Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations to PKD1 or PKD2, triggering progressive cystogenesis and typically leading to end-stage renal disease in midlife. The phenotypic spectrum, however, ranges from in utero onset to adequate renal function at old age. Recent patient data suggest that the disease is dosage dependent, where incompletely penetrant alleles influence disease severity. Here, we have developed a knockin mouse model matching a likely disease variant, PKD1 p.R3277C (RC), and have proved that its functionally hypomorphic nature modifies the ADPKD phenotype. While Pkd1^+/null mice are normal, Pkd1^RC/null mice have rapidly progressive disease, and Pkd1^RC/RC animals develop gradual cystogenesis. These models effectively mimic the pathophysiological features of in utero–onset and typical ADPKD, respectively, correlating the level of functional Pkd1 product with disease severity, highlighting the dosage dependence of cystogenesis. Additionally, molecular analyses identified p.R3277C as a temperature-sensitive folding/trafficking mutant, and length defects in collecting duct primary cilia, the organelle central to PKD pathogenesis, were clearly detected for the first time to our knowledge in PKD1. Altogether, this study highlights the role that in trans variants at the disease locus can play in phenotypic modification of dominant diseases and provides a truly orthologous PKD1 model, optimal for therapeutic testing.

Autosomal dominant polycystic kidney disease (ADPKD [MIM no. 173900 for PKD1 and 613095 for PKD2]) is one of the most common monogenic disorders, with a prevalence of 1:400 to 1:1,000 (1, 2). It is genetically heterogeneous and has been linked to 2 loci, PKD1 (16p13.3) and PKD2 (4q21), mutated in approximately 85% and 15% of cases, respectively (3–5). Typically, the disease manifests with progressive bilateral cystic kidney enlargement, leading to end-stage renal disease (ESRD) in midlife; while, symptomatic polycystic liver disease, hypertension, and an enhanced level of intracranial aneurysms are other disease complications (6, 7).

The pathogenesis underlying the polycystic kidney disease (PKD) phenotypes is still unclear; however, various cellular defects have been suggested. For example, cyst development has been strongly associated with defects to the primary cilia, as evident from model systems and syndromic PKD forms, categorizing PKD as a ciliopathy (8, 9). Structural and functional cilia defects, including length abnormalities, have been characterized in several types of PKD, including the infantile onset autosomal recessive form (ARPKD, MIM no. 263200) (8–12). However, no clear ciliary structural abnormalities have been described in PKD1 (13–15). The majority of ciliogenes appear to maintain the structural integrity of the cilia or regulate protein trafficking to and from the organelle, while the PKD1 and PKD2 proteins, polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively, are thought to form a complex on the primary cilium and act as a receptor (14, 16–19). One possibility is that the PC complex acts as a mechanosensor, signaling via a Ca²⁺ influx through PC-2, a TRP channel, after fluid flow detection by PC-1 (14, 16, 20). Additionally, urinary exosome-like vesicles (ELVs), which contain high levels of the polycystins, have been suggested to play a role in the mediation of signaling events at the primary cilia (11, 21, 22). However, the localization and function of PC-1 and PC-2 are still controversial, and various alternative cilia-related roles have been proposed (16, 23–26).

In addition to primary cilia defects, PKD cells exhibit many other cellular aberrations proposed to be associated with cystogenesis and/or cyst progression. These defects include, but are not limited to, dedifferentiation, increased proliferation and apoptosis, polarity defects, and altered gene expression that may be linked to increased intracellular cAMP, possibly associated with decreased intracellular Ca²⁺ (16, 27). The tubule segment that becomes cystogenic is also a point of debate in ADPKD. Traditionally, all tubule segments have been thought to be involved in disease, but the available data are limited and somewhat contradictory (28–30). Recently, treatments focused on lowering cAMP by targeting the arginine V2 vasopressin receptor (AVPR2), which is mainly expressed in the thick ascending limb of Henle and the collecting duct (CD), highlight the importance of clarifying this issue for the development of successful therapeutic interventions (28, 31, 32). Unfortunately, the currently available PKD1 rodent models are not ideal for the analysis of ADPKD pathogenesis, including determining the affected nephron segment or therapeutic testing. Pkd1-null animals die embryonically, and heterozygotes develop only very mild disease in old age, while conditional models do not reflect the disease development in human ADPKD due to the loss of all functional protein at one time (33–39). In addition, previously described hypomorphic mouse models develop very rapidly progressive disease, which is inadequate for therapeutic testing and does not reflect the gradual kidney enlargement typical of ADPKD (40, 41).

The severity of disease in ADPKD has been directly associated with the gene mutated; PKD1 on average leads to ESRD at approximately 54 years, while PKD2 leads to ESRD at approximately 74 years (42). The complete phenotypic spectrum, however, ranges from rare in utero–onset cases to patients with adequate renal function in old age; extreme heterogeneity that cannot be solely explained by genic effects (6, 43, 44). Some rare cases with early-onset ADPKD also develop tuberous sclerosis due to a contiguous deletion of PKD1 and TSC2 (MIM no. 600273), highlighting TSC2 as a likely modifier of the PKD1 phenotype (45–47). Causes of extreme intrafamilial phenotypic variation include mosaicism or bilineal inheritance of a pathogenic PKD1 and PKD2 variant, but these do not account for the majority of cases with extreme ADPKD phenotypes (45, 48, 49).

The mechanism by which a heterozygous mutation results in cyst development is controversial (50). Complete loss of the ADPKD proteins in mice results in embryonic lethality and renal cyst development by E14.5 (33, 35, 51). Likewise, the corresponding human genotype is thought to be incompatible with life (52). One proposed mechanism, consistent with cyst development in null animals and the focal nature of cystogenesis, is a cellular recessive one, where each cyst is a clonal development resulting from complete polycystin loss. Somatic mutations to the WT PKD1 or PKD2 allele in individual cyst linings support this 2-hit mechanism, while the frequency and timing of this inactivation may explain some of the observed variable expressivity (53–56). Consistent with this, a Pkd2 allele prone to somatic recombination results in progressive cystic disease, and PKD severity in conditional ADPKD mice differs radically depending on the age at complete polycystin inactivation (36–38, 57).

Although the cellular recessive mechanism is attractive, recent data indicate that the dosage level of the polycystins may be important. Homozygous Pkd1 and Pkd2 hypomorphs, with 13% to 33% correctly spliced Pkd1 or Pkd2, are viable but develop progressive cystic disease in the kidney, liver, and pancreas, despite the presence of some functional polycystin (40, 41, 58). Additionally, recently described atypical ADPKD families highlight the role of putative incompletely penetrant PKD1 or PKD2 alleles. If inherited alone, these alleles are associated with mild PKD, while homozygotes or compound heterozygotes are viable and present with typical to severe disease, and in trans inheritance with a null allele causes in utero–onset disease (29, 59–62). However, proving the importance of specific alleles and analyzing the molecular mechanism is not possible from the study of a number of small families.

Here, we have taken an experimental approach by developing a knockin mouse model of a clinically proposed incompletely penetrant PKD1 allele, p.R3277C (RC), to rigorously test a dose-dependent pathomechanism of cystogenesis and to investigate the role of these alleles in severe ADPKD. Furthermore, by generating models mimicking the pathophysiological features of typical and in utero–onset ADPKD, we have been able to study the process of cystogenesis in detail at the histological and molecular level, providing insights into pathogenesis and reagents to improve therapeutic options for this disorder.

Generation of a knockin mouse model mimicking a clinical PKD1 allele. The PKD1 p.R3277C allele was first identified in a consanguineous family of French ancestry in the United States, in which patients homozygous for the allele were viable but presented with mild to typical cystic disease (59). In 2 additional families with extreme intrafamilial disease variability, this allele segregated in trans with either a truncating or missense mutation, both resulting in very early-onset ADPKD (29, 59). These associated phenotypes suggested that the p.R3277C allele was incompletely penetrant. To date, 7 pedigrees (2 homozygous) have been identified with this variant, giving an ADPKD population allele frequency of 0.25% (ADPKD database [PKDB]; http://pkdb.mayo.edu). To rigorously test the role of such alleles in the variable expressivity of PKD1, we generated a knockin mouse model mimicking the exact codon usage seen in patients (Pkd1 c.9805.9807AGA>TGC). The targeting construct was cloned from a 129Sv/E phage library using recombineering (Figure 1A and ref. 63). Embryonic stem cell (ESC) clones were screened for homologous recombination (HR) by Southern blotting, and the mutation was verified by Sanger sequencing (Figure 1, B and C). To exclude effects of the loxP-flanked puromycin selection cassette, all male germline transmitting chimeras were crossed with an oocyte-expressing Cre female (129S1/Sv-Hprt^tm1(cre)Mnn/J) to excise the selection cassette at the zygote stage. Subsequent RT-PCR analysis of the kidneys from homozygous animals (Pkd1^RC/RC animals) showed no alteration in mRNA splicing or expression levels (Figure 1D). Likewise, PC-1 expression was unaltered between WT and Pkd1^RC/RC animals when analyzing kidney whole cell lysates or kidney-derived CD cells (in kidney, PC-1 expression in Pkd1^RC/RC was 94% ± 2.30% of WT, P = 0.450; in CD cells, PC-1 expression in Pkd1^RC/RC was 99% ± 2.30% of WT, P = 0.605; Figure 1E). This suggested that the defect in the action of this allele was likely at the level of functional protein.

Figure 1

Generation of the Pkd1 p.R3277C knockin mouse. (A) Diagram of the targeting construct used for injection into ESCs. Restriction sites flanking the construct are in green (Sb, SbfI; Mo, MboI). The Pkd1 p.R3277C mutation was introduced using the exact codon found in ADPKD probands (29, 59) (red). Restrictions sites used for introducing the mutation and the loxP-flanked (red triangles) selection cassette (yellow box) are in orange (A, AdhI; X, XhoI; S, SalI). The restriction site used to linearize the targeting construct is shown in purple (P, PacI). Restriction sites and probes used for Southern blotting are in blue (Bs, BspHI; M, MfeI). (B) Representative Southern blot of successful HR in ESCs. (C) Sequencing chromatogram of WT and heterozygous ESC clones and a homozygous animal. (D) RT-PCR analysis across the mutation and the loxP-containing intron of a WT and a homozygous animal (after Cre-lox recombination). A normally seen alternative splice product, skipping exon 31 (in-frame change), was not significantly different between WT and homozygous animals (asterisk) (105) (PCR product, 762 bp; splice variant, 669 bp). (E) Quantification of PC-1 protein levels in whole kidney and CD cell lysate. PC-1 protein levels between WT and Pkd1^RC/RC animals were not significantly different (WB not shown, see Figure 9 for more detail). Data are shown as a normalized ratio (loading control, γ-tubulin [TUBG1]) between WT and Pkd1^RC/RC animals (n = 3/group). Statistical values were obtained by Student’s t test; error bars indicate ± SD.

Mice homozygous for the Pkd1 p.R3277C allele develop mild but progressive PKD. We first set out to characterize the Pkd1 p.R3277C allele in homozygosity (Figures 2 and 3, Supplemental Figure 1, and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI64313DS1), mimicking the published consanguineous family with adult-onset ADPKD (59). From a heterozygous cross, we observed the expected Mendelian ratios of offspring (data not shown), implying that Pkd1 p.R3277C is not a fully penetrant allele. To analyze disease presentation and progression, we initially followed 4 WT and 6 Pkd1^RC/RC littermates by ultrasonography (US). At 3 months of age, cysts ranging from 0.3 to 2.5 mm in diameter were visible in all Pkd1^RC/RC animals (Figure 2A). Kidney volumes (KVs) were estimated from 3D M-mode renderings, and it was determined that Pkd1^RC/RC KVs increased progressively over time in most animals and were significantly increased compared with those of WT littermates at all investigated time points (Figure 2B and Supplemental Table 1); although, there was noticeable variability between these outbred animals (Figure 2B; e.g., animal no. 3 vs. no. 6). The increase in KV was directly correlated with the progressive but variable increase in cyst burden seen histologically at 3, 6, 9, and 12 months (Figure 3A and Supplemental Figure 1) and reflected in a steady increase in percentage of kidney weight per body weight (%KW/BW) (Figure 2C and Supplemental Table 1). At 12 months of age, we noted a slight decrease in KV and %KW/BW, which was likely associated with increased fibrosis (3.3% of kidney area at 3 months vs. 20.3% at 12 months; Table 1), highlighted by increased Masson Trichrome staining (Figure 3A and Supplemental Figure 1). A similar decrease in %KW/BW associated with increased fibrosis has been observed at earlier ages in other hypomorphic Pkd1 models and in late stages of human ADPKD (40, 41, 64). This steady increase in fibrosis seems mainly to correlate with cyst growth, although some fibrosis is evident when there is minimal cystogenesis. A number of factors, including epithelial paracrine release of proinflammatory and profibrotic mediators, epithelial production of extracellular matrix proteins, and epithelial-mesenchymal transition, likely promote the fibrosis (65).

Figure 2

Homozygosity of the Pkd1 p.R3277C allele results in progressive PKD with physiological characteristics comparable to those of human ADPKD. (A) Representative US M-mode images of Pkd1^RC/RC mice. Kidneys with the mildest (left) and most severe disease (right) are shown at 3 months. Kidney cysts are visible as echolucent spots (arrows). Scale bars: 10 mm (left); 12 mm (right). (B) Quantification of US at 3, 6, 9, and 12 months of each individual animal and the mean of all animals. Significant differences between WT and homozygotes were seen at all time points. KV increased in most animals progressively, but variability among mice was observed. (C–E) Graphic representations of %KW/BW, cAMP, and BUN measured at 3, 6, 9, and 12 months (Supplemental Table 1). (C) %KW/BW increased progressively with cyst burden until 9 months, after which a significant decrease was observed, likely correlated with increased fibrosis (Table 1). (D) Similar to that in patients with ADPKD, cAMP levels rose with disease burden (16). (E) Increased kidney damage due to cystogenesis/fibrosis resulted in a significantly elevated BUN after 9 months. The data in C–E were obtained from the same group of animals at each time point (WT, n = 4; Pkd1^RC/RC, n = 6). Statistical values were obtained by the Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001); error bars indicate ± SD.

Figure 3

Histology of homozygous Pkd1 p.R3277C animals mimics typical ADPKD. (A) Representative Masson Trichrome histology sections of Pkd1^RC/RC and WT kidneys at 1 to 12 months show that cysts developed progressively with age. At later time points, an increased level of fibrosis was noted (12 months; Table 1). Scale bars: 1 mm (kidney cross-section, WT, and Pkd1^RC/RC); 250 μm (Pkd1^RC/RC magnified region [images in second column]); 200 μm (WT magnified region [insets]). (B) Masson Trichrome histology sections of 12-month-old Pkd1^RC/RC and WT livers (no significant difference between percentage of liver weight [LW] per body weight [%LW/BW] of WT animals and homozygotes was noted). Four out of five homozygous animals showed ductal plate malformations, including microhamartomas of varying severity. These cystic lesions are progenitors of the liver cysts often found in patients with ADPKD (69). Scale bars: 100 μm.

Table 1

Summary of kidney composition by area, using lectin and fibrosis markersA

Analysis of the onset of cystogenesis showed that the Pkd1^RC/RC animals had histologically visible cysts as early as E16.5 (Supplemental Figure 2). This compares with approximately E14.5 in Pkd1^null/null mice and highlights that, although these animals are viable, the time of cyst initiation is not radically different from that in the null state (35, 51). Renal cystic enlargement was accompanied by an increase in renal cAMP, significant after 6 months, as described in other rodent models and patients with ADPKD (Figure 2D and refs. 16, 39, 66, 67). Additionally, increasing kidney damage, presumably due to both the development and expansion of cysts and fibrosis (Table 1), caused a measurable decline in kidney function, detected by a rise in blood urea nitrogen (BUN) above the physiological range (18.6–32.9 mg/dl) by 9 months (Figure 2E and refs. 66–68). Further, histological analysis showed that at 12 months most Pkd1^RC/RC animals developed multiple ductal plate malformations, seen as microhamartomas (Figure 3B). These cystic lesions have been reported previously in Pkd1^+/null animals, are characteristic of human ADPKD, and are the progenitors of the liver cysts found in the majority of patients (34, 69). Pkd1^RC/+ animals did not develop any renal cysts, have any significant differences in renal function from WT, or develop any liver phenotype by 1 year of age (data not shown).

This analysis of homozygous animals showed that the Pkd1 p.R3277C allele is pathogenic, as all analyzed animals developed PKD, dismissing the notion that it may be one of the many silent, nonsynonymous variants detected in PKD1 (PKDB, http://pkdb.mayo.edu). On the other hand, the viability of the homozygotes showed that the allele is not fully penetrant, proving for the first time to our knowledge the existence of incompletely penetrant PKD1 alleles and highlighting their role as a disease modifier.

The Pkd1 p.R3277C allele modifies the Pkd1^+/null phenotype to early-onset disease. To directly test the proposed modifier role of Pkd1 p.R3277C and to mimic the genotype associated with human in utero–onset disease, we crossed Pkd1^RC/+ animals with a well-established Pkd1-null model, Pkd1^+/del2 (Figures 4 and 5, Supplemental Figure 3, Supplemental Table 2, and refs. 51, 59). Again, we observed the expected Mendelian ratios of offspring (data not shown), but in this case the progression of disease was much more aggressive. Pkd1^RC/del2 animals showed enlarged kidneys by P1, visible from gross anatomy, and a significantly greater %KW/BW compared with that of WT littermates (3.05% vs. 1.29%; Figure 4, A and C, and Supplemental Table 2). The %KW/BW increased rapidly from approximately 3% at P1 to approximately 25% at P25. In comparison, the %KW/BW at P1 of Pkd1^RC/del2 animals was significantly higher than that of homozygous animals at 3 months (P = 0.01). At the histological level, cysts were visible as early as E15.5, and both the number and size of cysts increased in an exponential fashion to at least P25 (Figure 5, Figure 6B, Table 1, and Supplemental Figures 2 and 3). Interestingly, multiple glomerular cysts were detectable at the early time points, highlighting a clear similarity with human in utero–onset ADPKD (Figure 5, insets, and refs. 59, 70). The rapidly expansive PKD was also associated with premature death, first noted at P20, and with a median survival at P28; 80% of animals were dead by P50 (Figure 4B). The cause of death was likely associated with renal failure, as indicated by marked renal cystic enlargement (70.2% of kidney area by P25; Table 1), increased fibrotic index (24.2% by P25; Figure 5 and Table 1), and uremia evident by a significant increase in BUN at P12 (Figure 4D and Supplemental Table 2). Animals surviving beyond P25 exhibited further evidence of disease progression with significantly elevated BUN levels (~178 mg/dl [P25] vs. ~314 mg/dl [P180], P = 0.006; Supplemental Table 2) and a decline in %KW/BW, which is likely associated with an increase in fibrotic tissue (~25% [P25] vs. ~9% [P180]; Figure 4A, Figure 5, and Supplemental Table 2), as with the Pkd1^RC/RC mice at 12 months. The reason that approximately 20% of animals survived much longer is unclear, but it is of note that this outbred population had milder disease during the explosive cyst expansion period (P12–P25), suggesting a less critical role for PC-1 during adulthood. Consistent with a dosage-dependent disease mechanism, Pkd1^RC/del2 animals had a much more rapid rise in cAMP, with P1 mice having higher cAMP levels than 12-month-old Pkd1^RC/RC mice (P = 0.0042) and BUN levels above the physiological average as early as P18 (Figure 2; Figure 4, D and E; Supplemental Tables 1 and 2; and ref. 68). In addition, Pkd1^RC/del2 animals presented with osteopenia and left ventricular hypertrophy (LVH), known extrarenal phenotypes observed in other Pkd1 models and patients with ADPKD (Supplemental Figure 4 and refs. 71–73). A decrease in trabecular and cortical bone mass was observed in Pkd1^RC/del2 males and females at P25, likely correlating with reduced PC-1 function in osteoblasts, but possibly also associated with secondary hyperparathyroidism (Supplemental Figure 4A, data on females not shown, and refs. 71, 72). Heart weight was also significantly increased, with the myocardium of the left ventricle significantly thickened (Supplemental Figure 4B) and evidence of LVH, possibly due to hypertension in the P25 Pkd1^RC/del2 animals with advanced cystic disease. No intracranial aneurysms were noted.

Figure 4

Pkd1^RC/null mice develop in utero/early-onset ADPKD. (A) Gross images of WT and Pkd1^RC/del2 kidneys from P1 to P180, illustrating early-onset/rapidly progressive disease. After P25, the kidney size decreased, likely due to increased fibrosis (Figure 5 and Table 1). The ruler depicts centimeters. (B) Kaplan-Meier curve of WT and Pkd1^RC/del2 mice. Eighty percent of Pkd1^RC/del2 mice died before P50 (median survival, P28 [arrow]; n = 55/group). (C–E) Graphical representations of %KW/BW, BUN, and cAMP from P1 to P25 of Pkd1^RC/del2 animals (Supplemental Table 2). (C) %KW/BW was significantly increased as early as P1 (3%) and rapidly climbed to 25% by P25. (D) BUN levels rose beyond the physiological average as early as P18. (E) cAMP levels were significantly above normal by P1. Values in C–E were obtained from n ≥ 5 at each time point for both WT and Pkd1^RC/del2 mice. P180 data can be found in Supplemental Table 2. Statistical values were obtained by the Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001); error bars indicate ± SD.

Figure 5

Histology of Pkd1^RC/del2 mice highlights early-onset and rapidly progressive cystic disease. Representative Masson Trichrome sections of Pkd1^RC/del2 and WT kidneys from E14.5 to P180. Kidney cysts were clearly visible as early as E16.5 (a few cysts were present at E15.5; Supplemental Figure 2) and rapidly increased in number/size with age (Table 1). Glomerular cysts were present as early as E16.5 (insets) (59). The kidneys of animals with longer survival (P180) were highly fibrotic, with little obvious healthy tissue. Scale bars: 100 μm (E14.5 to P1); 500 μm (P12); 750 μm (P25 and P180); 50 μm (insets).

Figure 6

Pkd1 p.R3277C mice show a switch in tubular cyst origin corresponding to age. (A) IF lectin labeling of PT (LTA) and CD (DBA). During early kidney development (P1), cysts originated mainly from PT in Pkd1^RC/del2 and Pkd1^RC/RC animals, with only rare CD cysts (arrowheads). Later in development (P12), a switch to more CD cysts occurred that became more prominent after kidney development (P25, Pkd1^RC/del2; 3 months, Pkd1^RC/RC), with only a few remaining PT cysts (arrows). With disease progression, an increasing number of cysts seemed to dedifferentiate. Asterisks indicate a lack of labeling with tubule markers. Scale bars: 100 μm (P1); 200 μm (P12); 300 μm (P25); 500 μm (3 months and 12 months). (B) Quantification of cyst origin at P1, P12, and P25 for Pkd1^RC/del2 animals and (C) P1, P12, 3 months, and 12 months for Pkd1^RC/RC animals (n = 3; Table 1). Significance is based on cyst number. Statistical values were obtained by the Student’s t test (*P < 0.05, **P < 0.01). Data are not shown for distal tubule (PNA) and loop of Henle (THP), as they accounted for <1% of the total cyst number.

This compound heterozygous model clearly demonstrates the role of Pkd1 p.R3277C as a modifying allele. The Pkd1^+/del2 mice did not exhibit an ADPKD phenotype, lacking renal cysts and functional changes in kidneys at 1 year of age (data not shown), contrasting dramatically with the rapid disease progression in Pkd1^RC/del2 animals (51). These data prove that in trans inheritance of a null and this incompletely penetrant allele, without the requirement of other genetic events, results in early-onset ADPKD, suggesting a clear dosage pathomechanism of cyst initiation and/or progression.

Pkd1 p.R3277C mice show a switch in tubular cyst origin correspondent with age and an increase in proliferation. The models described here developed cysts due to a consistent reduction of PC-1, compared with conditional knockouts, in which only the segments engineered to be null for PC-1 became cystic. Consequently, answering the question of where and when cysts develop in our model is relevant to human PKD1. Using immunofluorescence (IF) markers for each tubule segment (lotus tetragonolobus agglutinin [LTA] – proximal tubule [PT], peanut agglutinin [PNA] – distal tubule, Tamm-Horsfall glycoprotein [THP] – loop of Henle, and Dolichos biflorus agglutinin [DBA] – CD), we examined the cystic origin throughout disease development. In Pkd1^RC/del2 animals at P1, 82% of cysts were of PT and 17% of cysts were of CD origin. However, by P25, these animals had only 6% PT and 76% CD cysts. Interestingly, by this time point, 18% of cysts (16% of kidney area) could not be recognized by any of the tubule markers used, including PNA and THP (Figure 6, A and B, and Table 1), suggesting that they had become dedifferentiated. The switch in cyst origin was also noticeable when looking at cyst area rather than cyst number, with the area occupied by PT cysts falling from 29.2% at P1 to 2.9% at P25. During the same period, CD cysts rose from 4.2% of kidney area to 51.3% (Table 1). In Pkd1^RC/RC mice, a similar switch was observed; at P1, 90% of cysts were of PT and 8% were of CD origin, while at 3 months, 76% were of CD and 15% were of PT origin (Figure 6, A and C, and Table 1). Interestingly, at 12 months of age, the percentage of PT cysts increased as compared with that at 3 months, from 15% to 29%; however, most of the PT cysts corresponded to mildly dilated tubules, whereas the majority of larger cysts were either of CD origin (56%, 13.6% of area) or dedifferentiated (15%, 8.5% of area; Figure 6, A and C, and Table 1). The PT dilatation at this stage may reflect acquired renal cystic disease associated with advanced renal insufficiency as seen in human ADPKD (74, 75).

This analysis of both models shows a consistent shift in cyst origin, with a predominance of PT kidney cysts during kidney development, which continues in the mouse until approximately 2 weeks postnatally, to a predominance of CD cysts once the kidney has fully developed (37). This is consistent with PT cysts predominating in early-onset ADPKD cases, with a similar shift from PT to CD cysts associated with completion of kidney development in ARPKD (28, 29, 39, 76, 77). The observation that the majority of adult cysts are of CD origin correlates with the AVPR2 antagonists showing some benefit in ADPKD (31, 32, 78).

PCNA staining of CD tubules and cysts in P18 Pkd1^RC/del2 animals highlighted that cyst expansion/size correlates with an increase in proliferation (Figure 7 and Supplemental Table 3). Proliferation was greatest in the largest cysts (5.50% vs. 3.09% in small cysts, P = 0.002), but, notably, even nondilated tubules showed increased proliferation compared with WT (1.71% vs. 0.94%, P = 0.009). The role of proliferation to cyst expansion in ADPKD is a controversial one, but when analysis is limited to tubular/cystic epithelia, increased cell division clearly occurs (36, 37, 79).

Figure 7

Cystogenesis of Pkd1^RC/del2 mice is associated with an increase in proliferation. (A) Representative images of kidney tissue from P18 Pkd1^RC/del2 mice, indicating increased proliferation in noncystic and cystic CDs (DBA) compared with that in WT. Reports on associations between cyst expansion and proliferation are controversial, but analysis of the CD showed a clear increase in cell division (36, 37, 79). Scale bars: 20 μm. (B) Graph summarizing data of 100 nondilated CD cysts and 20 small (<50 cells [S]), 10 medium (50–200 cells [M]), and 5 large (>200 cells) CD cysts (Supplemental Table 3) (n = 3/group). Statistical values were obtained by the Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001); error bars indicate ± SD.

The Pkd1 p.R3277C allele is associated with elongated CD primary cilia. In syndromic forms of PKD, the mutated proteins are proposed to regulate ciliary protein trafficking and, hence, influence ciliogenesis; therefore, structural ciliary abnormalities, including length defects, have been associated with these diseases (8–10). However, in PKD1, the majority of studies have not detected cilia length defects, but the data are not conclusive, as these studies were largely conducted by light microscopy using cultured cells (14, 15, 80, 81). To analyze whether PC-1 may have a role in ciliary maintenance, we analyzed renal CD and bile duct cilia length using scanning electron microscopy (SEM). Cilia of nondilated and cystic CDs were significantly longer in P25 Pkd1^RC/del2 animals (mean = 4.98 μm) and 12-month-old Pkd1^RC/RC animals (mean = 3.02 μm) compared with those in WT controls (mean = 1.98 μm; Figure 8A and Supplemental Table 4 [WT vs. Pkd1^RC/del2, P = 0.0012; WT vs. Pkd1^RC/RC, P = 5.65 × 10^–4]), with a significant difference between Pkd1^RC/del2 mice and their homozygous counterparts (P = 0.0066). Interestingly, these length defects were not related to cystic dilatation, since measurements in cystic and noncystic CDs in both animal models did not significantly differ (Supplemental Table 4). Furthermore, this observation was independent of age, since the length of WT cilia did not change from P25 to 12 months of age (Supplemental Table 4). Cilia within normal bile ducts did not vary significantly between genotypes (Figure 8B, Supplemental Table 5, and ref. 79). However, bile duct cilia were elongated in 1 microhamartoma that was found by SEM (P = 1.49 × 10^–6; Figure 8B and Supplemental Table 5). Therefore, within the kidney, the dosage dependence of cilia length abnormality indicates that the level of functional PC-1 is directly associated with the observed ciliary defect, whereas in the liver, cilia elongations seem related to bile duct abnormalities.

Figure 8

Primary cilia are elongated in Pkd1 p.R3277C CDs but not normal bile ducts. SEM analysis of primary cilia length (A) in the CDs (Supplemental Table 4) and (B) bile ducts (Supplemental Table 5) of WT, Pkd1^RC/del2, and Pkd1^RC/RC mice at the ages indicated (n = 3 animals/group). (A) Representative SEM images of CDs and individual cilia. Scale bars: 2 μm (bottom row); 10 μm (WT, top row); 15 μm (Pkd1^RC/del2 and Pkd1^RC/RC, top row) (left). Graph summarizing >100 cilia measurements (~40 cilia in nondilated, ~30 in dilated, and ~30 in cystic CDs) in mutant animals and >50 cilia of nondilated CDs in WT animals (P25 and 12 months). The level of functional Pkd1 inversely correlated with cilia length, indicating a role of PC-1 in cilia maintenance (right). (B) Representative SEM image of WT and Pkd1^RC/RC bile duct and one microhamartoma. Bile duct cilia of the microhamartoma were significantly elongated (6.24 ± 1.57 μm, P < 0.001) (left). Scale bars: 1 μm (WT and Pkd1^RC/RC, bottom row); 2 μm (microhamartoma, bottom row); 50 μm (Pkd1^RC/RC, top row); 100 μm (WT and microhamartoma, top row). Graph summarizing >50 cilia measurements of >5 bile ducts per animal (right). Statistical values were obtained by the Student’s t test (**P < 0.01, ***P < 0.001); error bars indicate ± SD.

The Pkd1 p.R3277C allele alters PC-1 cleavage and folding/maturation. PC-1 is a multidomain glycoprotein that is cleaved at a G protein–coupled receptor proteolytic site (GPS; Figure 9A and refs. 82, 83). After cleavage, the N-terminal product (NTP) and the C-terminal product (CTP) are thought to remain tethered and functionally act together (Figure 9A and ref. 83). It has also been reported that endogenous PC-1 is observed as 2 distinct glycoforms, endoglycosidase H–resistant (EndoH-resistant) (NTP**) and -sensitive (NTP*) forms (84). This implies a difference in protein trafficking of these glycoforms, as EndoH resistance is obtained in the trans-Golgi, in which high-mannose oligosaccharides are converted to complex saccharides by the addition of N-acetylglucosamine to form secreted or membrane-bound, mature, forms of the protein (85). We confirmed these findings by Western blot (WB) analysis of kidney membrane protein with 2 products found in the untreated sample, one being the EndoH stable form (no shift in size [NTP**]), and the other being the sensitive form that reduces in size upon EndoH treatment (loss of all N-linked glycosylation [NTP*]; Figure 9B). Treatment with peptide-N-Glycosidase F (PNGaseF), an enzyme that removes all N-linked sugars independent of maturation status, reduced the size of the 2 products to that of the EndoH-sensitive form, showing that the difference in size of the 2 untreated products is only due to variable glycosylation status. Analysis of urinary ELVs, showed that it is the EndoH-resistant PC-1 that is present in these vesicles (Figure 9B and refs. 21, 22). Comparison of ELV PC-1 with an exogenously expressed PC-1 GPS cleavage mutant (E2771K; Figure 9A) showed that the PNGaseF-treated full-length PC-1 was much larger than the treated ELV product, indicating that only the cleaved product is found in ELVs (NTP**; Figure 9C). Also, only the cleaved product (NTP) was observed in kidney tissue preparations, consistent with the significance of this cleavage event for normal function (83).

Figure 9

Pkd1 p.R3277C is a temperature-sensitive folding/maturation mutant. (A) The PC-1 protein (full-length [FL]) is cleaved into NTPs and CTPs (estimated sizes of glycosylated PC-1 are indicated in parentheses) (82–84). The positions of the incompletely penetrant variants (p.R3277C, p.R2220W) and the cleavage mutant p.E2771K are shown (29, 59, 83). NTP*, EndoH-sensitive, immature; NTP**, EndoH-resistant, mature. (B) WB of PC-1 showing the 2 glycoforms in kidney membranes and only the mature form in urinary ELVs. (C) WB of the exogenously expressed cleavage mutant Pkd1 p.E2771K (ex) and WT urinary ELVs (83). The endogenous (end) PC-1 protein is also seen at a low level. The endogenous, mature, cleaved PC-1 (NTP**) runs at a size similar to the ex.FL PC-1 p.E2771K (immature form). (D) WB and cleavage quantification (n = 6 transfections) of exogenously expressed WT and mutant forms of PC-1 shown in A. (E) WB and quantification of PC-1 levels in WT and Pkd1^RC/RC urinary ELVs at 1 and 12 months (n = 4 urine collections of 6 animals/group). ELVs from Pkd2^WS25/null mice show PC-1 levels in Pkd2 cystic kidneys (57). (F) EndoH assay of urinary Pkd1^RC/RC ELVs (analyzed on separate gels as indicated by the white line). (G) WB of Pkd1^RC/RC kidney membrane preparation and urinary ELVs compared with WT. (H) WB and quantification (n = 3 experiments) of PC-1 in cell media–isolated ELVs from primary CD cells isolated from WT and Pkd1^RC/RC P30 kidneys at 37°C and 33°C. PDCD6IP is an ELV expressed control protein (21). E, EndoH; P, PNGaseF. Statistical values were obtained by the Student’s t test (***P < 0.001); error bars indicate ± SD.

To analyze the possible pathomechanism of Pkd1 p.R3277C, we first investigated whether the allele altered PC-1 cleavage by using an exogenously expressed C-terminally tagged PC-1 construct (Figure 9A). WB using an antibody to the C-terminal V5 epitope tag highlighted significantly reduced levels of cleaved p.R3277C PC-1 compared with WT (~76% of WT, Figure 9D). However, we also tested a second suspected incompletely penetrant Pkd1 allele (p.R2220W) that was found in trans with PKD1 p.R3277C resulting in early-onset ADPKD, and this PC-1 product was far more resistant to cleavage (~26% of WT) (29). The p.R2220W variant is localized within the receptor for egg jelly domain (REJ), a domain previously shown to be important for GPS cleavage (83). These results suggested that inefficient cleavage may be a pathomechanism associated with some incompletely penetrant PC-1 alleles but may not be central to the reduced function of Pkd1 p.R3277C; hence, we investigated further possible mechanisms. Since PKD1 p.R3277C is 2 amino acids from the second transmembrane domain (TMII; Figure 9A), we tested whether TMII integrates appropriately into the lipid bilayer in the presence of this missense change. Using exogenous expression of a partial PC-1 construct with a C-terminal glycosylation reporter, we assayed successful TMII integration. However, no noticeable difference in membrane integration ability was found compared with WT (Supplemental Figure 5).

Since the cleaved, mature PC-1 is found in urinary ELVs, it was possible to use urinary ELVs from WT and mutant mice to assay possible folding or trafficking defects. We isolated ELVs from a 12-hour urine collection of 1- and 12-month-old Pkd1^RC/RC mice and found from WB analysis that they had an approximately 61% reduction of the mature, EndoH-resistant PC-1 glycoform compared with WT; all mutant PC-1 found in ELVs was EndoH stable, highlighting that the mutant protein trafficked appropriately (Figure 9, E and F). Analysis of ELVs collected from Pkd2^WS25/– mice that have a similar level of cystogenesis as the analyzed Pkd1^RC/RC mice showed unaltered levels of PC-1 NTP** compared with WT mice, highlighting that the reduction in PC-1 in the Pkd1^RC/RC mice is not due to the presence of cystic disease (57). To confirm these findings in the kidney, we performed WB of kidney membrane preparations of 1-month-old littermates. Consistent with the results from the urinary ELVs, Pkd1^RC/RC animal kidneys showed a reduced level of the EndoH stable PC-1 NTP** compared with WT (Figure 9G).

These results suggested that the reduction of EndoH stable PC-1 p.R3277C may result from a maturation defect between the ER and the trans-Golgi or from a folding defect within the ER (85). To investigate a possible folding defect, primary cells isolated from Pkd1^RC/RC kidney CDs were grown at a reduced temperature to create an environment more permissive for protein folding (33°C, compared with 37°C) (86). Indeed, the temperature shift increased the relative amount of the EndoH stable PC-1 glycoform in cellular ELVs to a level close to that in WT ELVs and significantly different from that at the higher temperature (Figure 9H, P = 0.00089 [Pkd1^RC/RC, 33°C vs. 37°C], P = 0.00017 [37°C, WT vs. Pkd1^RC/RC], P = 0.025 [33°C, WT vs. Pkd1^RC/RC]). Consequently, we propose that the Pkd1 p.R3277C allele is a temperature-sensitive folding mutant. However, accumulation of the mutant protein was not sufficient to trigger ER stress (Supplemental Figure 6). Therefore, due to the folding defect (and reduced cleavage), p.R3277C results in an approximately 60% reduction of mature PC-1 in Pkd1^RC/RC mice.

In this study, we set out to understand the etiology and pathomechanism associated with the phenotypic heterogeneity of PKD1, which at its extremes ranges from mild PKD in old age without renal insufficiency to in utero lethality (6, 43, 44). Emerging family data suggested that in trans inheritance of a null and an incompletely penetrant PKD1 allele could result in the devastating early-onset phenotype (29, 59–61). To rigorously test this theory, we developed and characterized a knockin mouse model mimicking a naturally occurring PKD1 variant, p.R3277C, highlighted by recent family studies (29, 59). Mirroring the human studies, Pkd1^RC/RC animals developed gradual cystic disease over 1 year, while Pkd1^RC/null mice had rapidly progressive disease, proving the significance of this combination of alleles. This simple pathomechanism is consistent with the description by Zerres (43), nearly 20 years ago, of a high recurrence risk of severe disease in siblings of an early-onset case in families with otherwise typical ADPKD. Data from a handful of additional families, highlighting other possible incompletely penetrant alleles, indicate a broader applicability, but studies of larger populations and better definition of these alleles are needed to determine the full importance of in trans inheritance to explaining early-onset disease (29, 59–62). Indeed, other mechanisms, such as modifier loci beyond the disease gene and environmental factors, also significantly influence disease variability. Early-onset severe ADPKD has been linked to contiguous deletion of PKD1 and TSC2 as well as co-inheritance of a PKD1 and a HNF1B or PKHD1 allele, highlighting the fact that digenic inheritance may account for some of the phenotypic variability seen in ADPKD (45–47, 62). Further, 3 SNPs in DKK3 have been associated with severity of renal disease in a candidate gene association study, while nongenetic factors, such as male hormones, caffeine, and smoking, have been suggested as risk factors in ADPKD (87–89). Careful analysis is now required to determine the significance of each of these elements with respect to disease severity and to identify other genetic and environmental disease modifiers.

Insights from our model systems into the etiology of in utero ADPKD and the functional analysis of the mutant PC-1 provide further clues to the underlying pathomechanism of typical ADPKD. Although cystogenesis is often considered a cellular recessive process, in our case both the Pkd1^RC/RC and Pkd1^RC/del2 animals have a Pkd1 mutation to both alleles, making it unlikely that a somatic Pkd1 mutation is required for cyst initiation. Instead, it seems more plausible that the dosage of PC-1 is underlying disease severity, and, by expression analysis of PC-1 in ELVs (an abundant site of polycystin expression that is readily isolated), we have precisely quantified the levels of mature protein associated with progressive adult disease (~40% in Pkd1^RC/RC) and inferred a level of mature protein associated with rapidly progressive, early-onset disease (approximately 20% in Pkd1^RC/del2 and ref. 21). Hence, it seems reasonable to propose that ADPKD severity is directly related to PC-1 dosage; adult-onset disease is usually caused by a 50% PC-1 reduction (haploinsufficiency), while further reductions due to variants on the “normal” allele cause severe early-onset presentations. The progressive PKD associated with transgenic miRNA reduction of Pkd1 by 60% to 70% is consistent with the dosage model presented here (90). Although, there is strong evidence of a second-hit mechanism, with detected somatic mutations in large cysts, we propose that they are not necessary for cyst initiation and instead may play a role in cyst progression by providing cysts a growth or survival advantage (53–56).

The combination of a dosage pathomechanism in ADPKD and the realization that variants of the disease gene inherited from the “normal” parent can significantly influence the PKD1 phenotype have profound implications for understanding the variable expressivity of ADPKD. We have focused on extreme early-onset disease through co-inheritance of an incompletely penetrant and fully penetrant allele, but some alleles may have a lesser effect on the level of functional PC-1 and result in less extreme effects, such as ESRD in early adulthood. While efforts to identify genetic modifiers of the PKD1 phenotype through genome-wide association studies and exome or whole-genome sequencing are underway, it seems plausible that variants to the “normal” PKD1 allele are a major disease modifier and that complex allelic inheritance is relevant more generally to the variable expressivity of ADPKD. Hence, better classification of variants with incompletely penetrant potential will be of increasing prognostic value. Further, it seems likely that this pathomechanism is important to the phenotypic variability of other haploinsufficient, dominant diseases, although the large array of “normal” variants cataloged at PKD1 indicate that it may be an extreme example (PKDB, http://pkdb.mayo.edu) (91, 92).

Our models not only allow the genetic mechanism of disease to be analyzed but also provide insight into the pathophysiology. We have shown that increased proliferation is an important factor in cyst expansion. Of further interest, in both models cyst initiation occurred embryonically (E15.5–E16.5), but PKD developed much more quickly in the model with the lower level of PC-1. It is not known whether this dosage difference influences the rate of cyst initiation or expansion, but in this study, it seems that dosage may be relevant in both instances. Previous comparisons of PKD1 and PKD2 concluded that it was the rate of cyst initiation rather than rate of expansion that makes PKD1 more severe (88). Interestingly, analysis of conditional Pkd1 models showed that PKD severity altered radically depending upon the time of gene inactivation, with a tipping point between rapid and slow expansion of disease roughly corresponding to the completion of kidney development (37). Hence, developing tubules with high proliferative indices may be more sensitive to PC-1 reduction/loss. The rate of epithelial cell proliferation during development may also explain the switch with completion of kidney development from predominately PT to CD cysts observed in our study and previously reported in human ADPKD (28–30). The proliferation of immature early tubules (S-shaped bodies) is very high and remains high throughout embryonic life (93). In later development, when the epithelium differentiates into nephron segments recognizable by light microscopy, the proliferative index decreases substantially but remains elevated in the distal nephron and CD, whereas it becomes very low in the PT (93). In pediatric and adult kidneys, proliferative indices are very low in all tubular segments but remain higher in the CD compared with those in PT (94). Thus, the underlying rate of epithelial cell proliferation may explain both the increased susceptibility of the developing kidney to cystogenesis and the switch from predominantly PT to predominantly CD cysts. Understanding this switch is important, since theoretical considerations have highlighted that embryonic cysts, by growing at faster rates compared with cysts initiated later in life, contribute majorly to the total cyst burden (95).

A major argument for the 2-hit hypothesis is that the cyst development in ADPKD is focal, each the result of an individual somatic mutation (50). However, in the models presented here in which somatic mutations to the normal allele are not likely to be important, cyst development is still focal. This implies that a threshold model is important where stochastic factors, such as variation in the PC-1 expression level from cell to cell and so-called third hits (perhaps to be considered second hits now), such as renal damage or other factors triggering proliferation, may be key to driving a cell or segment of tubule into cystic transformation (96–98).

The role primary cilia play in ADPKD cystogenesis remains controversial, with the majority of studies focused on downstream signaling defects (16). Little attention has been given to the structural appearance of ADPKD primary cilia, despite length and other structural defects detected in other PKD-related ciliopathies (8–15). Here, we report for the first time to our knowledge a clear PC-1 dosage-dependent increase in cilia length in normal and cystic CDs. Increased cilia length has been associated with enhanced cAMP levels and injury/inflammation, the latter of which may be associated with the observed increase in cilia length within the microhamartoma; however, within the kidney, it is of interest that we observed the length increase independent of the cystogenic state of the tubule (80, 99, 100). This highlights a potential direct role of PC-1 in cilia maintenance. Alternatively, the length increase could be a compensatory response to reduced functional PC-1, extending the platform hosting the flow or ELV detector when less PC-1 is present. Furthermore, the clear description of the processing of PC-1 into mature and immature EndoH forms presented here should stimulate further study of the localization of this large and complex protein. Here, we have shown that just the resistant form can be found in urinary ELVs; however, the glycoform found on primary cilia or the plasma membrane remains unknown (21).

One of the most valuable features of this study is likely the development of new Pkd1 models, which are well suited for therapeutic testing in comparison with current models. As described in the Introduction, the present fully inactivating models are unsatisfactory, and since conditional models lose all functional PC-1 at one time, with the affected kidney segment dictated by the Cre promoter, they do not reflect human disease development. Previously described hypomorphic models have approximately 70% to 80% PC-1 reduction, leading to very rapid disease with high levels of fibrosis early in life, similar to our approximately 80%-reduction model (Pkd1^RC/del2), and thus leaving a short therapeutic window (40, 41). Our 60%-reduction model (Pkd1^RC/RC) appears to best mimic human ADPKD (over a shorter timescale) with a 9-month progressive expansion and then a slight reduction in KV associated with fibrosis as ESRD approaches (101). Other similarities include those observed physiologically, with increased cAMP and elevation of BUN indicating renal insufficiency after 9 months, and histologically, with progressive cyst development and expansion, and a switch from PT to CD cysts following renal development. The temperature-sensitive nature of p.R3277C suggests that interventions to promote more efficient folding of PC-1 may increase the level of functional protein. This indicates that, in addition to the current experimental therapies that focus on cAMP and other signaling defects, chaperone-enhancing or proteasome-inhibiting strategies may also be effective in a subset of patients with missense mutations to PKD1 or PKD2 (102, 103). Last, a dosage model of ADPKD suggests that approaches to increase the level of the normal PKD1 (PKD2) allele in patients with typical ADPKD, even by a modest amount, could have a dramatic clinical benefit.

View Supplemental data

We thank the Mayo Gene Targeting Core, especially Jan van Deursen, Ming Li, and Wei Zhou, for help in generating the chimeric knockin mice; Jennifer Westendorf and Meghan McGee-Lawrence for assisting with the bone morphology analysis; Bruce Knudsen for training K. Hopp at US analysis of mouse kidneys; the Mayo Clinic Electron Microscopy Core, especially Scott Gamb and Jason Bakeberg, for training K. Hopp to use SEM on kidney tissue; Xiaofang Wang for her assistance with the cAMP assays; and Tatyana Masyuk for help with the PCNA staining. This work was supported by an AHA predoctoral fellowship to K. Hopp (11Pre5250020), NIDDK grant DK058816, the Mayo Translational PKD Center (DK090728), and the Zell Family Foundation.

Address correspondence to: Peter C. Harris, Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. Phone: 507.266.0541; Fax: 507.266.9315; E-mail: harris.peter@mayo.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(11):4257–4273. doi:10.1172/JCI64313.

See the related article at Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease.

1. Iglesias CG, Torres VE, Offord KP, Holley KE, Beard CM, Kurland LT. Epidemiology of adult polycystic kidney disease, Olmsted County, Minnesota: 1935–1980. Am J Kidney Dis. 1983;2(6):630–639.
   View this article via: PubMed Google Scholar
2. Dalgaard OZ. Bilateral polycystic disease of the kidneys; a follow-up of two hundred and eighty-four patients and their families. Acta Med Scand Suppl. 1957;328:1–255.
   View this article via: PubMed Google Scholar
3. Mochizuki T, et al. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science. 1996;272(5266):1339–1342.
   View this article via: PubMed CrossRef Google Scholar
4. Rossetti S, et al. Comprehensive molecular diagnostics in autosomal dominant polycystic kidney disease. J Am Soc Nephrol. 2007;18(7):2143–2160.
   View this article via: PubMed CrossRef Google Scholar
5. [No authors listed]. The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. The European Polycystic Kidney Disease Consortium. Cell. 1994;77(6):881–894.
   View this article via: PubMed CrossRef Google Scholar
6. Harris PC, Torres VE. Polycystic kidney disease, autosomal dominant. In: Pagon RA, et al., eds. GeneReviews. Seattle, Washington, USA: University of Seattle; 1993(updated 2011):NBK1246.
7. Torres VE, Harris PC, Pirson Y. Autosomal dominant polycystic kidney disease. Lancet. 2007;369(9569):1287–1301.
   View this article via: PubMed CrossRef Google Scholar
8. Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med. 2011;364(16):1533–1543.
   View this article via: PubMed CrossRef Google Scholar
9. Gascue C, Katsanis N, Badano JL. Cystic diseases of the kidney: ciliary dysfunction and cystogenic mechanisms. Pediatr Nephrol. 2011;26(8):1181–1195.
   View this article via: PubMed CrossRef Google Scholar
10. Tammachote R, et al. Ciliary and centrosomal defects associated with mutation and depletion of the Meckel syndrome genes MKS1 and MKS3. Hum Mol Genet. 2009;18(17):3311–3323.
    View this article via: PubMed CrossRef Google Scholar
11. Woollard JR, et al. A mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation. Kidney Int. 2007;72(3):328–336.
    View this article via: PubMed CrossRef Google Scholar
12. Masyuk TV, et al. Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat. Gastroenterology. 2003;125(5):1303–1310.
    View this article via: PubMed CrossRef Google Scholar
13. Xu C, 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(3):F930–F945.
    View this article via: PubMed CrossRef Google Scholar
14. Nauli SM, et al. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet. 2003;33(2):129–137.
    View this article via: PubMed CrossRef Google Scholar
15. Kurbegovic A, Cote O, Couillard M, Ward CJ, Harris PC, Trudel M. Pkd1 transgenic mice: adult model of polycystic kidney disease with extrarenal and renal phenotypes. Hum Mol Genet. 2010;19(7):1174–1189.
    View this article via: PubMed CrossRef Google Scholar
16. Torres VE, Harris PC. Autosomal dominant polycystic kidney disease: the last 3 years. Kidney Int. 2009;76(2):149–168.
    View this article via: PubMed CrossRef Google Scholar
17. Yoder BK, Hou X, Guay-Woodford LM. The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol. 2002;13(10):2508–2516.
    View this article via: PubMed CrossRef Google Scholar
18. Qian F, Germino FJ, Cai Y, Zhang X, Somlo S, Germino GG. PKD1 interacts with PKD2 through a probable coiled-coil domain. Nat Genet. 1997;16(2):179–183.
    View this article via: PubMed CrossRef Google Scholar
19. Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB. Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr Biol. 2002;12(11):R378–380.
    View this article via: PubMed CrossRef Google Scholar
20. Hanaoka K, et al. Co-assembly of polycystin-1 and -2 produces unique cation-permeable currents. Nature. 2000;408(6815):990–994.
    View this article via: PubMed CrossRef Google Scholar
21. Hogan MC, et al. Characterization of PKD protein-positive exosome-like vesicles. J Am Soc Nephrol. 2009;20(2):278–288.
    View this article via: PubMed CrossRef Google Scholar
22. Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A. 2004;101(36):13368–13373.
    View this article via: PubMed CrossRef Google Scholar
23. Sharif-Naeini R, et al. Polycystin-1 and -2 dosage regulates pressure sensing. Cell. 2009;139(3):587–596.
    View this article via: PubMed CrossRef Google Scholar
24. Steigelman KA, et al. Polycystin-1 is required for stereocilia structure but not for mechanotransduction in inner ear hair cells. J Neurosci. 2011;31(34):12241–12250.
    View this article via: PubMed CrossRef Google Scholar
25. Bataille S, et al. Association of PKD2 (polycystin 2) mutations with left-right laterality defects. Am J Kidney Dis. 2011;58(3):456–460.
    View this article via: PubMed CrossRef Google Scholar
26. Battini L, et al. Loss of polycystin-1 causes centrosome amplification and genomic instability. Hum Mol Genet. 2008;17(18):2819–2833.
    View this article via: PubMed CrossRef Google Scholar
27. Yamaguchi T, Wallace DP, Magenheimer BS, Hempson SJ, Grantham JJ, Calvet JP. Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype. J Biol Chem. 2004;279(39):40419–40430.
    View this article via: PubMed CrossRef Google Scholar
28. Torres VE, Harris PC. Mechanisms of Disease: autosomal dominant and recessive polycystic kidney diseases. Nat Clin Pract Nephrol. 2006;2(1):40–55.
    View this article via: PubMed CrossRef Google Scholar
29. Vujic M, et al. Incompletely penetrant PKD1 alleles mimic the renal manifestations of ARPKD. J Am Soc Nephrol. 2010;21(7):1097–1102.
    View this article via: PubMed CrossRef Google Scholar
30. Baert L. Hereditary polycystic kidney disease (adult form): a microdissection study of two cases at an early stage of the disease. Kidney Int. 1978;13(6):519–525.
    View this article via: PubMed CrossRef Google Scholar
31. Higashihara E, et al. Tolvaptan in autosomal dominant polycystic kidney disease: three years’ experience. Clin J Am Soc Nephrol. 2011;6(10):2499–2507.
    View this article via: PubMed CrossRef Google Scholar
32. Torres VE, et al. Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease. Nat Med. 2004;10(4):363–364.
    View this article via: PubMed Google Scholar
33. Wilson PD. Mouse models of polycystic kidney disease. Curr Top Dev Biol. 2008;84:311–350.
    View this article via: PubMed Google Scholar
34. Lu W, et al. Late onset of renal and hepatic cysts in Pkd1-targeted heterozygotes. Nat Genet. 1999;21(2):160–161.
    View this article via: PubMed CrossRef Google Scholar
35. Lu W, et al. Perinatal lethality with kidney and pancreas defects in mice with a targetted Pkd1 mutation. Nat Genet. 1997;17(2):179–181.
    View this article via: PubMed Google Scholar
36. Lantinga-van Leeuwen IS, Leonhard WN, van der Wal A, Breuning MH, de Heer E, Peters DJ. 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(24):3188–3196.
    View this article via: PubMed CrossRef Google Scholar
37. Piontek K, Menezes LF, Garcia-Gonzalez MA, Huso DL, Germino GG. A critical developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1. Nat Med. 2007;13(12):1490–1495.
    View this article via: PubMed CrossRef Google Scholar
38. Takakura A, Contrino L, Beck AW, Zhou J. Pkd1 inactivation induced in adulthood produces focal cystic disease. J Am Soc Nephrol. 2008;19(12):2351–2363.
    View this article via: PubMed CrossRef Google Scholar
39. Starremans PG, 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(12):1394–1405.
    View this article via: PubMed CrossRef Google Scholar
40. Jiang ST, et al. Defining a link with autosomal-dominant polycystic kidney disease in mice with congenitally low expression of Pkd1. Am J Pathol. 2006;168(1):205–220.
    View this article via: PubMed CrossRef Google Scholar
41. Lantinga-van Leeuwen IS, et al. Lowering of Pkd1 expression is sufficient to cause polycystic kidney disease. Hum Mol Genet. 2004;13(24):3069–3077.
    View this article via: PubMed CrossRef Google Scholar
42. Hateboer N, et al. Comparison of phenotypes of polycystic kidney disease types 1 and 2. Lancet. 1999;353(9147):103–107.
    View this article via: PubMed CrossRef Google Scholar
43. Zerres K, Rudnik-Schoneborn S, Deget F. Childhood onset autosomal dominant polycystic kidney disease in sibs: clinical picture and recurrence risk. German Working Group on Paediatric Nephrology (Arbeitsgemeinschaft fuer Padiatrische Nephrologie). J Med Genet. 1993;30(7):583–588.
    View this article via: PubMed CrossRef Google Scholar
44. Bergmann C, Bruchle NO, Frank V, Rehder H, Zerres K. Perinatal deaths in a family with autosomal dominant polycystic kidney disease and a PKD2 mutation. N Engl J Med. 2008;359(3):318–319.
    View this article via: PubMed CrossRef Google Scholar
45. Consugar MB, et al. Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome. Kidney Int. 2008;74(11):1468–1479.
    View this article via: PubMed CrossRef Google Scholar
46. Sampson JR, et al. Renal cystic disease in tuberous sclerosis: role of the polycystic kidney disease 1 gene. Am J Hum Genet. 1997;61(4):843–851.
    View this article via: PubMed CrossRef Google Scholar
47. Brook-Carter PT, et al. Deletion of the TSC2 and PKD1 genes associated with severe infantile polycystic kidney disease--a contiguous gene syndrome. Nat Genet. 1994;8(4):328–332.
    View this article via: PubMed Google Scholar
48. Pei Y, et al. Bilineal disease and trans-heterozygotes in autosomal dominant polycystic kidney disease. Am J Hum Genet. 2001;68(2):355–363.
    View this article via: PubMed CrossRef Google Scholar
49. Connor A, et al. Mosaicism in autosomal dominant polycystic kidney disease revealed by genetic testing to enable living related renal transplantation. Am J Transplant. 2008;8(1):232–237.
    View this article via: PubMed Google Scholar
50. Harris PC. What is the role of somatic mutation in autosomal dominant polycystic kidney disease? J Am Soc Nephrol. 2010;21(7):1073–1076.
    View this article via: PubMed CrossRef Google Scholar
51. Muto S, et al. Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant. Hum Mol Genet. 2002;11(15):1731–1742.
    View this article via: PubMed CrossRef Google Scholar
52. Paterson AD, Wang KR, Lupea D, St George-Hyslop P, Pei Y. Recurrent fetal loss associated with bilineal inheritance of type 1 autosomal dominant polycystic kidney disease. Am J Kidney Dis. 2002;40(1):16–20.
    View this article via: PubMed CrossRef Google Scholar
53. Brasier JL, Henske EP. Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis. J Clin Invest. 1997;99(2):194–199.
    View this article via: JCI PubMed CrossRef Google Scholar
54. Qian F, Watnick TJ, Onuchic LF, Germino GG. The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease type I. Cell. 1996;87(6):979–987.
    View this article via: PubMed CrossRef Google Scholar
55. Pei Y, et al. Somatic PKD2 mutations in individual kidney and liver cysts support a “two-hit” model of cystogenesis in type 2 autosomal dominant polycystic kidney disease. J Am Soc Nephrol. 1999;10(7):1524–1529.
    View this article via: PubMed Google Scholar
56. Koptides M, Hadjimichael C, Koupepidou P, Pierides A, Constantinou Deltas C. Germinal and somatic mutations in the PKD2 gene of renal cysts in autosomal dominant polycystic kidney disease. Hum Mol Genet. 1999;8(3):509–513.
    View this article via: PubMed CrossRef Google Scholar
57. Wu G, et al. Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell. 1998;93(2):177–188.
    View this article via: PubMed CrossRef Google Scholar
58. Kim I, et al. Polycystin-2 expression is regulated by a PC2-binding domain in the intracellular portion of fibrocystin. J Biol Chem. 2008;283(46):31559–31566.
    View this article via: PubMed CrossRef Google Scholar
59. Rossetti S, et al. Incompletely penetrant PKD1 alleles suggest a role for gene dosage in cyst initiation in polycystic kidney disease. Kidney Int. 2009;75(8):848–855.
    View this article via: PubMed CrossRef Google Scholar
60. Pei Y, et al. A missense mutation in PKD1 attenuates the severity of renal disease. Kidney Int. 2012;4(4):412–417.
    View this article via: PubMed Google Scholar
61. Losekoot M, et al. Neonatal onset autosomal dominant polycystic kidney disease (ADPKD) in a patient homozygous for a PKD2 missense mutation due to uniparental disomy. J Med Genet. 2012;49(1):37–40.
    View this article via: PubMed CrossRef Google Scholar
62. Bergmann C, et al. Mutations in multiple PKD genes may explain early and severe polycystic kidney disease. J Am Soc Nephrol. 2011;22(11):2047–2056.
    View this article via: PubMed CrossRef Google Scholar
63. Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 2003;13(3):476–484.
    View this article via: PubMed CrossRef Google Scholar
64. Ishikawa I, Saito Y. Volume changes in autosomal dominant polycystic kidneys after the initiation of hemodialysis. Nephron. 1993;65(4):649–650.
    View this article via: PubMed CrossRef Google Scholar
65. Norman J. Fibrosis and progression of autosomal dominant polycystic kidney disease (ADPKD). Biochim Biophys Acta. 2011;1812(10):1327–1336.
    View this article via: PubMed Google Scholar
66. Gattone VH. Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist. Nat Med. 2003;9(10):1323–1326.
    View this article via: PubMed CrossRef Google Scholar
67. Smith LA, et al. Development of polycystic kidney disease in juvenile cystic kidney mice: insights into pathogenesis, ciliary abnormalities, and common features with human disease. J Am Soc Nephrol. 2006;17(10):2821–2831.
    View this article via: PubMed CrossRef Google Scholar
68. Mazzaccara C, et al. Age-related reference intervals of the main biochemical and hematological parameters in C57BL/6J, 129SV/EV and C3H/HeJ mouse strains. PLoS One. 2008;3(11):e3772.
    View this article via: PubMed CrossRef Google Scholar
69. Bae KT, et al. Magnetic resonance imaging evaluation of hepatic cysts in early autosomal-dominant polycystic kidney disease: the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease cohort. Clin J Am Soc Nephrol. 2006;1(1):64–69.
    View this article via: PubMed CrossRef Google Scholar
70. Reeders ST, et al. Prenatal diagnosis of autosomal dominant polycystic kidney disease with a DNA probe. Lancet. 1986;2(8497):6–8.
    View this article via: PubMed CrossRef Google Scholar
71. Xiao Z, Zhang S, Cao L, Qiu N, David V, Quarles LD. Conditional disruption of Pkd1 in osteoblasts results in osteopenia due to direct impairment of bone formation. J Biol Chem. 2010;285(2):1177–1187.
    View this article via: PubMed CrossRef Google Scholar
72. Boulter C, Mulroy S, Webb S, Fleming S, Brindle K, Sandford R. Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene. Proc Natl Acad Sci U S A. 2001;98(21):12174–12179.
    View this article via: PubMed CrossRef Google Scholar
73. Chapman AB, Johnson AM, Rainguet S, Hossack K, Gabow P, Schrier RW. Left ventricular hypertrophy in autosomal dominant polycystic kidney disease. J Am Soc Nephrol. 1997;8(8):1292–1297.
    View this article via: PubMed Google Scholar
74. Ishikawa I, Horiguchi T, Shikura N. Lectin peroxidase conjugate reactivity in acquired cystic disease of the kidney. Nephron. 1989;51(2):211–214.
    View this article via: PubMed CrossRef Google Scholar
75. Vandeursen H, Van Damme B, Baert J, Baert L. Acquired cystic disease of the kidney analyzed by microdissection. J Urol. 1991;146(4):1168–1172.
    View this article via: PubMed Google Scholar
76. Nakanishi K, Sweeney WE Jr, Zerres K, Guay-Woodford LM, Avner ED. Proximal tubular cysts in fetal human autosomal recessive polycystic kidney disease. J Am Soc Nephrol. 2000;11(4):760–763.
    View this article via: PubMed Google Scholar
77. Ahrabi AK, et al. Glomerular and proximal tubule cysts as early manifestations of Pkd1 deletion. Nephrol Dial Transplant. 2010;25(4):1067–1078.
    View this article via: PubMed CrossRef Google Scholar
78. Wang X, Wu Y, Ward CJ, Harris PC, Torres VE. Vasopressin directly regulates cyst growth in polycystic kidney disease. J Am Soc Nephrol. 2008;19(1):102–108.
    View this article via: PubMed CrossRef Google Scholar
79. Stroope A, et al. Hepato-renal pathology in Pkd2^ws25/- mice, an animal model of autosomal dominant polycystic kidney disease. Am J Pathol. 2010;176(3):1282–1291.
    View this article via: PubMed CrossRef Google Scholar
80. Besschetnova TY, Kolpakova-Hart E, Guan Y, Zhou J, Olsen BR, Shah JV. Identification of signaling pathways regulating primary cilium length and flow-mediated adaptation. Curr Biol. 2010;20(2):182–187.
    View this article via: PubMed CrossRef Google Scholar
81. Xu C, et al. Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells. Am J Physiol Renal Physiol. 2009;296(6):F1464–F1476.
    View this article via: PubMed CrossRef Google Scholar
82. Hughes J, et al. The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains. Nat Genet. 1995;10(2):151–160.
    View this article via: PubMed CrossRef Google Scholar
83. Qian F, et al. Cleavage of polycystin-1 requires the receptor for egg jelly domain and is disrupted by human autosomal-dominant polycystic kidney disease 1-associated mutations. Proc Natl Acad Sci U S A. 2002;99(26):16981–16986.
    View this article via: PubMed CrossRef Google Scholar
84. Newby LJ, Streets AJ, Zhao Y, Harris PC, Ward CJ, Ong AC. Identification, characterization, and localization of a novel kidney polycystin-1-polycystin-2 complex. J Biol Chem. 2002;277(23):20763–20773.
    View this article via: PubMed CrossRef Google Scholar
85. Vagin O, Kraut JA, Sachs G. Role of N-glycosylation in trafficking of apical membrane proteins in epithelia. Am J Physiol Renal Physiol. 2009;296(3):F459–F469.
    View this article via: PubMed CrossRef Google Scholar
86. Wang X, Koulov AV, Kellner WA, Riordan JR, Balch WE. Chemical and biological folding contribute to temperature-sensitive DeltaF508 CFTR trafficking. Traffic. 2008;9(11):1878–1893.
    View this article via: PubMed CrossRef Google Scholar
87. Liu M, et al. Genetic variation of DKK3 may modify renal disease severity in ADPKD. J Am Soc Nephrol. 2010;21(9):1510–1520.
    View this article via: PubMed CrossRef Google Scholar
88. Harris PC, et al. Cyst number but not the rate of cystic growth is associated with the mutated gene in autosomal dominant polycystic kidney disease. J Am Soc Nephrol. 2006;17(11):3013–3019.
    View this article via: PubMed CrossRef Google Scholar
89. Rossetti S, Harris PC. Genotype-phenotype correlations in autosomal dominant and autosomal recessive polycystic kidney disease. J Am Soc Nephrol. 2007;18(5):1374–1380.
    View this article via: PubMed CrossRef Google Scholar
90. Wang E, et al. Progressive renal distortion by multiple cysts in transgenic mice expressing artificial microRNAs against Pkd1. J Pathol. 2010;222(3):238–248.
    View this article via: PubMed CrossRef Google Scholar
91. Li D, et al. The roles of two novel FBN1 gene mutations in the genotype-phenotype correlations of Marfan syndrome and ectopia lentis patients with marfanoid habitus. Genet Test. 2008;12(2):325–330.
    View this article via: PubMed CrossRef Google Scholar
92. Pereira R, Halford K, Sokolov BP, Khillan JS, Prockop DJ. Phenotypic variability and incomplete penetrance of spontaneous fractures in an inbred strain of transgenic mice expressing a mutated collagen gene (COL1A1). J Clin Invest. 1994;93(4):1765–1769.
    View this article via: JCI PubMed CrossRef Google Scholar
93. Nadasdy T, Lajoie G, Laszik Z, Blick KE, Molnar-Nadasdy G, Silva FG. Cell proliferation in the developing human kidney. Pediatr Dev Pathol. 1998;1(1):49–55.
    View this article via: PubMed CrossRef Google Scholar
94. Nadasdy T, Laszik Z, Blick KE, Johnson LD, Silva FG. Proliferative activity of intrinsic cell populations in the normal human kidney. J Am Soc Nephrol. 1994;4(12):2032–2039.
    View this article via: PubMed Google Scholar
95. Grantham JJ, Cook LT, Wetzel LH, Cadnapaphornchai MA, Bae KT. Evidence of extraordinary growth in the progressive enlargement of renal cysts. Clin J Am Soc Nephrol. 2010;5(5):889–896.
    View this article via: PubMed CrossRef Google Scholar
96. Bastos AP, et al. Pkd1 haploinsufficiency increases renal damage and induces microcyst formation following ischemia/reperfusion. J Am Soc Nephrol. 2009;20(11):2389–2402.
    View this article via: PubMed CrossRef Google Scholar
97. Nishio S, et al. Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation. J Clin Invest. 2005;115(4):910–918.
    View this article via: JCI PubMed Google Scholar
98. Raj A, Rifkin SA, Andersen E, van Oudenaarden A. Variability in gene expression underlies incomplete penetrance. Nature. 2010;463(7283):913–918.
    View this article via: PubMed CrossRef Google Scholar
99. Verghese E, Weidenfeld R, Bertram JF, Ricardo SD, Deane JA. Renal cilia display length alterations following tubular injury and are present early in epithelial repair. Nephrol Dial Transplant. 2008;23(3):834–841.
    View this article via: PubMed CrossRef Google Scholar
100. Wann AK, Knight MM. Primary cilia elongation in response to interleukin-1 mediates the inflammatory response. Cell Mol Life Sci. 2012;69(17):2967–2977.
     View this article via: PubMed CrossRef Google Scholar
101. Grantham JJ, et al. Volume progression in polycystic kidney disease. N Engl J Med. 2006;354(20):2122–2130.
     View this article via: PubMed CrossRef Google Scholar
102. Fedeles SV, et al. A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation. Nat Genet. 2011;43(7):639–647.
     View this article via: PubMed CrossRef Google Scholar
103. Zode GS, et al. Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma. J Clin Invest. 2011;121(9):3542–3553.
     View this article via: JCI PubMed CrossRef Google Scholar
104. Bakeberg JL, et al. Epitope-Tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin. J Am Soc Nephrol. 2011;22(12):2266–2277.
     View this article via: PubMed CrossRef Google Scholar
105. Xu H, Shen J, Walker CL, Kleymenova E. Tissue-specific expression and splicing of the rat polycystic kidney disease 1 gene. DNA Seq. 2001;12(5–6):361–366.
     View this article via: PubMed Google Scholar
106. Zhang W, et al. Inhibition of tumor growth progression by antiandrogens and mTOR inhibitor in a Pten-deficient mouse model of prostate cancer. Cancer Res. 2009;69(18):7466–7472.
     View this article via: PubMed CrossRef Google Scholar
107. Fukudome H. A combined SEM and TEM study on the basal labyrinth of the collecting duct in the rat kidney. Arch Histol Cytol. 2001;64(3):339–351.
     View this article via: PubMed CrossRef Google Scholar
108. Nims N, Vassmer D, Maser RL. Transmembrane domain analysis of polycystin-1, the product of the polycystic kidney disease-1 (PKD1) gene: evidence for 11 membrane-spanning domains. Biochemistry. 2003;42(44):13035–13048.
     View this article via: PubMed CrossRef Google Scholar