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
) 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
in these two studies was driven under different tissue-specific promoters (Mx1
, 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 (9
). 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.