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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Dev Dyn. 2011 Apr 4;240(6):1493–1501. doi: 10.1002/dvdy.22625

Ectopic expression of Cux1 is associated with reduced p27 expression and increased apoptosis during late stage cyst progression upon inactivation of Pkd1 in collecting ducts

Binu M Paul 1,3, Dianne Vassmer 1,2, Aaron Taylor 1,3, Lynn Magenheimer 3, Carol G Carlton 1,3, Klaus B Piontek 4, Gregory G Germino 5, Gregory B Vanden Heuvel 1,3,6
PMCID: PMC3092830  NIHMSID: NIHMS279690  PMID: 21465620

Abstract

Polycystic kidney diseases (PKD) are inherited disorders characterized by fluid-filled cysts primarily in the kidneys. We previously reported differences between the expression of Cux1, p21 and p27 in the cpk and Pkd1 null mouse models of PKD. Embryonic lethality of Pkd1 null mice limits its study to early stages of kidney development. Therefore, we examined mice with a collecting duct specific deletion in the Pkd1 gene. Cux1 was ectopically expressed in the cyst lining epithelial cells of newborn, P7 and P15 Pkd1CD mice. Cux1 expression correlated with cell proliferation in early stages of cystogenesis, however, as the disease progressed, fewer cyst lining cells showed increased cell proliferation. Rather, Cux1 expression in late stage cystogenesis was associated with increased apoptosis. Taken together, our results suggest that increased Cux1 expression associated with apoptosis is a common feature of late stage cyst progression in both the cpk and Pkd1CD mouse models of PKD.

Keywords: Cux1, polycystic kidney disease, apoptosis, p27

Introduction

The Cux1 transcription factor is involved in the regulation of cell proliferation, differentiation and development (1–5). Cux1 is a murine homologue of the Drosophila gene Cut. Cut is required for the proper development of malpighian tubules in Drosophila which are the insect excretory organs that serve as their primitive kidney (2, 6–9).

Cux1 is highly expressed in the developing kidney, with highest expression restricted to the nephrogenic zone (6). As development proceeds, the levels of Cux1 decrease with only low levels of Cux1 detected in adult kidneys (10). Cux1 regulates the cell cycle by transcriptionally repressing the cyclin dependent kinase inhibitors (CKI) p21 and p27 (11, 12).

High rates of cell proliferation are one of the striking features of cyst epithelial cells in polycystic kidney disease (PKD), a life-threatening genetic disease. PKD can be inherited in two different forms: an autosomal recessive form (ARPKD), or an autosomal dominant form (ADPKD), both characterized by fluid-filled cysts primarily in the kidneys (13). ADPKD results from mutations in either of the two genes, PKD1 or PKD2 (13–16), while mutations in a single locus, PKHD1, are responsible for ARPKD (17).

Polycystin1, the protein product of PKD1 co-localizes with complexes involved in cell-to-cell and cell-to-extracellular matrix interactions. These complexes in turn have a regulatory role in cell proliferation (18). Polycystin1 also interacts with Polycystin2, the protein product of PKD2, to induce p21 (19), a transcriptional target of repression by Cux1 (12).

Several murine models have been described for PKD. A well characterized murine model of PKD is the cpk mouse model. The disease is transmitted in a recessive fashion and it shows a striking resemblance to human ARPKD in terms of cyst localization and disease progression (20, 21). A targeted mutation in the Pkd1 gene (Pkd1 null) has also been described. The Pkd1 null mice which are homozygous for this mutation present with kidney cysts and die embryonically (22).

Cux1 is upregulated in the kidneys of both the cpk and the Pkd1 null mouse models (11). Cells from human ADPKD kidneys also show increased expression of Cux1 (7). Analysis of cpk and the Pkd1 null mouse models showed a striking difference between the expression of Cux1, p21, p27, as well as, cell proliferation and apoptosis. Kidneys from Pkd1 null embryos showed increased expression of Cux1. However, in the kidneys of cpk mice, Cux1 upregulation was not observed until late stages of cystogenesis. While p21 was not detected in embryonic kidneys from Pkd1 null mice, Cux1 and p21 were co-expressed in cyst lining cells in cpk mice. In contrast to the reduced expression of p27 in kidneys from Pkd1 null embryos, we saw an increase in p27 expression in the cpk kidneys during late stages of cystogenesis. Apoptosis was also increased in the cpk kidneys during late stages of cystogenesis (11).

These results suggested a model in which cystogenesis proceeds through different mechanisms in the Pkd1 null mice and cpk mice. However, since the Pkd1 null mice died embryonically, our analysis of cystogenesis in that mouse model was restricted to the earliest stages of cystogenesis. In order to analyze the role of Cux1 in ADPKD beyond the embryonic stages of cystogenesis, we examined a mouse model with a collecting duct specific deletion of the Pkd1 gene. Early stages of cystogenesis in this mouse model showed an increase in Cux1 expression that correlated with increased cell proliferation. In more advanced stages of cystogenesis, the increased expression of Cux1 was associated with an increase in apoptosis.

Results

Our previous studies with cpk and Pkd1 null mice suggested different mechanisms of PKD progression in these mouse models. These studies showed that increased expression of Cux1 was primarily associated with cell proliferation in the Pkd1 null mice. In contrast, increased expression of Cux1 in the cpk mice during late stages of cyst progression was associated with apoptosis (11).

Embryonic lethality of Pkd1 null mice limited our studies to the early stages of cystogenesis. Therefore, in the present study, we have examined an ADPKD mouse model with a conditional deletion of the Pkd1 gene in the kidneys. We crossed the Pkd1cond mice with Hoxb7/Cre mice to generate a kidney specific deletion of the Pkd1 gene. Hoxb7/Cre is active in the mesonephric duct of the kidney as early as embryonic day 9.5 and its expression continues in the mesonephric duct derivatives of the kidney, which include collecting ducts and ureteral epithelia (24). Mice in which the Pkd1 gene was disrupted using Hoxb7/cre were designated Pkd1CD (Pkd1collecting duct) mice.

Morphological evaluation of the Pkd1CD mice

We analyzed Pkd1CD mice at various ages, beginning at postnatal day 0 (P0). Mice with Hoxb7/Cre+/ Pkd1cond/wt or Hoxb7/Cre+/ Pkd1wt/wt genotypes were used as controls. Morphological analysis of kidney sections from newborn Pkd1CD mice revealed microscopic cysts. These microscopic dilations were derived from both cortical and medullary collecting ducts (Figure 1A). In mice, nephrogenesis continues until about postnatal day 7 (P7) (23). Therefore, we analyzed Pkd1CD mice at this time point. By P7, Pkd1CD mice had a slight bulging of their flanks, which was visible only upon careful examination. The kidneys of these mice were larger and cystic compared to their age-matched control littermates (Figure 1B). The kidneys also had more and larger cysts, with less normal parenchyma, compared to cystic kidneys isolated from newborn Pkd1CD mice (Figure 1D). Although nephrogenesis is completed by P7 in mice, the process of elongation and maturation of already formed nephrons continues until about a week after the completion of nephrogenesis. In order to analyze kidneys at this time point, we analyzed Pkd1CD mice at P15. By P15, Pkd1CD mice presented with huge bilateral masses on their flanks (Figure 1C). The kidneys of these mice were larger and grossly cystic in comparison to age-matched control littermates, or when compared to Pkd1CD mice at P0 and P7. The kidneys were crowded by cystic tissue and very little normal renal parenchyma was preserved (Fig 1E). Since HoxB7/Cre specifically deletes the Pkd1 gene in the ureteric bud derivatives of the kidney, we expected all the cysts in the Pkd1CD mice to have originated from the collecting ducts. Labeling of kidney sections from newborn, P7 and P15 Pkd1CD mice with Dolichus Biflorus Agglutinin (DBA), confirmed the collecting duct origin of the cysts (Figure 2A-F).

Figure 1. Morphological evaluation of the Pkd1CD mouse kidneys.

Figure 1

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A: Bright field images of newborn kidneys from a Pkd1CD mouse and an age-matched control littermate. Microscopic cyst dilations (arrows) can be seen in the Pkd1CD kidney. Scale bar= 500µm. B and C: Gross appearance of littermate kidneys (control and Pkd1CD) at P7 and P15 respectively. The Pkd1CD kidneys are large and severely cystic compared to the controls. Pkd1CD kidney at P15 is also larger compared to the Pkd1CD kidney at P7. D, E: H & E stained kidney sections from control and Pkd1CD mice at P7 and P15 respectively. Arrows point toward cysts and arrowheads show normal renal tissue. By P15, the Pkd1CD kidneys lost most of the normal renal parenchyma and the kidneys were crowded with cystic tissue. Scale bar= 100µm.

Figure 2. All the cysts in the Pkd1CD kidneys originated from collecting ducts.

Figure 2

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Kidney sections from Pkd1CD mice were labeled with DBA (green, A, C and E) and merged with DAPI staining (blue, B, D and F). Arrows show some collecting duct derived cysts labeled with DBA. Arrowheads show normal collecting ducts labeled with DBA. A, B: Kidney sections from a newborn Pkd1CD mouse. C, D: Cystic kidney from a Pkd1CD mouse at P7. E, F: Cystic kidneys from a Pkd1CD mouse at P15. Scale bar= 100µm.

Analysis of PKD severity in the Pkd1CD mice

To evaluate disease progression in Pkd1CD mice, we determined the ratio of kidney weight to body weight (KW/BW) at P7 and at P15 (Figure 3A). Kidneys harvested from Pkd1CD mice were significantly larger compared with kidneys from control mice, both at P7 and P15. To further analyze the Pkd1CD cystic phenotype, isolated cystic kidneys were examined morphologically. Histological analysis showed that the cystic index (the percentage of cystic area) increased between P7 and P15 (Figure 3B). The developmental stage of the renal cysts in Pkd1CD mice was determined by counting the number of cells lining the cysts (see materials and methods). The results showed that cystic kidneys in P7 mice were mainly composed of early and intermediate stage cysts, while advanced stage cysts were also seen in the cystic kidneys of P15 mice (Figure 3C). Cystic kidney disease is directly correlated with reduced renal function and high BUN levels. Accordingly, Pkd1CD mice at P7 and P15 showed higher BUN values compared to controls, indicative of decreased renal function (Figure 3D).

Figure 3. Analysis of PKD severity in the Pkd1CD mouse kidneys.

Figure 3

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A: KW/BW of P7 and P15 Pkd1CD mice compared to their respective age-matched controls. KW/BW was significantly increased in P7 (p value <0.0001) and P15 (p value 0.03) Pkd1CD mice compared to their age-matched controls. B: Cystic index of kidneys from Pkd1CD mice at P7 and P15 are shown. Cystic index was significantly higher in P15 kidneys compared to P7 kidneys from Pkd1CD mice (p value 0.02). C: Cysts classified according to their developmental stages are shown. Early stage cysts had up to 50 cyst-lining epithelial cells. Cysts which had 51–200 cyst-lining epithelial cells were defined as intermediate cysts. Advanced stage cysts were lined with more than 200 cyst-linging epithelial cells. By P7, Pkd1CD kidneys were comprised mainly of small cysts. Medium cysts were also seen in these cystic kidneys. However, no advanced cysts were seen in these kidneys. The Pkd1CD kidneys at P15 showed more medium cysts and also a small percentage of advanced stage cysts. D: BUN values from the Pkd1CD mice were compared with their age-matched controls. Increased BUN values were observed in Pkd1CD mice at both P7 (p value <0.0001) and P15 (p value <0.0001) compared to their respective age matched controls. Between P7 and P15, BUN values did not show a great difference.

Cux1 is ectopically expressed in the Pkd1CD mice

Cux1 is highly expressed during normal kidney development with the highest level of expression seen in the nephrogenic zone of the kidney (6, 11). Since Cux1 is a cell cycle regulatory gene and increased cell proliferation is a hallmark characteristic of PKD, we analyzed the expression pattern of Cux1 at various stages of cystogenesis in the Pkd1CD mice. As expected, high levels of Cux1 were seen in the nephrogenic zone of newborn control kidneys (Suppl. Figure 1A-B), as well as in the Pkd1CD kidneys (Figure 4 A-B). Cux1 was also ectopically expressed in the cyst lining epithelium of kidneys from Pkd1CD mice (Figure 4A-B). The continuation of the proliferative phase of kidney development at P7 correlated with continued expression of Cux1 in the kidneys of control mice (Suppl. Figure 1C-D). Cystic kidneys from P7 Pkd1CD mice showed increased expression of Cux1 (Figure 4C-D), compared to the controls. By P15, control kidneys showed little Cux1 expression (Suppl. Figure 1). In contrast, cystic kidneys from Pkd1CD mice continued to show high and ectopic expression of Cux1 (Figure 4E-F).

Figure 4. Ectopic expression of Cux1 in the Pkd1CD mouse kidneys.

Figure 4

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Kidney sections from Pkd1CD mice were labeled with Cux1 (green, A, C and E) and merged with DAPI staining (blue, B, D and F). A, B: Kidney section from a newborn Pkd1CD mouse. Cux1 labeling was seen in the nephrogenic zone (*) and cysts (arrows). C, D: Cystic kidney section from a P7 Pkd1CD mouse. Arrows point toward Cux1 positive nuclei in the cysts. Arrowheads show normal tubules positive for Cux1. E, F: Cystic kidney section from a P15 Pkd1CD mouse. Arrows point toward Cux1 positive nuclei in the cysts. All the cyst-lining epithelial cells were Cux1 positive. Scale bar= 50µm.

Early and late stage of cystogenesis in the Pkd1CD mice is associated with increased cell proliferation and increased Cux1 expression

Increased cell proliferation is one of the characteristic features of PKD. We have previously shown that increased expression of Cux1 is associated with increased cell proliferation in human ADPKD cystic epithelia and in several mouse models of PKD (7, 11). We analyzed cell proliferation and its association with Cux1 in the Pkd1CD mice by labeling kidney sections for Cux1 and the cell proliferation marker PCNA. PCNA staining co-localized with Cux1 in the nephrogenic zone and in the cyst lining cells of newborn and P7 Pkd1CD mice (Figure 5). By P15, the nephrogenic zone is essentially gone, however, the cyst lining cells expressed PCNA and Cux1 (Figure 5 G-I). Kidney sections from control newborn mice showed high levels of cell proliferation, which were associated with Cux1 expression. In contrast, kidney sections from P7 and P15 control mice showed little PCNA or Cux1 expression (Suppl. Figure 2A-I).

Figure 5. Early and late stage cystogenesis in Pkd1CD mice is associated with increased cell proliferation.

Figure 5

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Kidney sections from Pkd1CD mice were co-labeled with Cux1 (green, A, D and G), and PCNA (red, B, E and H). A merge between Cux1 and PCNA is also shown (C, F and I). A, B, C: Newborn Pkd1CD kidney section. Cux1 and PCNA co-localized in the nephrogenic zone (*). Arrows point toward nuclei in the cysts which show co-localization of Cux1 and PCNA. Arrowheads show normal tubules where Cux1 and PCNA did not co-localize. D, E, F: Kidney section from a P7 Pkd1CD mouse. Arrows point toward nuclei in the cysts which show co-localization of Cux1 and PCNA. Arrowheads show cystic nuclei in Cux1 and PCNA did not co-localize. G, H, I: Kidney section from a P15 Pkd1CD mouse. All of the cyst-lining epithelial cells were positive for Cux1. However, only some of them showed co-localization with PCNA (arrowheads). Arrows point toward the co-localization of Cux1 and PCNA in cyst-lining epithelial cells. Scale bar= 50µm.

Late stage of cystogenesis in the Pkd1CD mice is associated with increased apoptosis and increased Cux1 expression

Apoptosis is another pathological feature seen in PKD (24). We used the TUNEL assay to analyze apoptosis in kidney sections from Pkd1CD mice. Kidney sections from newborn Pkd1CD mice were mostly TUNEL negative (Figure 6A), while kidney sections from P7 and P15 Pkd1CD mice showed increased apoptosis (Figure 6 B,C). In contrast, age-matched littermate controls showed little or no apoptosis (Suppl Figure 3).

Figure 6. Late stage cystogenesis in the Pkd1CD mice is associated with increased apoptosis.

Figure 6

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The terminal deoxynucleotidal transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) method was used to identify apoptotic cells in newborn (A), P7 (B) and P15 (C) kidneys from Pkd1CD mice. A: Newborn Pkd1CD kidney section. Very few apoptotic nuclei (arrows) were seen in the newborn kidneys. B: Kidney section from a P7 Pkd1CD mouse showing cyst-lining epithelial cells positive for TUNEL labeling (arrows). C: Kidney section from a P15 Pkd1CD mouse showing cyst-lining epithelial cells that are TUNEL positive (arrows). Scale bar= 50µm.

Increased expression of Cux1 correlates with the down regulation of p27 in the Pkd1CD mice

Since p27 and p21 are targets of transcriptional repression by Cux1, we analyzed the levels of these proteins in total kidney lysates from the Pkd1CD mice. Compared to controls, p27 levels were reduced in the kidneys from both P7 and P15 Pkd1CD mice (Fig 7A-B). p21 is normally downregulated very early during kidney development. Accordingly, we were unable to detect p21 expression in the Pkd1CD mice (data not shown).

Figure 7. Downregulation of p27 in the Pkd1CD mouse kidneys.

Figure 7

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A: Total kidney lysates were prepared from P7 and P15 Pkd1CD mice and their respective age matched controls. A western blot analysis was done on these lysates using antibodies raised against p27 and β-tubulin (loading control). A loss of p27 protein expression was seen in the Pkd1CD mice. B: Relative intensity of p27 bands compared to the loading control is shown.

Discussion

Polycystic kidney disease is a systemic disorder characterized by fluid-filled renal cysts together with several extra-renal features. Autosomal dominant polycystic kidney disease (ADPKD) results from mutations in one of two genes: PKD1, which encodes the polycystin-1 protein, and PKD2, which encodes the polycystin-2 protein. Increasing evidence suggests that PKD is a developmental disorder (22, 25). Aberrant cell proliferation is a pathological feature of PKD and micropolyps or foci of proliferating cells can be found populating the kidneys of human PKD patients and experimental animal models of PKD (7, 26–28). The role of polycystins in regulating the cell cycle has been described in which polycystin 1, in cooperation with polycystin 2, functions to regulate the cyclin kinase inhibitor p21 by signaling through the JAK-STAT pathway (19).

Cux1 is a homeobox gene that regulates the cell cycle by transcriptionally repressing the cyclin kinase inhibitors p21 (12) and p27 (11, 29). In the developing kidney, Cux1 is highly expressed in the nephrogenic zone, an area of high cell proliferation, where it functions to repress p27, thereby keeping cells in the cell cycle. As nephrons mature, the levels of Cux1 decrease, and cells move out of the cell cycle and terminally differentiate.

Our previous studies showed the ectopic expression of Cux1 in the Pkd1 null and cpk mouse models of polycystic kidney disease (11), and in cells obtained from the renal cysts of ADPKD patients (7). Comparative studies of the expression of Cux1 and its correlation with cyst progression were done in the Pkd1 null and cpk mouse models. Kidneys from the Pkd1 null mice showed increased expression of Cux1, which correlated with increased cell proliferation. In contrast, increased expression of Cux1 during late stages of cyst progression in the cpk mice was associated with apoptosis. These studies suggested a difference in the mechanism of cyst progression between these animal models. However, the embryonic lethality of Pkd1 null mice limited our studies to the embryonic stages of cystogenesis. Analysis of the Pkd1CD mice has allowed us to examine cyst progression in a postnatal ADPKD mouse model.

Microscopic cysts derived from both cortical and medullary collecting ducts were observed in the kidneys from newborn Pkd1CD mice. Even though the deletion of the Pkd1 gene was restricted to the collecting ducts, Pkd1CD mice developed severe PKD as early as P7 where the entire kidney was crowded by cystic tissue, and ectopic expression of Cux1 was seen in the kidneys of newborn and P7 Pkd1CD mice, where it was associated with cell proliferation. Cux1 was widely expressed in the cyst lining cells from P15 Pkd1CD kidneys, however, it did not co-localize with PCNA in many of the cyst lining cells. This apparent uncoupling of Cux1 expression and cell proliferation is similar to what was previously seen in the cpk mice. However, the kidneys of cpk mice did not show increased expression of Cux1 until late stages of cystogenesis.

Consistent with the increased expression of Cux1, we saw downregulation of p27 in the Pkd1CD kidneys, similar to what we previously reported for the Pkd1 null mouse. This observation also contrasts the previously reported upregulation of p21 and p27 in cystic kidneys from cpk mice.

Apoptosis is associated with several mouse models of cystic disease (30). While cystic kidneys from newborn Pkd1CD mice did not show higher levels of apoptosis than controls, there was an increase in apoptosis as the disease progressed, similar to our observations in the cpk mouse model. In the cpk mice, there was co-localization of p21 and Cux1 that was associated with increased apoptosis, suggesting contradictory signals to proliferate or arrest (11). However, we did not see ectopic expression of p21 in the Pkd1CD mice. While conditional deletion of Pkd1 using Ksp Cre did not show significant changes in apoptosis (31), we did see some TUNEL labeling at very advanced stages of cyst growth, although it seemed to be restricted to only a small subset of large cysts. Thus, although there appears to be apoptosis in the very large cysts, it is probably not involved in the cystogenic process, but in the loss of already damaged cells.

In comparing the Pkd1 null, Pkd1CD, and cpk mouse models, the time point examined must be carefully considered. For example, cystogenesis can only be examined embryonically in the Pkd1 null mouse model, at a time point corresponding to continued nephrogenesis and abundant cell proliferation. In contrast, in the cpk and the Pkd1CD mouse models, cystogenesis can also be examined postnatally, at a time point where nephrogenesis and maturation of the nephrons have been completed. Thus, one explanation for the differences in cell proliferation, apoptosis, and the expression of Cux1 observed between the Pkd1 null, cpk and the Pkd1CD mouse models is the relative stage of development. In both the embryonic Pkd1 null and postnatal day 7 Pkd1CD mice, expression of Cux1 is associated with cell proliferation, which is at a time point when Cux1 is normally expressed and associated with cell proliferation. In contrast, at more advanced stages of cystic disease progression, such as observed in postnatal day 15 Pkd1CD and cpk mice, the ectopic expression of Cux1 is associated with apoptosis, which is at a time point when cell proliferation is tapering off in the kidney, and Cux1 is downregulated. One possibility is that the ectopic expression of Cux1 in cells no longer competent to proliferate results in apoptosis.

Taken together, our studies support our previous conclusion that differences exist in the mechanism of cyst progression in the Pkd1CD and cpk mouse models of PKD. The association between the expression of Cux1 and cell proliferation is well established. However, the association between Cux1 and apoptosis remains to be elucidated.

Experimental Procedures

Animals

Pkd1cond mice

The Pkd1cond mouse line has been described previously. This mouse line has loxP sites flanking exons 2 through 4 of the murine Pkd1 gene, thereby allowing the inactivation of the gene specifically in the tissue of interest. The Pkd1cond allele is fully functional and mice homozygous for this allele are viable and healthy (32).

Hoxb7/Cre mice

The Hoxb7/Cre transgenic mouse line (STOCK Tg(Hoxb7-cre)13Amc/J) was purchased from Jackson Laboratory (Bar Harbor, ME) and the stock colonies are maintained at the University of Kansas Medical Center. The Cre recombinase in this transgenic mouse is expressed under the control of the mouse Hoxb7 enhancer and promoter. The expression of Hoxb7/ Cre can be detected in the mesonephric duct of the kidney as early as embryonic day 9.5. Thereafter, Cre expression can be seen in the mesonephric duct derivatives of the kidney, which include the collecting duct and the ureteral epithelia (33).

Breeding strategy

We crossed mice heterozygous for the Pkd1cond allele to Hoxb7/Cre+ mice. Brother-sister matings were set up between F1 progeny having the Pkd1cond/wt; Hoxb7/Cre+ genotype. A total of 59 out of 266 pups were Pkd1CD (homozygous for the Pkd1cond allele and heterozygous for the Hoxb7/cre transgene). All protocols were approved by the University of Kansas Medical Center Animal Care and Use Committee. The University of Kansas Medical Center is fully accredited by the American Association of the Accreditation of Laboratory Animal Care.

Characterization of cystic phenotype

We analyzed the offspring of F1 crosses at postnatal day 0 (P0, newborn), P7, and P15 stages. Kidneys were harvested from newborn (P0), 7-day old (P7), 12-day old (P12) and 15-day old (P15) mice. Kidneys were weighed and the total kidney weight (KW) was measured as a percentage of body weight (BW) for each mouse. Harvested kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5µm) of cystic kidneys, stained with Haematoxylin and eosin (H & E), were utilized to stage the cysts, as described previously (7). Cysts with up to 50 cyst-lining epithelial cells were defined as early stage, intermediate stage if there were 51–200 cyst-lining epithelial cells, and advanced stage cysts if there were more than 200 cyst lining epithelial cells. The cystic index (ratio of cystic area in the kidney to the total kidney area) was measured in H & E stained cystic kidney sections using ImageJ (NIH) software.

Serum chemistry

Blood was collected by exsanguination, following decapitation and immediately centrifuged at 2000 g to collect serum. Blood urea nitrogen (BUN) in these samples was measured using QuantiChrom™ Urea Assay Kit (BioAssay Systems, Hayward, CA).

Immunofluorescence (IF)

Immunofluorescence was performed as previously described (11). Kidney sections were incubated with 1M ammonium chloride to quench autofluorescence, washed in PBST, and blocked in 10% normal goat serum (NGS) or 10% normal horse serum (NHS) for 1 hour at room temperature. Rabbit anti-Cux1 (1:50, Santa Cruz), mouse anti-PCNA (1:3000, Sigma), mouse anti-p21 (1:100, AbCam), primary antibody was applied to sections and incubated for 1 hour at room temperature. Bound anti-Cux1 antibodies were detected using biotinylated goat anti-rabbit (1:400) secondary antibody (Vector) followed by FITC-avidin (5µg/ml, Vector). PCNA and p21 antibodies were detected using horse anti-mouse Texas red or FITC-conjugated secondary antibody (Vector, 1:400). To identify collecting ducts, kidney sections were incubated with biotinylated dolichos biflorus agglutinin (20ug/ml DBA, Vector) for 1 hour at room temperature, followed by incubation with FITC-conjugated avidin. Sections were then washed in PBST, mounted with Vectashield medium with DAPI (Vector) and images were captured with an Optronics Magnafire digital camera.

Western blot analysis

Whole tissue lysates were prepared from frozen kidneys. Protein (40ug) was loaded onto 4–15% or 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked in 5% milk in PBST. Membranes were probed with anti-p27 antibody (1:100, AbCam), or anti-β-tubulin antibody (1:1000, Sigma), followed by PBST washes and HRP-peroxidase secondary antibody (1:10,000) application.

TUNEL assay

Sections were processed for Terminal deoxynucleotidal transferase (TdT)-mediated dUTP-nick-end labeling (TUNEL) with the TUNEL Apoptosis Detection Kit (Upstate) according to manufacturer's instructions. Sections were counterstained with DAPI, cover-slipped, and visualized on a fluorescence microscope. Images were captured with an Optronics Magnafire digital camera.

Statistics

Two-tailed t-tests were performed in all statistical studies.

Supplementary Material

Supp Fig S1. Supplemental figure 1: Cux1 expression in the control kidneys.

Kidney sections from control mice were labeled with Cux1 (green, A, C and E) and merged with DAPI staining (blue, B, D and F). A, B: Kidney section from a newborn mouse. Cux1 labeling was seen in the nephrogenic zone (*) and tubules (arrows). C, D: Kidney section from a P7 control mouse. Cux1 staining in the nephrogenic zone (*) is decreased as compared to the newborn mouse. Cux1 was also seen in some tubules (arrows). E, F: Kidney section from a P15 control mouse. Cux1 levels are much reduced by P15 compared to the newborn and P7 control kidneys. Arrows point toward Cux1 positive nuclei. Scale bar= 50µm.

Supp Fig S2. Supplemental figure 2: Decreased Cux1 expression and cell proliferation in the control kidneys.

Kidney sections from control mice were co-labeled with Cux1 (green, A, D and G), and PCNA (red, B, E and H). A merge between Cux1 and PCNA is also shown (C, F and I). A, B, C: Newborn control kidney section. Cux1 and PCNA co-localized in the nephrogenic zone (*). Arrows point toward tubules which show no co-localization between Cux1 and PCNA. D, E, F: Kidney section from a P7 control mouse. Very few tubules stained positive for PCNA. Cux1 and PCNA co-localized in some tubules (arrowheads). Arrows point toward tubules in which Cux1 and PCNA did not co-localize. G, H, I: Kidney section from a P15 control mouse. Very few tubules stained positive for PCNA. Cux1 and PCNA co-localized in some tubules (arrowheads). Arrows point toward tubules in which Cux1 and PCNA did not co-localize. Scale bar= 50µm.

Supp Fig S3. Supplemental figure 3: Decreased apoptosis in the control kidneys.

Kidney sections from control mice were stained with TUNEL kit (green, A, C and E) to label apoptotic nuclei and were merged with DAPI (blue, B, D and F) staining. Very few apoptotic nuclei (arrows) were seen in control mice. A, B: Newborn control kidney section. C, D: Kidney section from a P7 control mouse. E, F: Kidney section from a P15 control mouse. Scale bar= 50µm.

Acknowledgements

We thank Rosetta Barkley and Jennifer Brantley for their expert technical assistance. We thank Dr. Madhulika Sharma for many helpful discussions and expert technical advice. This work was supported by NIH grants RO1-DK58377 and P50-DK57301 (G.B.V.H) and RO1-DK48006 (G.G.G.) and a KUMC biomedical research training program fellowship (B.M.P).

References

  • 1.Truscott M, Harada R, Vadnais C, Robert F, Nepveu A. p110 CUX1 cooperates with E2F transcription factors in the transcriptional activation of cell cycle-regulated genes. Mol Cell Biol. 2008;28:3127–3138. doi: 10.1128/MCB.02089-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Sansregret L, Goulet B, Harada R, Wilson B, Leduy L, Bertoglio J, Nepveu A. The p110 isoform of the CDP/Cux transcription factor accelerates entry into S phase. Mol Cell Biol. 2006;26:2441–2455. doi: 10.1128/MCB.26.6.2441-2455.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Nishio H, Walsh MJ. CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription. Proc Natl Acad Sci U S A. 2004;101:11257–11262. doi: 10.1073/pnas.0401343101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Harada R, Vadnais C, Sansregret L, Leduy L, Bérubé G, Robert F, Nepveu A. Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the activation of DNA replication genes. Nucleic Acids Res. 2008;36:189–202. doi: 10.1093/nar/gkm970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Aleksic T, Bechtel M, Krndija D, von Wichert G, Knobel B, Giehl K, Gress TM, Michl P. CUTL1 promotes tumor cell migration by decreasing proteasome-mediated Src degradation. Oncogene. 2007;26:5939–5949. doi: 10.1038/sj.onc.1210398. [DOI] [PubMed] [Google Scholar]
  • 6.Vanden Heuvel GB, Bodmer R, McConnell KR, Nagami GT, Igarashi P. Expression of a cut-related homeobox gene in developing and polycystic mouse kidney. Kidney Int. 1996;50:453–461. doi: 10.1038/ki.1996.336. [DOI] [PubMed] [Google Scholar]
  • 7.Alcalay NI, Sharma M, Vassmer D, Chapman B, Paul B, Zhou J, Brantley JG, Wallace DP, Maser RL, Vanden Heuvel GB. Acceleration of polycystic kidney disease progression in cpk mice carrying a deletion in the homeodomain protein Cux1. Am J Physiol Renal Physiol. 2008;295:F1725–F1734. doi: 10.1152/ajprenal.90420.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Nepveu A. Role of the multifunctional CDP/Cut/Cux homeodomain transcription factor in regulating differentiation, cell growth and development. Gene. 2001;270:1–15. doi: 10.1016/s0378-1119(01)00485-1. [DOI] [PubMed] [Google Scholar]
  • 9.Goulet B, Watson P, Poirier M, Leduy L, Bérubé G, Meterissian S, Jolicoeur P, Nepveu A. Characterization of a tissue-specific CDP/Cux isoform, p75, activated in breast tumor cells. Cancer Res. 2002;62:6625–6633. [PubMed] [Google Scholar]
  • 10.Ledford AW, Brantley JG, Kemeny G, Foreman TL, Quaggin SE, Igarashi P, Oberhaus SM, Rodova M, Calvet JP, Vanden Heuvel GB. Deregulated expression of the homeobox gene Cux-1 in transgenic mice results in downregulation of p27(kip1) expression during nephrogenesis, glomerular abnormalities, and multiorgan hyperplasia. Dev Biol. 2002;245:157–171. doi: 10.1006/dbio.2002.0636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Sharma M, Brantley JG, Alcalay NI, Zhou J, Heystek E, Maser RL, Vanden Heuvel GB. Differential expression of Cux-1 and p21 in polycystic kidneys from Pkd1 null and cpk mice. Kidney Int. 2005;67:432–442. doi: 10.1111/j.1523-1755.2005.67099.x. [DOI] [PubMed] [Google Scholar]
  • 12.Coqueret O, Berube G, Nepveu A. The mammalian Cut homeodomain protein functions as a cell-cycle-dependent transcriptional repressor which downmodulates p21WAF1/CIP1/SDI1 in S phase. EMBO J. 1998;17:4680–4694. doi: 10.1093/emboj/17.16.4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Igarashi P, Somlo S. Genetics and pathogenesis of polycystic kidney disease. J Am Soc Nephrol. 2002;13:2384–2398. doi: 10.1097/01.asn.0000028643.17901.42. [DOI] [PubMed] [Google Scholar]
  • 14.Low SH, Vasanth S, Larson CH, Mukherjee S, Sharma N, Kinter MT, Kane ME, Obara T, Weimbs T. Polycystin-1, STAT6, and P100 function in a pathway that transduces ciliary mechanosensation and is activated in polycystic kidney disease. Dev Cell. 2006;10:57–69. doi: 10.1016/j.devcel.2005.12.005. [DOI] [PubMed] [Google Scholar]
  • 15.Peters DJ, Breuning MH. Autosomal dominant polycystic kidney disease: modification of disease progression. Lancet. 2001;358:1439–1444. doi: 10.1016/S0140-6736(01)06531-X. [DOI] [PubMed] [Google Scholar]
  • 16.Torres VE, Harris PC. Mechanisms of Disease: autosomal dominant and recessive polycystic kidney diseases. Nat Clin Pract Nephrol. 2006;2:40–55. doi: 10.1038/ncpneph0070. [DOI] [PubMed] [Google Scholar]
  • 17.Guay-Woodford LM, Desmond RA. Autosomal recessive polycystic kidney disease: the clinical experience in North America. Pediatrics. 2003;111:1072–1080. doi: 10.1542/peds.111.5.1072. [DOI] [PubMed] [Google Scholar]
  • 18.Glassberg KI. Normal and abnormal development of the kidney: a clinician's interpretation of current knowledge. J Urol. 2002;167:2339–2350. [PubMed] [Google Scholar]
  • 19.Bhunia AK, Piontek K, Boletta A, Liu L, Qian F, Xu PN, Germino FJ, Germino GG. PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2. Cell. 2002;109:157–168. doi: 10.1016/s0092-8674(02)00716-x. [DOI] [PubMed] [Google Scholar]
  • 20.Schieren G, Pey R, Bach J, Hafner M, Gretz N. Murine models of polycystic kidney disease. Nephrol Dial Transplant. 1996;11 Suppl 6:38–45. doi: 10.1093/ndt/11.supp6.38. [DOI] [PubMed] [Google Scholar]
  • 21.Hou X, Mrug M, Yoder BK, Lefkowitz EJ, Kremmidiotis G, D'Eustachio P, Beier DR, Guay-Woodford LM. Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease. J Clin Invest. 2002;109:533–540. doi: 10.1172/JCI14099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lu W, Peissel B, Babakhanlou H, Pavlova A, Geng L, Fan X, Larson C, Brent G, Zhou J. Perinatal lethality with kidney and pancreas defects in mice with a targetted Pkd1 mutation. Nat Genet. 1997;17:179–181. doi: 10.1038/ng1097-179. [DOI] [PubMed] [Google Scholar]
  • 23.Bernhardt WM, Schmitt R, Rosenberger C, Münchenhagen PM, Gröne HJ, Frei U, Warnecke C, Bachmann S, Wiesener MS, Willam C, Eckardt KU. Expression of hypoxia-inducible transcription factors in developing human and rat kidneys. Kidney Int. 2006;69:114–122. doi: 10.1038/sj.ki.5000062. [DOI] [PubMed] [Google Scholar]
  • 24.Woo D. Apoptosis and loss of renal tissue in polycystic kidney diseases. Engl J Med. 1995;333:18–25. doi: 10.1056/NEJM199507063330104. [DOI] [PubMed] [Google Scholar]
  • 25.Baert L. Hereditary polycystic kidney disease (adult form): a microdissection study of two cases at an early stage of the disease. Kidney Int. 1978;13:519–525. doi: 10.1038/ki.1978.75. [DOI] [PubMed] [Google Scholar]
  • 26.Grantham JJ, Geiser JL, Evan AP. Cyst formation and growth in autosomal dominant polycystic kidney disease. Kidney Int. 1987;31:1145–1152. doi: 10.1038/ki.1987.121. [DOI] [PubMed] [Google Scholar]
  • 27.Wilson PD. Aberrant epithelial cell growth in autosomal dominant polycystic kidney disease. Am J Kidney Dis. 1991;17:634–637. doi: 10.1016/s0272-6386(12)80338-6. [DOI] [PubMed] [Google Scholar]
  • 28.Gardner KD, Jr, Glew RH, Evan AP, McAteer JA, Bernstein J. Why renal cysts grow. Am J Physiol. 1994;266:F353–F359. doi: 10.1152/ajprenal.1994.266.3.F353. [DOI] [PubMed] [Google Scholar]
  • 29.Sharma M, Brantley JG, Vassmer D, Chaturvedi G, Baas J, Vanden Heuvel GB. The homeodomain protein Cux1 interacts with Grg4 to repress p27 kip1 expression during kidney development. Gene. 2009;439:87–94. doi: 10.1016/j.gene.2009.03.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Edelstein CL. What is the role of tubular epithelial cell apoptosis in polycystic kidney disease (PKD)? Cell Cycle. 2005;4:1550–1554. doi: 10.4161/cc.4.11.2185. [DOI] [PubMed] [Google Scholar]
  • 31.Shibazaki S, Yu Z, Nishio S, Tian X, Thomson RB, Mitobe M, Louvi A, Velazquez H, Ishibe S, Cantley LG, Igarashi P, Somlo S. Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1. Hum Mol Genet. 2008;17:1505–1516. doi: 10.1093/hmg/ddn039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Piontek KB, Huso DL, Grinberg A, Liu L, Bedja D, Zhao H, Gabrielson K, Qian F, Mei C, Westphal H, Germino GG. A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo. J Am Soc Nephrol. 2004;15:3035–3043. doi: 10.1097/01.ASN.0000144204.01352.86. [DOI] [PubMed] [Google Scholar]
  • 33.Yu J, Carroll TJ, McMahon AP. Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney. Development. 2002;129:5301–5312. doi: 10.1242/dev.129.22.5301. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig S1. Supplemental figure 1: Cux1 expression in the control kidneys.

Kidney sections from control mice were labeled with Cux1 (green, A, C and E) and merged with DAPI staining (blue, B, D and F). A, B: Kidney section from a newborn mouse. Cux1 labeling was seen in the nephrogenic zone (*) and tubules (arrows). C, D: Kidney section from a P7 control mouse. Cux1 staining in the nephrogenic zone (*) is decreased as compared to the newborn mouse. Cux1 was also seen in some tubules (arrows). E, F: Kidney section from a P15 control mouse. Cux1 levels are much reduced by P15 compared to the newborn and P7 control kidneys. Arrows point toward Cux1 positive nuclei. Scale bar= 50µm.

NIHMS279690-supplement-Supp_Fig_S1.tif (6.4MB, tif)
Supp Fig S2. Supplemental figure 2: Decreased Cux1 expression and cell proliferation in the control kidneys.

Kidney sections from control mice were co-labeled with Cux1 (green, A, D and G), and PCNA (red, B, E and H). A merge between Cux1 and PCNA is also shown (C, F and I). A, B, C: Newborn control kidney section. Cux1 and PCNA co-localized in the nephrogenic zone (*). Arrows point toward tubules which show no co-localization between Cux1 and PCNA. D, E, F: Kidney section from a P7 control mouse. Very few tubules stained positive for PCNA. Cux1 and PCNA co-localized in some tubules (arrowheads). Arrows point toward tubules in which Cux1 and PCNA did not co-localize. G, H, I: Kidney section from a P15 control mouse. Very few tubules stained positive for PCNA. Cux1 and PCNA co-localized in some tubules (arrowheads). Arrows point toward tubules in which Cux1 and PCNA did not co-localize. Scale bar= 50µm.

NIHMS279690-supplement-Supp_Fig_S2.tif (4.3MB, tif)
Supp Fig S3. Supplemental figure 3: Decreased apoptosis in the control kidneys.

Kidney sections from control mice were stained with TUNEL kit (green, A, C and E) to label apoptotic nuclei and were merged with DAPI (blue, B, D and F) staining. Very few apoptotic nuclei (arrows) were seen in control mice. A, B: Newborn control kidney section. C, D: Kidney section from a P7 control mouse. E, F: Kidney section from a P15 control mouse. Scale bar= 50µm.

NIHMS279690-supplement-Supp_Fig_S3.tif (6.3MB, tif)

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