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Hum Mol Genet. 2009 July 15; 18(14): 2523–2531.
Published online 2009 April 2. doi:  10.1093/hmg/ddp147
PMCID: PMC2701325

Renal injury is a third hit promoting rapid development of adult polycystic kidney disease

Abstract

The ‘two-hit’ model is a widely accepted genetic mechanism for progressive cyst formation in autosomal dominant polycystic kidney disease. We have previously shown that adult inactivation of Pkd1 using the Mx1Cre+ allele causes a late onset of focal cystic disease. An explanation for the delayed appearance of cysts is the requirement for an additional independent factor, or ‘third hit’. Here we show that renal injury leads to massive cystic disease in the same mouse line. Cysts are labeled with a collecting duct/tubule marker, Lectin Dolichos biflorus Agglutinin, which correlates with the site of Cre-mediated recombination in the collecting system. 5-Bromo-2′-deoxyuridine labeling reveals that cyst-lining epithelial cells are comprised of regenerated cells in response to renal injury. These data demonstrate, for the first time, a role for polycystin-1 in kidney injury and repair and indicate that renal injury constitutes a ‘third hit’ resulting in rapid cyst formation in adulthood.

INTRODUCTION

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenetic disorder, occurring in 1:500 of the population (1,2). The disease results in end-stage renal disease in ~50% of subjects by the 6th decade. Mutations in the PKD1 (85%) and PKD2 (15%) genes, encoding polycystin-1 (PC1) and -2 (PC2) respectively, are responsible for this disease. Although every cell in ADPKD patients carries one germline mutated allele and one normal PKD allele, cyst formation occurs in only 5% of nephrons. The reasons for the focal nature of the disease are incompletely understood. However, the leading genetic hypothesis is a ‘two-hit’ model (3), which proposes that a germline mutation (first hit) in one allele of a PKD gene is not sufficient to result in cyst formation, but loss of both functional copies of a PKD gene upon a somatic mutation (second hit) later in life in a single cell is required for cyst formation. Germline homozygous deletion of Pkd1 in mice results in numerous large cysts in kidney and pancreas during embryogenesis (4,5), indicating that Pkd1 is necessary for normal kidney development and further supporting the ‘two-hit’ hypothesis. However, Pkd1 inactivation induced in adult life, shown by us and others, causes delayed cystogenesis (68). In our inducible Pkd1 knockout (IKO) mouse model, which utilizes the Cre/loxP system and an interferon (IFN)-inducible Mx1 promoter, inactivation of Pkd1 in 1-week-old developing kidneys leads to rapid, widespread cyst formation in distal nephron segments, consistent with the ‘two-hit’ hypothesis. However, when Pkd1 was inactivated in 5-week-old mice, there was only focal and slow progression of polycystic kidney disease (PKD). The discordance between rapid cyst growth in neonates versus much slower cyst growth in adult mice prompted us to search for a ‘third hit’, such as renal injury as a cause for rapid cyst development in adult life.

RESULTS

Renal ischemia–reperfusion injury causes extensive tubular damage in Pkd1 IKO mice

To investigate the impact of loss of Pkd1 on tubular injury, we performed unilateral renal ischemia reperfusion injury (IRI) on the Pkd1 IKO and their littermate control mice. All mice were injected with polyinosinic-polycytidylic acid (pI:pC) to induce the expression of Cre recombinase and to inactivate Pkd1 at 5 weeks of age. Unilateral renal IRI was performed at 8 weeks of age. The mice were then sacrificed 48 h later. Extensive tubular damage including hyaline casts, epithelial cell detachment from the basement membrane, tubular dilatation and cellular cast formation were observed in both the inner cortex and the outer stripe of the outer medulla in the kidneys from all Pkd1 IKO (n = 10), heterozygous Pkd1null/+ (n = 5) and littermate control (n = 7) mice, indicating successful IRI (Fig. 1A–C, G–I). As expected, contralateral (non-ischemic) kidneys were histologically normal (Fig. 1D–F, J–L). Interestingly, we found widespread and considerably more severe tubular damage in the inner stripe of outer medulla of Pkd1 IKO kidneys (Fig. 2B) compared with littermate kidneys from mice without Pkd1 inactivation (Fig. 2A). In contrast, contralateral (non-ischemic) kidneys were histologically normal in mice from all three genotypes (Fig. 2D–F). The inner stripe of the outer medulla contains distal straight tubules, descending thin limb of the loop of Henle and collecting ducts, and is not classically a site of severe injury in the renal IRI model (911). Consistent with these results, heterozygous Pkd1null/+ kidneys showed slightly more severe tubular damage in the medulla, when compared with littermate kidneys from mice without Pkd1 inactivation (Fig. 2C).

Figure 1.
Extensive tubular damage and cell proliferation were seen in the renal corticomedullary junction response to ischemia–reperfusion injury. Sex-matched 5-week-old littermate mice were injected with pI:pC to induce Cre recombinase expression and ...
Figure 2.
Pkd1 IKO mice are susceptible to ischemia–reperfusion injury. Tubular damage in the inner stripe of outer medulla was significantly higher in Pkd1 IKO mice (b) compared with control littermates (a, c). (df) Control kidney sections from ...

Renal ischemia–reperfusion injury causes extensive cell proliferation in Pkd1 IKO mice

In contrast to adult kidney, the developing kidney is characterized by a very high rate of epithelial cell proliferation. Abnormal cell proliferation is also a major feature of cystic kidneys in ADPKD (12). Acute renal injury reactivates developmental signaling pathways and triggers epithelial cell proliferation (9,13). To verify that our model of unilateral, renal IRI causes cell proliferation, mice subjected to IRI were injected with 5-bromo-2′-deoxyuridine (BrdU) 48 h later, and were sacrificed 2 h after BrdU injection. BrdU incorporation, a measure of DNA synthesis, was visualized by immunohistochemistry. In agreement with previous studies (9), numerous BrdU-labeled cells were detected at the corticomedullary junction in injured kidneys from Pkd1 IKO, heterozygous Pkd1null/+ and their littermate control mice (Fig. 1M–O), in contrast to the contralateral (non-ischemic) kidneys (Fig. 1P–R), indicating that renal IRI stimulus resulted in epithelial cell proliferation. Consistent with the more severe histological injury (Fig. 2B), we observed a significantly higher number of BrdU-labeled cells in tubules labeled with Dolichos biflorus Agglutinin (DBA), a collecting duct/tubule marker, in Pkd1 IKO kidneys (Fig. 2I and J) compared with littermate control kidneys (Fig. 2G and H) (12.5 ± 4.1 versus 0.9 ± 0.9 BrdU+/100 DBA+ cells; P < 0.005) (Fig. 2G–K). There was a trend toward a higher number of BrdU-positive cells in heterozygous Pkd1null/+ kidney, when compared with control kidney, which did not reach statistical significance. These findings demonstrate that Pkd1 inactivation in adult collecting tubules/ducts enhances susceptibility to ischemic injury.

Renal ischemia–reperfusion injury results in rapid cyst formation in Pkd1 IKO mice

To determine whether renal IRI promotes cyst development, we subjected additional groups of animals to unilateral, renal IRI and allowed the kidney to repair. Small animal magnetic resonance imaging (MRI) was used to monitor cyst progression. Unilateral renal IRI was performed at 8 weeks of age (15 IKO and 12 control mice, Table 1). Sex-matched littermate heterozygous Pkd1null/+ mice (n = 7) were also included to determine the effect of Pkd1 haploinsufficiency on cyst development. In stark contrast to the mice not subjected to injury (6), Pkd1 IKO mice subjected to renal IRI developed multiple cysts that appear as light gray signals in the medulla of the injured kidneys 4 weeks later (Fig. 3B, left kidney, arrow). Cystic lesions were absent in the contralateral (non-ischemic) kidneys (Fig. 3B, right kidney, arrowhead) that served as an internal control. These experiments provide direct evidence that renal injury promotes cyst formation in mice with Pkd1 inactivation induced in adult life. Importantly, kidneys from littermate control mice without Pkd1 mutation did not develop cysts (Fig. 3A, C), ruling out the possibility that cyst development is simply a result of renal injury. While renal cysts were completely absent by MRI 6 weeks after renal injury in littermate control mice and only few cysts were observed in Pkd1 IKO contrateral (non-ischemic) kidney (Supplementary Material, Fig. S1, arrowhead), a striking progression of renal cystic disease was evident in Pkd1 IKO mice (Fig. 3E, Supplementary Material, Fig. S1). The presence of renal cysts detected by MRI was confirmed by H&E staining (Fig. 3G). Of note, only one out of seven heterozygous Pkd1null/+ mice showed a single cyst in the ischemic kidney, indicating that Pkd1 inactivation in one allele is not sufficient to evoke extensive cystogenesis after renal injury. In contrast, contralateral kidneys were histologically normal but showed hypertrophy due to hyperfiltration (data not shown). Cysts in IKO mice after IRI were positive for a collecting duct/tubule marker, DBA but not for a proximal tubule marker, Lotus tetragonolobus lectin (LTL), which correlates with the site of Cre-mediated recombination in the collecting ducts/tubules for the Mx1Cre transgenic line (Fig. 3H). To determine the cyst volume, which we calculated it as the percentage of cystic area versus whole kidney area in a minimum of three sections, MRI images of injured kidneys from four individual Pkd1 IKO mice 4 and 6 weeks following IRI were analyzed using an imaging analysis software. The cyst volume of all four Pkd1 IKO kidneys increased over the period (Fig. 3I and j, Table 2). To compare the severity of PKD between the injured, not injured and sham-operated Pkd1 IKO mice, we determined the cyst volume in four H&E-stained sections of 14-week-old kidneys from each of 12 IKO mice and found a dramatic increase in cyst volume in the injured Pkd1 IKO kidneys 6 weeks following IRI (Fig. 3K). To investigate the time course of cystogenesis in response to IRI, we dissected mice 1 and 2 week(s) after IRI. Tubule dilatation was observed as early as 1 week after IRI and widespread small cysts were detected after another week (Supplementary Material, Fig. S2). To verify the findings in unilateral IRI model, we performed bilateral renal IRI on Pkd1 IKO or control mice. As expected, the progression of PKD in Pkd1 IKO kidneys can be detected by MRI at 2 (Fig. 4B), 5 (Fig. 4E) and 11 weeks (Fig. 4H) after bilateral IRI. Only Pkd1 IKO mice (n = 6) developed extensive cyst formation in both kidneys (Fig. 4B, E, H), in comparison with littermate non-IKO control (n = 5) or heterozygous Pkd1null/+ mice (n = 2). Severe renal cystic disease in Pkd1 IKO kidney can also be seen by histology (Fig. 4I). Similar to human ADPKD, Pkd1 IKO mice exhibited massively enlarged kidney, compared with control kidneys (Fig. 4J). Analyses of a total of 54 MRI images from both kidneys of three Pkd1 IKO mice at 2, 5 and 11 weeks following bilateral IRI revealed that there is a remarkable increase (~8-fold) in cyst volume in the Pkd1 IKO kidneys in the 9-week period we investigated (Fig. 4K and L, Table 2).

Figure 3.
Unilateral ischemia reperfusion injury causes severe cystic disease in adult mice with Pkd1 inactivation. Sex-matched 5-week-old littermate mice were injected with pI:pC and subjected to IRI 3 weeks later. Representative magnetic resonance imaging (MRI) ...
Figure 4.
The enlargement of polycystic kidney is accelerated in response to bilateral ischemia–reperfusion injury. Representative MRI images were taken at 2 (ac), 5 weeks (df) and 11 weeks (gi) after bilateral IRI. The progression ...
Table 1.
The total number of mice subjected to IRI
Table 2.
The time course of cyst volume (%) in Pkd1 IKO kidneys after IRI

Cell proliferation in response to renal injury is increased in Pkd1 IKO mice

Recent studies show that repairing cells in the kidney in response to IRI derive from proliferation of surviving tubular epithelial cells (14). To explore the role of epithelial cell proliferation on injury-induced cystogenesis and to determine whether cyst-lining epithelial cells are comprised of regenerated cells after kidney injury, we injected BrdU on the 2nd and 3rd days after unilateral IRI to label proliferating cells. Nine weeks later, kidneys were harvested and the paraffin-embedded kidney sections were co-stained with anti-BrdU antibody and DBA. The intensity of BrdU labeling in DBA-positive cyst-lining epithelial cells and dilated tubules in IKO mice was weak, and barely detectable in some cysts (Fig. 5A and B), which is in marked contrast to the strong signals observed at 48 h post-IRI (Fig. 5C), suggesting a dilution of BrdU following multiple rounds of cell proliferation. This set of experiments suggests that cyst-lining cells are derived from tubular epithelia that have proliferated in response to IRI (14). To investigate the time course of cell proliferation in collecting ducts/tubules in response to IRI, kidney sections were co-stained with an antibody against Ki67, a cell proliferation marker, and the collecting duct/tubule marker DBA. Approximately 900 DBA-labeled cells in six non-overlapping areas of two kidney sections from each of three experimental and three control mice were counted for Ki67 at four time points [48 h, 1, 2 and 7 week(s)]. We defined the percentage of Ki67 and DBA double-labeled cells versus total of DBA-labeled cells as tubular cell proliferation index (tPI). In injured kidney from Pkd1 IKO mice, about 60% of collecting ducts/tubules were positively labeled for Ki67 48 h after IRI and this population dropped by ~50% 1 week after IRI (Fig. 5D, Table 3). Compared with control mice, IKO mice tend to display a higher tPI at 48 h after IRI, but did not reach statistical significance. A significant increase in tPI was seen, however, in Pkd1 IKO kidney at 1 week (~28%) and 2 weeks (~21%) after IRI when small cysts are widespread (Supplementary Material, Fig. S2). The tubular proliferation index remained at ~9% in Pkd1 IKO kidneys 7 weeks after IRI (Fig. 5D, Table 3). In contrast, injured kidney from control mice without Pkd1 inactivation had no Ki67 positive cells at these stages (Fig. 5D, Table 3).

Figure 5.
Cell proliferation in Pkd1 IKO kidneys post-IRI. (ac) Kidney sections were double labeled with anti-BrdU antibody (red) and DBA (green). Diluted BrdU signals were detected in cyst-lining epithelial cells in IKO mice 9 weeks after IRI (a, b), ...
Table 3.
The time course of tubular proliferation index (%) in Pkd1 IKO kidneys after IRI

DISCUSSION

Because adult inactivation of Pkd1 in the kidney results in only focal and late onset of PKD, we recently proposed the ‘third hit’ hypothesis for ADPKD (6). Here we have tested renal IRI as a ‘third hit’ to cause rapid cyst development in adult life.

Two days after renal IRI we observed extensive tubular damage in both the inner cortex and the outer stripe of the outer medulla in the kidneys from all Pkd1 IKO, heterozygous Pkd1null/+, and littermate control mice. However, Pkd1 IKO mice exhibited widespread and more severe tubular damage in the inner stripe of outer medulla in kidneys, where Mx1Cre-mediated recombination (Pkd1 inactivation) takes place in this mouse line. The data suggest that Pkd1 expression normally protects these nephron segments from ischemic injury. Pkd1 may act either to protect tubular epithelia from injury or to accelerate repair, or both.

To see whether injury-induced cell proliferation in Pkd1 IKO kidney leads to cyst formation, we monitored the progression of PKD by MRI. In contrast to the contralateral (non-ischemic) kidney of Pkd1 IKO mice that served as an internal control, Pkd1 IKO injured kidney developed severe widespread cystic disease. To verify this observation after unilateral IRI, we performed bilateral IRI on Pkd1 IKO mice. As expected, Pkd1 IKO mice exhibited massively enlarged polycystic kidneys. Our study clearly demonstrates that IRI promotes renal cyst formation in cells that have received ‘two hits’ in Pkd1. Defects of primary cilia are implicated in the pathogenesis of PKD (15) and that PC1-deficient cells cannot respond to fluid flow shear stress through their primary cilia (16). Recently, Patel et al. (17) reported that adult Kif3A knockout mice exhibit mild cystic phenotype 2.5 weeks after 45 min of ischemia followed by reperfusion. In our mouse model, widespread small cysts were observed as early as 2 weeks after 25 min of ischemia followed by reperfusion, which is a milder ischemic condition. It is noteworthy that the nephron segments developing cystic lesions are collecting ducts/tubules in our Pkd1 model, and proximal tubule and thick ascending limbs of the loops of Henle in the Kif3a model. One possible explanation for the difference in phenotype is that the role of PC1 in adult collecting tubules/ducts in the kidney is either cilia independent or PC1 may serve more functions than a mechanosensor on primary cilia. However, further studies are required to address the role of PC1 on cilia in adult life because the Cre recombinase used to inactivate Pkd1 and Kif3a in these two studies was driven under different tissue-specific promoters (Mx1 and Ksp, respectively), which may have different activity in specific nephron segments. Nevertheless, we have established an excellent animal model for PKD1-disease that accounts for ~85% of human ADPKD cases.

The mechanism(s) for injury-induced cystogenesis in Pkd1 IKO mice is unknown. It is possible that Pkd1-deficient cells, unlike normal tubular epithelial cells, fail to switch off the normal renal injury-induced repair program. Instead, they continue to proliferate, resulting in cyst formation. The BrdU tracing study revealed that BrdU-incorporated cells in response to IRI in Pkd1 IKO kidney continue to proliferate. Consistent with this finding, the tubular proliferation index in collecting ducts/tubules fail to reduce to baseline after IRI. These results, in contrast to a recent report (8), support the classic model that increased cell proliferation is a major requirement of cyst development in human ADPKD (12). Notably the number of Ki67 positive cells in Pkd1 IKO kidney was similar to that in littermate control kidney 48 h after IRI despite the BrdU-labeled cells were significantly higher in Pkd1 IKO kidney. BrdU incorporation occurs during DNA synthesis in S phase, whereas Ki67 is expressed in all phases of the cell cycle (G1, S, G2, M). The Ki67-positive cells in collecting ducts/tubules in the injured wild-type kidneys may not be actively cycling because distal nephron segments including collecting tubules are not usually affected by IRI, in contrast to the S3 segment of the proximal tubule (911). Accordingly, we speculate that polycystin-1 may regulate G1 to S transition in mature renal epithelial cells. When PC1 is disrupted, distal tubular epithelial cells entering G1 phase as a response to injury will proceed to S phase, and the cell cycle progresses. An alternative explanation is that there is a defect in the control of the length of S phase in Pkd1 IKO kidney, such as a prolonged S phase that contributes to the increase in BrdU-labeled cells. Overall, these data suggested that tubular epithelial cells with Pkd1 inactivation have enhanced susceptibility to injury.

The data reported here provide the first experimental evidence supporting the ‘third hit’ hypothesis that renal injury or other genetic or non-genetic insults reactivating renal developmental programs and/or increasing cell proliferation promotes rapid cyst formation in mature kidneys in a orthologous model of human cystic disease. Several cross-sectional studies including the CRISP study have demonstrated considerable heterogeneity in the size of cysts within and between human ADPKD kidneys (18). Results from the current study suggest that in humans with ADPKD, subclinical kidney injury may be an important factor determining disease progression in adults. Preventing kidney injury and targeting the developmental pathways reactivated in repairing kidney represent important areas of possible intervention in the at risk population.

MATERIALS AND METHODS

Generation of inducible Pkd1 knockout mice

Inducible Pkd1 knockout mice were generated by crossing Pkd1flox/+ mice with a transgenic mouse line that expresses Cre recombinase under the control of an interferon-inducible Mx1 promoter (Mx1Cre mice). The resultant Mx1Cre+Pkd1flox/+ mice were bred with either Pkd1flox/+ mice or Pkd1null/+ mice to generate Mx1Cre+Pkd1flox/flox mice or Mx1Cre+Pkd1null/flox mice, respectively. Pkd1flox/+, Pkd1flox/flox or Mx1Cre+Pkd1+/+ mice were used as controls, and Pkd1null/flox, Pkd1null/+ or Mx1Cre+Pkd1null/+ mice were used as heterozygous Pkd1null/+.

Induction of Cre expression

Mice were intraperitoneally injected with 250 µg of IFN inducer pI:pC (Sigma) for 5 consecutive days at 5 weeks of age to induce the expression of Cre recombinase.

Renal IRI model

Studies were performed according to the animal experimental guidelines issued by the Animal Care and Use Committee at Harvard University. Animals were anesthetized with pentobarbital sodium (60 mg/kg body weight, intraperitoneally) prior to surgery. Body temperatures were controlled at 36.5–37.5°C throughout the procedure. Kidneys were exposed through flank incisions, and mice were subjected to ischemia by clamping the left renal pedicle or both left and right renal pedicle with nontraumatic microaneurysm clamps (Roboz, Rockville, MD, USA), which were removed after 25 min (males) or 35 min (females). One milliliter of 0.9% NaCl was administered subcutaneously 2 h after surgery. Sham operation was done in the same manner but without clamping of the renal pedicles. To label proliferating cells, mice were injected with BrdU (Sigma-Aldrich) intraperitoneally (50 mg/kg body wt) 48 h after IRI, and sacrificed 2 h later. Some mice were injected with BrdU on the 2nd and 3rd days after IRI and sacrificed more than 5 weeks after surgery.

Histology and immunohistochemistry

Paraffin-embedded sections (4 µm) were dewaxed, rehydrated through graded alcohols and boiled in 10 mm citrate (pH 6.0) (VECTOR) for 30 min. The sections were then placed in the staining dish at room temperature and allowed to cool for 1–2 h. Sections were incubated with 10% goat serum for 30 min and incubated with anti-BrdU antibody (1:100, BD Biosciences) or Ki67 (1:100, Vector) overnight at 4°C or for 1 h at room temperature. After washing with PBS, sections were incubated with secondary antibody for 1 h at room temperature. For double staining with tubules markers, Lectin DBA (VECTOR) and Lotu Tetragonolobus Lectin (LTL) (VECTOR), a dilution of 1:500 was used. After washing with PBS, sections were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen).

MRI experiments

MRI measurements were performed with a 4.7 T Bruker Avance horizontal bore system. The mice were anesthetized with 1% isoflurane in an oxygen mixture. After the mouse was fully anesthetized, the entire mouse was then placed in a special home-made body holder in order to minimize motion artifacts. Animals were imaged with a RARE imaging sequence (TR = 2800 ms, effective TE = 55 ms) in the coronal plane with a slice thickness of 0.75 mm and a slice number sufficient to both kidneys, with a matrix size of 256 × 192 and field of view of 3 × 3 cm2.

Measurement of cyst volume

The cyst volume was quantified in whole kidney sections using Image Pro Plus v5 software (Media Cybernetics) and calculated as (cystic area/total kidney area) × 100%. In MRI images of unilateral IRI kidneys, seven sections per kidney at each time point were analyzed for each mouse. In MRI images of bilateral IRI kidneys, three sections for each kidney and each time point were analyzed for each mouse. In H&E staining sections, four sections were analyzed for each mouse.

Tubular cell proliferation index

The tPI was measured by counting Ki67-positive cells in DBA-positive tubular epithelial cells and calculated as the percentage of Ki67 and DBA double-labeled cells versus total DBA-labeled cells. Six non-overlapping areas of two kidney sections at each time point were analyzed for each mouse.

Statistic analysis

The significance of differences between groups was determined by Student’s t-test. A P-value of <0.05 was considered statistically significant.

FUNDING

This work was supported by grants from the National Institutes of Health (DK51050 and DK40703) to J.Z., DK73628 to B.H. and DK074030 to the Harvard Center of Polycystic Kidney Disease. A.T. was a recipient of a postdoctoral fellowship from the American Heart Association.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

[Supplementary Data]

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Kambiz Zandi-Nejad for assistance in manuscript preparation and scientific suggestions. The authors also thank Drs Patrick Starremans, Jordan Kreidberg and other members of the Polycystic Kidney Disease Center at several Harvard institutions for scientific discussions and support.

Conflict of Interest statement. None declared.

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