (33). Because all cells inherit two mutatedgenes and only portions of proximal tubules develop cysts, theremay be local factors contributing to cyst formation in inv/invand possibly in humans and other murine models with inheritedcystic renal diseases. Second, individual collecting duct cystsin inv/inv newborns contain a mixture of both principal andintercalated cells in a distribution pattern similar to +/+.This suggests that, in the inv/inv model, collecting duct cystsdo not arise from clonal proliferation of a single mutated principleor intercalated cell because we did not identify collectingduct cysts composed entirely of DBA-positive principle cellsor DBA-negative intercalated cells.
Finally, we found that one cyst potentially may involve contiguousbut heterogeneous tubule segments, such as cystic collectingducts that uniformly lack DBA staining at the transition todistal connecting segments. Alternatively, this heterogeneousstaining pattern may not represent one cyst formed across twodistinct tubule segments but may in fact represent the lossof DBA staining in one cystic collecting duct. We favor theformer explanation given the sharply demarcated staining patternconsistently lost at distal connecting segments (future distalconvoluted tubules) of both cystic and normal collecting ducts.
The diversity of cystic animal models suggests many proteinsinteract to ensure tubule integrity. Currently available murinemodels with PKD and NPHP (1) have supported the concept thatmany gene mutations may be responsible for cystogenesis. Theinv mouse represents one model with renal cysts similar to humanand other murine forms of ARPKD and NPHP but also has left-rightasymmetry defects and cardiopulmonary (29) and hepatobiliarypathology (28). Cilia are postulated to establish left-rightasymmetry (14) and have been implicated in renal cystogenesis(15). During early embryonic mouse development, asymmetricalcurrents generated by the vortical motion of nodal cilia arebelieved to establish the left-right axis. Praetorius and Spring(42) mechanically bent monocilia of cultured Madine Darby caninekidney (MDCK) cells and detected increased intracellular calciumthat propagated as a wave from the perturbed cell to neighboringcells by diffusion of a second messenger via gap junctions.
The first connection of cilia to cysts was found in studiesof TgN737Rpw that is mutated in orpk mice. Partial deletionof TgN737Rpw results in cystic kidneys (43), but mice with Tg7372–3bGalknockout allele have left-right asymmetry and defective cilia(44). Polaris, the product of TgN737Rpw, has been localizedto axoneme of MDCK cilia (21), suggesting an intact cilia functionis required for renal and axis development. Cystin, encodedby Cys1 and defective in cpk mice, is found in cilia (18). Althoughcysts of inv/inv exhibit structurally normal nonmotile ciliasimilar to the cpk mouse, functionally inv/inv motile ciliahave been reported to generate slow, turbulent nodal flow (45).A subset of motile cilia containing left-right dynein appearsto generate nodal flow that is sensed by nonmotile cilia containingpolycystin-2 (46). Patients with ADPKD who inherit a PKD2 mutationhave defective polycystin-2 but normal left-right axis (2).Individuals with situs inversus due to a defect in ciliary dyneinarms do not develop renal cysts (15), and therefore, just havingnonfunctional motile cilia cannot cause cysts. How defectivecilia induce NPHP is unclear; however, data from several modelsclearly implicate ciliary function in the pathogenesis of renalcystic disease. The inv/inv mouse will be useful for the studyof cilia defects and appears to represent a good model systemfor early infantile nephronophthisis.
Acknowledgments
We thank Michael Goheen, Barry Babin, Michael Sanford, OlgaCabello, and Doug Kojetin for technical assistance and HeatherWard for reading the manuscript. Figures were prepared withthe assistance of Jill Goodwin and Brent Gann. C.L.P. acknowledgesNIH K08 DK02785, Polycystic Kidney Disease Foundation grant99023, National Kidney Foundation of Indiana, Indiana UniversitySchool of Medicine Biomedical Research Grant, Clarian HealthValues Fund VFR21, and Ralph W. and Grace M. Showalter Foundation.Funds were also received from Deutsche Forschungsgemeinschaft(J.N.), NIH PO1 HL49953 (P.A.O.), Indiana University StrategicDirections Initiative (K.W.D.), and the Indiana Genomics Initiativegrant from the Lilly Foundation to Indiana University Schoolof Medicine. Parts of this manuscript were presented in posterform at the national meetings of the American Society of Nephrologyand the American Society for Cell Biology.
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Received for publication November 29, 2001.
Accepted for publication April 19, 2004.
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Top
Abstract
Introduction
). PNA-labeled proximal tubules from inv/inv mice showed variable luminal widening and cysts (Figure 5, B through D). The interstitium appeared relatively disorganized and more abundant (Figure 5, B and C) when sections were dual-labeled with phalloidin. Similarly labeled +/+ kidneys had evenly dispersed glomeruli and uniform tubules with little intervening interstitium (Figure 5A). 
2–3bGal knockout allele have left-right asymmetry and defective cilia (44). Polaris, the product of TgN737Rpw, has been localized to axoneme of MDCK cilia (21), suggesting an intact cilia function is required for renal and axis development. Cystin, encoded by Cys1 and defective in cpk mice, is found in cilia (18). Although cysts of inv/inv exhibit structurally normal nonmotile cilia similar to the cpk mouse, functionally inv/inv motile cilia have been reported to generate slow, turbulent nodal flow (45). A subset of motile cilia containing left-right dynein appears to generate nodal flow that is sensed by nonmotile cilia containing polycystin-2 (46). Patients with ADPKD who inherit a PKD2 mutation have defective polycystin-2 but normal left-right axis (2). Individuals with situs inversus due to a defect in ciliary dynein arms do not develop renal cysts (15), and therefore, just having nonfunctional motile cilia cannot cause cysts. How defective cilia induce NPHP is unclear; however, data from several models clearly implicate ciliary function in the pathogenesis of renal cystic disease. The inv/inv mouse will be useful for the study of cilia defects and appears to represent a good model system for early infantile nephronophthisis. 
) mice compared with +/+ control littermates (
) during the first week of life. (A) Cystic kidneys of P7 inv/inv mice weighed more than twice as much as +/+ kidneys. (B) Body weight of +/+ mice progressively increased whereas little weight gain was noted in inv/inv mice (n = number of mice per time point available).
