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.

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 subjected to IRI 4 weeks later. These mice were sacrificed 48 h after injury. Representative H&E staining of kidney sections were shown (a–l). Extensive tubular damage was seen in the renal corticomedullary junction in control (a, g), Pkd1 IKO (b, h) and Pkd1 heterozygous (c, i) mice. (d–f, g–i) Control kidney sections from not injured (non-IR) mice. Sections from ischemic kidney 48 h post-IRI (m–o) and contralateral non-ischemic kidneys (p–r) were labeled with anti-BrdU antibody (red). Autofluorescence (green) was used to illuminate the tubular structures. There is no significant difference in the number of BrdU-positive cells between control, Pkd1 IKO and Pkd1 heterozygous. The genotypes shown here are: control, Pkd1^flox/+; IKO, Mx1Cre⁺Pkd1^null/flox; Het, Pkd1^null/+.

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). (d–f) Control kidney sections from not injured (non-IR) mice. (g–j) Sections were double labeled with anti-BrdU antibodies (red) and a collecting duct/tubule marker DBA (green). Compared with control mice (g, h), the number of BrdU-positive tubular epithelial cells (represented by DBA labeling) in medulla were higher in Pkd1 IKO mice (i, j). Asterisk denotes dilated tubule (k). Quantitation of the number of BrdU-positive cells per hundred DBA-positive tubular epithelial cells in IKO mice. Data are mean ± SD with three or four mice for each genotype group. *P < 0.005. C, control, I, IKO, H, Het.

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) images were taken at 4 (a–c) and 6 weeks (d–f) after unilateral IRI. No cysts were observed in Pkd1^flox/flox (control) (a, d) and Pkd1^null/+ (Het) (c, f) mice. A striking polycystic phenotype is found in the ischemic kidney (arrow) but not in the contralateral (non-ischemic, arrowhead) kidney from Mx1Cre⁺Pkd1^flox/flox (IKO) mice (b, e). All mice shown here are females. Representative H&E staining sections from kidneys 7 weeks after unilateral IRI showing extensive cystic lesions in Mx1Cre⁺Pkd1^flox/flox (IKO) ischemic kidney (g). Panels were digitally assembled to show the whole kidney. All mice shown here are males. Mx1Cre⁺Pkd1^null/flox (IKO) ischemic kidney sections were stained with DBA (red) and LTL (green) (h). Panels were digitally assembled to show the cortex and medulla. The total cyst volume was measured in seven serial MRI images of injured kidneys from each of four Pkd1 IKO mice at 4 and 6 weeks following IRI (i, j). The renal cyst volume of each of the four IKO mice (i) and their average (j) both indicate a steady rate of disease progression over 2 weeks. The total cyst volume was also measured in four serial H&E stained kidney sections from each of five injured (IRI), four not injured (no IRI) and three sham-operated (Sham) Pkd1 IKO mice (k).

Figure 4.

The enlargement of polycystic kidney is accelerated in response to bilateral ischemia–reperfusion injury. Representative MRI images were taken at 2 (a–c), 5 weeks (d–f) and 11 weeks (g–i) after bilateral IRI. The progression of polycystic phenotype was observed in the both ischemic kidneys (arrows) in Pkd1 IKO mice, while the injured kidneys in their littermate control and Pkd1^null/+ mice were recovered from injury. (j) Gross appearance of massively enlarged polycystic (right) and normal (left) kidneys from respective IKO and control mice 15 weeks after bilateral IRI. Representative H&E staining sections of these kidneys are shown in the right panels. The cyst volume was measured in six independent MRI images of both kidneys from each of three Pkd1 IKO mice at 2, 5 and 11 weeks following IRI (k, l). The cyst volume in each mouse (n = 3) (k) and the average cystic volume of a Pkd1 IKO kidney (n = 6) (l) steadily increase with time.

Figure 5.

Cell proliferation in Pkd1 IKO kidneys post-IRI. (a–c) 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), in contrast to the strong BrdU signals at 48 h after IRI (c). Dashed lines outline the tubule in (c). Photos were taken under the same exposure time for (b) and (c). The tubular cell proliferation index (percentage of Ki67 positive cells in DBA positive cells) was measured in six non-overlapping areas of two kidney sections from each of three experimental and three control mice at 48 h, 1, 2 and 7 week(s) following IRI (d).