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Genes to Cells (2006) 11, 1213-1224. doi:10.1111/j.1365-2443.2006.01011.x
© 2006 Blackwell Publishing or its licensors
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Sustained cell proliferation of renal epithelial cells in mice with inv mutation

Noriyuki Sugiyama and Takahiko Yokoyama*

Department of Anatomy and Developmental Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
A tubule system is an important component of the nephron, which is the structural and functional unit of the kidney. Expansion of renal tubules results in renal cysts. Hereditary forms of renal cystic diseases suggest that tubular size is determined genetically. The inv was discovered as a mutant with renal cysts and situs inversus. Inv/inv, inv{Delta}C::GFP (inv{Delta}C) mouse was created by the introduction of the inv gene lacking the C-terminus (inv{Delta}C) into inv/inv mice. The mouse develops multiple renal cysts without situs abnormality, giving us an opportunity to study inv function in renal tubular structure maintenance. In the present study, we showed that inv suppresses cyst progression in a dose-dependent manner and that the inv{Delta}C cystic kidneys showed increased cell proliferation and apoptosis. Cell cycle regulators for G1-S progression were activated in the cystic kidney. Furthermore, cDNA microarray and semiquantitative RT-PCR analysis showed that growth-related genes maintained a high level of expression in the cystic kidney at 4 weeks of age whereas they were decreased in control kidneys, suggesting that cells in inv{Delta}C kidney are still active in the cell cycle. One of the inv protein functions may provide a stop signal for renal epithelial cell proliferation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Tubes are an essential structure for many organs. However, little is known about how these tubular structures such as size are maintained. In the kidney, a tubule system is important to make up the nephron that is the structural and functional unit of the kidney. Expanding of the renal tubules results in renal cysts. Several forms of renal cystic diseases are hereditary, suggesting that tubular size is determined genetically. Analysis of these hereditary mutants will give us an opportunity to study how renal epithelial cells determine their size and tubular structure.

The inv mouse was discovered as a mutant that shows a reversal of left–right asymmetry, jaundice and multiple renal cysts (Yokoyama et al. 1993; Mochizuki et al. 1998). Most inv/inv mutant mice die before 7 days of age, probably because of cardiovascular malformations (Morishima et al. 1998). The inv gene encodes 15 repeats of the ankyrin motif and two calmodulin-binding IQ domains (Mochizuki et al. 1998; Yasuhiko et al. 2001). In the inv mouse, a region encoding all but the first 91 amino acids of inv protein is deleted (Mochizuki et al. 1998). The introduction of a full length inv cDNA completely rescues all the inv phenotypes, not only situs inversus but also kidney cyst formation, indicating that loss of the inv gene causes both abnormalities (Mochizuki et al. 1998). Although situs inversus and renal cysts appear to be unrelated, it is now known that primary cilia have an important role in both phenotypes (Igarashi & Somlo 2002; Nonaka et al. 2002; Nauli et al. 2003). The inv/inv, inv{Delta}C::GFP (inv{Delta}C) mouse was created by introduction of the inv gene lacking the C-terminus fused with GFP into inv/inv mice (Watanabe et al. 2003). The inv{Delta}C mouse develops renal cysts, but does not show any situs abnormality and jaundice, and has a longer survival. Therefore, inv{Delta}C mice have enabled us to study renal function and gene expression during cyst development in mice with an inv mutation, excluding other associated abnormalities such as jaundice and cardiovascular anomalies.

Recently, mutations in INV have been found to be responsible for human nephronophthisis type 2 (NPHP2) (Otto et al. 2003). NPHP is a group of progressive renal disorders characterized by a variable number of cysts associated with cortical tubular atrophy and renal interstitial fibrosis, and is classified into six variants (NPHP1-6) according to onset and associated lesions (Smith & Graham 1945; Fanconi et al. 1951; Hildebrandt 1999; Otto et al. 2005; Chang et al. 2006). The inv protein interacts with nephrocystin-1 (a gene product of NPHP1) (Hildebrandt et al. 1997; Otto et al. 2003). Nephrocystin-1 interacts with nephrocystin-3 and -4 that are products of NPHP3 and NPHP4, respectively (Olbrich et al. 2003; Mollet et al. 2005). Pcy mice have been found to have a mutation in nphp3 (Olbrich et al. 2003). It is expected that inv protein forms a complex and functions cooperatively with other NPHP gene products.

The inv protein is localized in the cilia of renal epithelia cells (Morgan et al. 2002a; Watanabe et al. 2003), like other cyst-related proteins including polycystin-1 (PC-1), polycystin-2 (PC-2) (Pazour et al. 2002; Yoder et al. 2002), fibrocystin (Ward et al. 2003), cystin (Hou et al. 2002), polaris (Taulman et al. 2001) and nephrocystins (Otto et al. 2003). PC-1 and PC-2 are products of the PKD1 and PKD2 genes that are mutated in most cases of autosomal dominant polycystic kidney disease (ADPKD) (Harris 2002; Igarashi & Somlo 2002). PC-1 and PC-2 make a complex (Qian et al. 1997) and have been suggested to have a role in the mechanosensory Ca2+ channel (Hanaoka et al. 2000; Nauli et al. 2003) and the inv protein binds to calmodulin in a Ca2+ dependent manner (Yasuhiko et al. 2001; Morgan et al. 2002b). Fibrocystin is a product of the human PKHD1 gene that is responsible for autosomal recessive polycystic kidney disease (ARPKD) (Ward et al. 2002, 2003). Although no structural and functional abnormalities of the renal cilia and Ca2+ entry in inv mutants have been detected (Shiba et al. 2005), ciliary localization of inv protein suggests that development of cystic kidneys in inv mutants shares a common pathway (or pathways) with other PKD mutants.

Altered gene expression related to proliferation and growth has been reported in cell lines derived from human PKD patients and inherited PKD mouse pcy and cpk mutants (Nakamura et al. 1993; Ebihara et al. 1995; Gattone et al. 2002; Husson et al. 2004; Lee et al. 2004). Increased apoptosis and cell proliferation have also been reported in murine and human PKD mutants (Harding et al. 1992; Rankin et al. 1992; Nadasdy et al. 1995; Woo 1995; Ostrom et al. 2000; Couillard et al. 2002; Nishio et al. 2005). However, little has been known about cell proliferation, apoptosis and gene expression in the kidney of inv mutants, although morphological characterization of renal cysts in inv mutant was reported (Phillips et al. 2004). Characterization about renal cysts in inv mutants will help to find a common pathway for renal cyst formation in PKD mutants.

In the present study, we characterized formation and development of renal cysts in inv{Delta}C mice, and showed a relationship between inv expression and cyst development. We also studied cell proliferation, apoptosis and expression patterns of growth related genes in the cystic kidney of inv{Delta}C mice, and showed that renal cells of inv{Delta}C mice showed increased cell proliferation, which is probably due to increased G1-S progression. One of the inv protein functions may provide a stop signal for renal epithelial cell proliferation, which maintains the renal tubular architecture.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Correlation between survival and renal function of inv{Delta}C mice and the number of inv{Delta}C transgenic alleles

We designated inv/inv mice carrying one inv{Delta}C transgenic allele as heterozygous inv{Delta}C mice and those carrying two inv{Delta}C transgenic alleles as homozygous inv{Delta}C mice. We used +/+ or +/inv mice carrying the inv{Delta}C transgene as controls. We first screened heterozygous or homozygous inv{Delta}C mice by intensity of PCR amplified bands using EGFP primers (Fig. 1A). To confirm inv{Delta}C transgene homozygosity, inv{Delta}C mice that were identified as homozygous inv{Delta}C by PCR were mated with mice carrying no inv{Delta}C allele. More than 20 offspring from each homozygous inv{Delta}C candidate mouse were genotyped and it was confirmed that all of them were carrying the inv{Delta}C transgene. The mice that were confirmed as homozygous inv{Delta}C mouse were mated with each other and the homozygous inv{Delta}C line was maintained. To obtain heterozygous inv{Delta}C mice, we mated homozygous inv{Delta}C mice with inv mice. Survival, total body weight, kidney weight, serum urea nitrogen and creatinine of heterozygous and homozygous inv{Delta}C mice are shown in Fig. 1B–F. Heterozygous inv{Delta}C mice started to die at 2 weeks of age and > 50% had died by 4 weeks of age (Fig. 1B). In contrast, homozygous inv{Delta}C mice started to die after 4 weeks of age. The 50% survival rate in heterozygous inv{Delta}C mice was seen at 26 days and that of homozygous inv{Delta}C mice at 64 days (P < 0.05). Body weight growth was retarded from 2 weeks of age in heterozygous inv{Delta}C mice and from 3 weeks of age in homozygous inv{Delta}C mice (Fig. 1C). The kidney weight of heterozygous inv{Delta}C mice started to increase from the age of 2 weeks compared with that of control mice (Fig. 1D) and reached 387.9 ± 29.6 mg (mean ± SE, maximum 652.2 mg) at 4 weeks of age. Kidney weight of homozygous inv{Delta}C mice was between that of the controls and heterozygous inv{Delta}C mice (Fig. 1C,D,G) and reached 566.6 ± 38.4 mg (maximum 672.3 mg) at 10 weeks of age (data not shown). In gross appearance, the kidney in heterozygous and homozygous inv{Delta}C mice aged 3 weeks had many cysts of various sizes that were visible from the surface (Fig. 1G). At 4 weeks after birth, serum concentrations of creatinine and urea nitrogen were markedly elevated in heterozygous inv{Delta}C mice, but not in homozygous inv{Delta}C mice (Fig. 1E,F). Serum concentration of bilirubin as a bile functional marker was not changed in inv{Delta}C mice (data not shown). Expression of inv{Delta}C or inv mRNA in the kidney of inv{Delta}C (heterozygous and homozygous) and heterozygous inv mice (inv/+) and wild (+/+) at 3 weeks of age were determined by semiquantitative RT-PCR. Inv or inv{Delta}C mRNA expression of wild-type (+/+), heterozygous inv{Delta}C and homozygous inv{Delta}C mice was 1.8 ± 0.2, 0.52 ± 0.05 and 0.2 ± 0.02, respectively, times inv mRNA expression of inv/+mice, when compared to inv expression of inv/+mice (Fig. 1H). The ratio between inv heterozygous (inv/+) and wild-type (+/+) mice and between heterozygous and homozygous inv{Delta}C mice corresponded to the number of inv and inv{Delta}C transgenes.


Figure 1
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  		Figure 1  Identification, survival rate, total body weight, kidney weight, serum urea nitrogen and creatinine of heterozygous and homozygous inv{Delta}C mice. (A) PCR identification for mice carrying one ({Delta}C/+) or two alleles ({Delta}C/{Delta}C) of the inv{Delta}C transgene. (B) Survival rate of heterozygous inv{Delta}C mice (n = 12) and homozygous inv{Delta}C mice (n = 16). Log rank test was used to compare survival curves and the difference was considered significant at P < 0.05. (C) Total body weight, (D) kidney weight, (E) serum creatinine and (F) serum urea nitrogen of control (x), heterozygous inv{Delta}C (•) and homozygous inv{Delta}C mice ({circ}). Error bars = S.E.; *Significantly different from controls of the same age using Student's t-test with P < 0.01. (G) Gross appearance of representative kidneys from control (right), heterozygous inv{Delta}C (left) and homozygous inv{Delta}C mice (middle). Scale bar = 5 mm. (H) Inv mRNA expression levels of wild, inv/+heterozygous mutant, heterozygous inv{Delta}C and homozygous inv{Delta}C mice. Lower panels show inv and hprt expression level of each mouse analyzed by semiquantitative RT-PCR at 3 weeks of age. Upper panel shows densitometry results of three separate experiments. Each point represents average+/– SE. Different superscripts were significantly different at the 0.05 level (multicomparison by Tukey–Kramer post hoc test).

 
Histology, apoptosis and cell proliferationin kidneys of inv{Delta}C mice

Histology, apoptosis and cell proliferation were studied in heterozygous inv{Delta}C mice. Renal cysts in the inv{Delta}C mice were observed as expanding tubules in the cortex and as pouches in the medulla near the renal pelvis at age 1 week (Fig. 2A,B). At 3 weeks after birth, cysts occupied both the cortex and the medulla and showed flattened epithelia (Fig. 2D,E). Some glomeruli were hyalinized but, in general, glomerular structure was preserved. Prevalance of DBA-positive collecting ducts, LTA-positive proximal tubules (Laitinen et al. 1987) and unstained tubules in control kidney were 45.2, 27.8 and 27%, respectively (Fig. 2C,F, Table 1), which is not significantly different from inv{Delta}C kidneys. We measured the minimum outer diameter of the tubules. Tubule diameter in control kidneys at 1 week of age was &lE; 50 µm (Table 1). We defined a tubule of diameter &lE; 50 µm as a normal tubule, a tubule with a diameter between 50 and 100 µm as a small cyst, and tubule of diameter &lE; 100 µm as a large cyst. At 1 week old, some LTA-positive and non-stained tubules in inv{Delta}C kidney were enlarged and defined as large cysts, but this was not significantly different from the control kidneys. The inv{Delta}C kidney, but not the control kidney, at 3 weeks of age had LTA-positive, DBA-positive and non-stained large cysts. Remarkably, some tubules consisted of both lectin-stained and non-stained epithelial cells (Fig. 2F arrow).


Figure 2
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  		Figure 2  Renal histology and lectin staining of kidneys in heterozygous inv{Delta}C mice aged 1 week (A–C) and 3 weeks (D–F). (A,B,D,E) Hematoxylin & eosin-stained kidneys. (C,F) LTA (green) and DBA (red) staining of cystic kidneys at 1 (C) and 3 (F) weeks of age. Scale bars = 1 mm (A,D) and 100 µm (B,C,E,F). DBA positive- and non-staining (arrows) cells are observed in the same tubule.

 

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  		Table 1  Tubular diameters and lectin staining
 
TUNEL-positive cells were observed in < 1% of tubules in the kidneys of control and inv{Delta}C mice at 1 week of age (0.10 ± 0.07% in control; 0.16 ± 0.09% in inv{Delta}C mice) (Fig. 3C). Although TUNEL-positive tubules were barely detectable in the kidney of 3-week-old control mice (Fig. 3A,C), > 10% of tubules in the kidneys of 3-week-old inv{Delta}C mice contained TUNEL-positive cells (Fig. 3B,C). The percentage of TUNEL-positive tubules per counted tubules, in triplicate groups of kidneys, was 14.7 ± 2.37% in inv{Delta}C mice and 0.16 ± 0.09% in control mice (P < 0.01) (Fig. 3C). Interestingly, many TUNEL-positive tubules in inv{Delta}C mice at 3 weeks of age contained &gE; 75% TUNEL-positive epithelial cells (Fig. 3B,D).


Figure 3
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  		Figure 3  Apoptotic cells and cell proliferation in kidneys of control and heterozygous inv{Delta}C mice. (A, B) TUNEL-positive cells (brown staining) were increased in inv{Delta}C (B) compared with control mice of 3-week-old (A). Asterisks show cysts in which most epithelial cells were TUNEL-positive; arrowheads, isolated TUNEL-positive cells. Scale bars = 100 µm. (C) The percentage rate is the number of TUNEL-positive cells per number of renal epithelial cells in tubules containing an apoptotic cell (cells). (D) The percentage rate is the number of tubules containing apoptotic cells per number of examined tubules. At least 200 tubules in each three kidneys were examined. Each bar represents the mean ± SE. *Significantly different from controls of the same age using Welch's t-test with P < 0.01. (E–J) BrdU incorporation (brown staining) in kidneys from control mice (E,H) and heterozygous inv{Delta}C (F,I) at 1 (E,F) and 3 (H,I) weeks old. Scale bars = 100 µm. (G,J) A number of tubules containing BrdU-positive cells per number of counted tubules at (G) 1 and ( J) 3 weeks old. Tubules in the kidney of heterozygous inv{Delta}C mice were classified based on diameter. Each bar represents the mean ± SE. *Significantly different P < 0.05 level (Welch's t-test).

 
BrdU-positive cells were observed in tubular epithelial cells, endothelial cells, glomerular cells and interstitial cells of control and inv{Delta}C kidneys at 1 week old (Fig. 3E,F). We allowed BrdU incorporation for 30 min. The percentage of BrdU-positive epithelial cells per total epithelial cells was 7.3 ± 0.67% in control mice and 8.5 ± 0.59% in inv{Delta}C mice (Fig. 3G). BrdU incorporation in renal epithelial cells increased as cyst of inv{Delta}C mice enlarged (Fig. 3G). Percentage of BrdU positive of cells was 8.3 ± 0.64% in normal tubules, 10.6 ± 0.98% in small cysts (P < 0.05) and 14.1 ± 1.99% in large cysts of inv{Delta}C mice (P < 0.05) (Fig. 3G). BrdU-positive cells were not detected after 2 h BrdU incorporation in 3-week-old control and inv{Delta}C mice (data not shown). Therefore, we allowed mice to incorporate BrdU for 2 days. BrdU-positive staining was seen in both interstitial and renal tubular epithelial cells in control kidneys at 3 weeks of age (Fig. 3H), but most BrdU staining was observed in the tubular epithelial cells in the kidneys of inv{Delta}C mice (Fig. 3I). The number of BrdU-positive epithelial cells per total epithelial cells in control mice was 11.1 ± 0.80% in normal tubules and 10.5 ± 3.17% in small cysts, whereas in inv{Delta}C mice the number of cells was 23.4 ± 1.59% in normal tubules, 40.2 ± 2.98% in small cysts and 39.4 ± 2.67% in large cysts of inv{Delta}C mice (P < 0.05) (Fig. 3J).

Analysis of cell cycle regulators expression and phosphorylation

Since the cystic kidney showed increased BrdU incorporation, we then examined expression and phosphorylation of cell cycle regulators. Kidneys from three inv{Delta}C and control mice were isolated and examined by Western blot analysis. As shown in Fig. 4, cyclin D1 and D3 expression were increased in the cystic kidneys. Expression of cdk4 and 6, which are a partner of cyclin D, were also elevated in the cystic kidneys. In addition, phosphorylation both at serine 795 and serine 807/811 of Rb protein was increased in the cystic kidneys.


Figure 4
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  		Figure 4  Immunoblot analysis of whole kidney lysate from 3-week-old control and inv{Delta}C mice for expression of cyclin D1 and D3, cdk4 and 6, and phosphorylated Rb protein.Three control and three inv{Delta}C mice were examined. Note that expression of cyclinD1 and D3, cdk4 and 6 is higher in inv{Delta}C mouse kidneys than in control mice. Phosphorylated Rb protein was also detected with anti-phospho-Rb. Inv{Delta}C mice have stronger signal for phosphorylated Rb protein both at serine795 and 807/811 sites than control mice do.

 
Expression of growth factors and growth-related genes in control and inv{Delta}C cystic kidney

To search growth factors and growth related genes that show increased expression in the cystic kidney, we performed cDNA microarray analysis. cDNA microarray analysis showed that 1326 genes were up-regulated and 3209 genes were down-regulated among 39 000 genes examined in cystic kidneys compared with gene expression of control kidneys. Four hundred and fifty-five genes were up-regulated by over fourfold. The whole results of cDNA microarray are available at the NCBI Gene Expression Omnibus website (Accession No. GSE4462 [NCBI GEO] ).

We selected four growth-related genes and six growth factors as well as several genes that showed high expression in cDNA microarray analysis, and performed semiquantitative RT-PCR. Three control and three inv{Delta}C kidneys were examined at 1 week and 4 weeks after birth. Selected growth-related genes included transforming growth factor ß induced (Tgfßi), c-myc, core promoter element binding protein (Copeb) and early growth response 2 (Egr2). Selected growth factors included transforming growth factor ß2 (Tgfß2), Wnt4 and 11, hepatocyte growth factor (Hgf) and bone morphgenic protein 2 (Bmp2) as well as epidermal growth factor (Egf) that is implicated in renal cyst formation. Results of semiquantitative RT-PCR and cDNA microarray were well correlated but not identical. Semi-quantitative RT-PCR confirmed cDNA microarray results, although the changes observed with cDNA microarray and semiquantitative RT-PCR were not always in agreement quantitatively (data not shown).

Temporal expression patterns of four growth-related genes and six growth factors were further examined in the kidneys of control and heterozygous inv{Delta}C mice. The hprt gene was used for normalization, and the ratio of each gene expression level in control and inv{Delta}C mice was compared to that of control newborn mice. Figure 5 shows the expression pattern of the genes of control and inv{Delta}C kidneys at newborn and 4 weeks of age. All examined genes except egf showed higher expression at newborn than at 4 weeks of age. However, expression of the growth-related genes stayed at relatively high levels at 4 weeks of age in inv{Delta}C kidneys compared to control mouse kidneys. This discrepancy made these genes appear up-regulated in inv{Delta}C kidneys.


Figure 5
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  		Figure 5  Temporal expression patterns of growth-related genes and growth factors in control and inv{Delta}C mice. RT-PCR was performed on three control and three cystic kidneys at newborn and 4-week-old and normalized using Hprt. The graph of semi-quantitative RT-PCR shows logarithmic mean ± SE. Upper row (Tgfßi, c-myc, Copeb, Egr2) shows growth-related genes, which decreased their expression in control mouse kidneys at 4 weeks old compared to newborn, but remained at high levels in heterozygous inv{Delta}C mouse kidneys. Lower row shows growth factors (Tgfß2, Wnt4, Hgf, Bmp2, Wnt 11, Egf). Ratios were calculated gene expression level in cystic kidney against expression level in normal kidney at newborn. The values are mean ± SD. *Significantly different from controls of the same age using Student's t-test with P < 0.01.

 
Growth factors showed various patterns for their expression in inv{Delta}C kidneys (Fig. 5). Expression of Tgfß2 increased at 4 weeks of age compared to that at newborn in inv{Delta}C kidneys. Wnt4, like growth related genes, maintained relatively higher level of expression at 4 weeks of age in inv{Delta}C kidneys than that in control kidneys, whereas Hgf, Bmp2, Wnt11 and egf decreased their expression at 4 weeks of age and showed no statistical difference compared to those in control kidneys. Different from other growth factors, Egf did not change its expression between newborn and 4 weeks of age in both inv{Delta}C and control kidneys.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Renal cysts are detected at embryonic day 18.5 in inv mice, but at age 1 week in heterozygous inv{Delta}C mice. Homozygous inv{Delta}C mice survive longer than heterozygous inv{Delta}C mice. Deterioration of renal function and increased kidney weight were less severe in homozygous inv{Delta}C mice than in heterozygous inv{Delta}C mice. Inv{Delta}C mRNA in the kidney of homozygous inv{Delta}C mice was expressed twice as much as that of heterozygous inv{Delta}C mice. Expression of inv{Delta}C transgene in heterozygous inv{Delta}C mice, although one fifth of the control mouse inv level, is enough to delay cyst development. Although it is unclear whether low level expression or a lack of the C-terminus of the inv gene is responsible for failure to prevent cyst development completely, these results show that the amount of inv mRNA is an important factor for renal cyst formation and development in inv{Delta}C kidneys. In human NPHP2 patients, age of onset of end-stage renal disease ranges from newborn to 2 years (Smith & Graham 1945; Fanconi et al. 1951; Hildebrandt 1999). The difference may reflect not only protein structures, but also amount or stability of inv mRNA expression. It would also be interesting to know if expression level of inv mRNA is affected in other PKD mutants. Prolonged survival of inv{Delta}C mice will be a useful animal model to find therapeutic agents for NPHPs.

Increased apoptosis and cell proliferation have been reported in various inherited PKD mutants (Harding et al. 1992; Rankin et al. 1992; Ostrom et al. 2000; Couillard et al. 2002; Nishio et al. 2005). Interestingly, apoptotic cells from inv{Delta}C kidneys were not isolated, but rather formed a cluster in renal cystic tubules. The results suggest that apoptosis in one area occurs simultaneously. The inv{Delta}C kidneys contain more BrdU-positive cells than control kidneys, suggesting that more renal epithelial cells are still in the cell cycle. Larger cysts contained more BrdU incorporated cells, suggesting that cell proliferation and cyst formation are linked. Our results showed that cyclin D1 and D3, cdk4/6 and Rb phosphorylation, which regulate G1-S progression, were activated in inv{Delta}C kidneys. Thus, it is likely that cell proliferation in inv{Delta}C kidneys is due to activation of these G1-S regulators.

Semi-quantitative RT-PCR fairly correlated with cDNA microarray analysis results. As control, we used +/+ or inv/+ mice carrying the inv{Delta}C transgene. Thus, altered gene expression in inv{Delta}C mice does not result from transgene integration or inv{Delta}C mRNA expression, but rather reflects direct or indirect renal cell response to mutated inv gene. High level of expression of growth related genes in 4-week-old inv{Delta}C mice supported that cells in inv{Delta}C kidney are still in the cell cycle. Increased expression of growth related genes and high BrdU incorporation in cystic kidneys suggest that renal cells of inv{Delta}C mice retain the character of premature (or developing) kidney or they are unable to maintain the character of mature (developed) kidney. The c-myc gene is well known to be up-regulated in cystic kidneys of pkd1 (Muto et al. 2002) and pcy mice (Gattone et al. 1996). Moreover, c-myc transgenic mice develop renal cysts (Trudel et al. 1991). High level of c-myc expression may also be an important factor for renal cyst formation in inv{Delta}C mice.

What stimulates G1-S transition in inv{Delta}C kidneys is unclear. Table 2 summarizes renal mRNA expression of inv{Delta}C mice examined in the present study, and previously reported results in pcy mice, human ADPKD cells and cpk mice. Among examined growth factors, Tgfb2 and Wnt4 showed high level of expression in 4-week-old inv{Delta}C kidneys compared to control kidneys. Tgfb2 is reported to control apoptosis in podocytes (Wu et al. 2005). Tgfb2 may have some role in increased apoptotic cells in inv{Delta}C kidneys. A recent report suggested that inv gene acts on wnt pathway to determine canonical or non-canonical pathway (Simons et al. 2005). Loss or mutation of inv fail to switch the wnt signal to the non-canonical from the canonical pathway, and continue to stimulate the canonical pathway that activate c-myc transcription.


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  		Table 2  Comparison of renal mRNA expression among inv{Delta}C mice in the present study, pcy mice (Nakamura et al. 1993; Gattone et al. 1996), human ADPKD cells (Husson et al. 2004; Lee et al. 2004) and cpk mice (Gattone et al. 2002)
 
The present study suggested that one of the inv protein functions provide a stop signal for renal epithelial cell proliferation. Loss or mutation in the inv gene may fail to provide this stop signal by unknown mechanisms, resulting in sustained high proliferation in renal epithelial cells. High cell proliferation and altered gene expression in both inv{Delta}C mice and other PKD mutants may suggest that they share a common pathway for renal cyst formation. To elucidate the mechanism that governs this cell cycle may give us a clue as to how a tubular structure is maintained. In addition, suppression of G1-S progression will be a therapeutic approach for NPHPs.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Mouse husbandry and identification of mice carrying the inv{Delta}C::GFP (inv{Delta}C) transgene

FVB/N, inv/inv, and mice carrying the inv{Delta}C::GFP (inv{Delta}C) transgenic gene (Watanabe et al. 2003) were maintained in a pathogen-free state in an animal facility of Kyoto Prefectural University of Medicine, Japan. The experimental procedures were permitted by the Committee for Animal Research, Kyoto Prefectural University of Medicine.

Transgenic mice were identified by PCR with specific primers for enhanced green fluorescent protein (EGFP) (5'-gac acc ctg gtg aac cgc atc gag ctg aag-3' and 5'-ggc gag ctg cac gct gcc gtc ctc-3'). Inv/inv, inv{Delta}C mice were obtained by mating male and female +/inv, inv{Delta}C::GFP mice. We used +/+ or +/inv mice carrying the inv{Delta}C transgene as controls through all the present experiments.

Histological analysis and lectin staining

For morphological evaluation, mouse kidneys were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and embedded in paraffin wax. Sections (4 µm thick) were stained with hematoxylin and eosin (H&E) according to standard protocols.

Lectin staining was performed using fluorescein isothiocyanate-coupled Lotus tetragonolobus agglutinin (LTA) and rhodamine-coupled dolichos biflorus agglutinin (DBA) (Vector Laboratories, Burlingame, CA, USA). Kidney samples for lectin staining were frozen in liquid nitrogen into Tissue Tek OCT compound (Miles; Elkhart, IN, USA) and stored at –70 °C until sectioned. Sections were performed at 10 µm, and fixed in 4% PFA in 0.1 M phosphate buffer, pH 7.5 at room temperature for 20 min. FITC conjugated LTA and rhodamine conjugated DBA were used at a concentration of 50–100 µg/mL in PBS. After incubation for 20 min, the samples were washed in PBS, then examined under an Olympus microscope IX70 equipped with digital camera (Olympus, DP70).

Assays for cell proliferation and apoptosis

Cell proliferation was assayed by using bromodeoxyuridine (BrdU) incorporation. Briefly, mice were injected intraperitoneally with a solution containing BrdU (Sigma) at a dose of 50 mg/kg/body weight. The kidneys were embedded in paraffin wax. Sections (4 µm) were cut and BrdU incorporation was detected with the specific antibody provided in the kit (Amersham).

The terminal deoxynucleotidyl tansferase-mediated dUTP nick-end labeling (TUNEL) method was used to detect apoptosis. Sections were treated with 20 µg/mL proteinase K for 15 min at 37 °C and treated with hydrogen peroxide/methanol solution. Fragmented DNA was labeled using a reaction mixture, according to the manufacturer's instructions (Roche). Bound probes were detected using 3,3'-diaminobenzidine as a substrate.

Biochemical analysis

Blood was obtained by cardiac puncture. Serum urea nitrogen and creatinine concentration were determined by the Mitsubishi Kagaku Bio-Clinical Laboratories.

RNA isolation

Kidney samples for microarray analysis were obtained from inv{Delta}C and control mice at 4 weeks of age. RNA was isolated with TRIzol Reagent (Invitrogen), then further purified with the RNeasy Mini kit (Qiagen). Total RNA for RT-PCR was isolated by TRIzol Reagent.

Microarray analysis

Comparison of gene expression in kidneys in 4-week-old inv{Delta}C and control mice was carried out by the KURABO Industries, Biomedical Department. The Affymetrix Murine Genome 430 2.0 set was used to compare gene expression. A signal log ratio for each probe set was generated using comparison files from AffymetrixMAS 5.0 and converted into fold change according to the MAS 5.0 documentation.

Semi-quantitative RT-PCR

For RT-PCR, total RNA (5 µg) was subjected to reverse transcription using oligo-dT primer and superscript II reverse transcriptase (Invitrogen) at 42 °C for 50 min, followed by RNase H treatment (Takara). Semi-quantitative RT-PCR was carried out on an Applied Biosystems GeneAmp PCR system 9700. Hprt was used as the control gene for normalization. Each PCR amplification was performed in a 20 µL reaction mixture containing 40 ng cDNA and 2 ng/mL of each primer by use of the Ex-Taq system (Takara). Blots were quantified by densitometric analysis with the ImageJ 1.32 program (NIH).

Immunoblot analysis

Kidneys were homogenized with a Polytron in ice-cold tris-lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 100 mM NaF, 1 mM EDTA, 1 mM Na3VO4, and mammalian cell protease inhibitor mixture (1 : 100 v/v, P8349, Sigma)) and sonicated with Astrason ultrasonic processor for 30 s on ice. Bradford reagent (B6916, Sigma) was used to determine protein concentration. The homogenate was added in an equal volume of 2x SDS PAGE sample buffer (4% SDS, 20% glycerol, 0.1 M Tris-HCl (pH 6.8), bromophenolblue, and 12% 2-mercaptoethanol) and sonicated for 30 s on ice. The samples were heated at 95 °C for 5 min and cleared by centrifugation at 10 000x g for 10 min at 4 °C. SDS-PAGE and immunoblotting were carried out by standard procedures. To confirm that protein concentration of each sample was equal, sample were subjected to SDS-PAGE and the Coomassie Brilliant Blue gel stain. Sample of total protein (10 µg) were separated by 7.5 or 10% SDS-PAGE and transferred to a PVDF membrane. Cyclin D1, cyclin D3, cdk4, and cdk6 proteins were detected with monoclonal antibodies against human cyclin D1 (DCS6, 1 : 2000, Cell Signaling Technology), cyclin D3 (DCS22., 1 : 2000, Cell Signaling Technology), cdk4 (DCS156., 1 : 2000, Cell Signaling Technology) & cdk6 (DCS83, 1 : 2000, Cell Signaling Technology). Phospho-Rb (ser795) and phospho-Rb (ser807/811) proteins were detected with rabbit polyclonal antibodies against phospho-Rb (ser795) (1 : 1000, Cell Signaling Technology #9301) and phospho-Rb (ser807/811) (1 : 1000, Cell Signaling Technology #9308). Internal control protein was used actin and detected with goat polyclonal antibody against human actin (I-19, 1 : 1000, Santa Crus Biotechnology, INC.). Secondary anti-mouse, -rabbit, or -goat IgG-conjugated horseradish peroxidase secondary antibodies were from Cell Signaling Technology. The signals were visualized with the ECL–plus-detection system (GE Healthcare Bio-Sciences KK).

Statistical analysis

Comparison of survival between heterozygous and homozygous inv{Delta}C mice was performed by log rank test. The multiple comparison of the amount of inv mRNA between inv{Delta}C homozygous, inv{Delta}C heterozygous, inv heterozygous mutant and wild-type mice was performed by the Tukey–Kramaer post hoc test method. The multiple comparison of BrdU incorporation between inv{Delta}C heterozygous mice and control mice was performed by the Tukey–Kramaer post hoc test method. Student's t-test was used for other statistical analyses to compare between inv{Delta}C and control mice. Statistical analysis was performed by using Statcel2 software (OMS publishing).


   Acknowledgements
 
This research was partially supported by the Mitsubishi Foundation and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (15370095) to T.Y. and for Young Scientists (17790558) to N.S. We are grateful to Drs Hiroshi Hamada and Daisuke Watanabe (Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University) for providing inv/inv mice expressing the inv-GFP and inv{Delta}C-GFP.


   Footnotes
 
Communicated by: Yo-ichi Nabeshima

* Correspondence: E-mail: tyoko{at}koto.kpu-m.a.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 8 May 2006
Accepted: 27 June 2006




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