Renal damage in obstructive nephropathy is decreased in Skp2-deficient mice

Abstract

Ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor p27 mediated by SCF-Skp2 ubiquitin ligase is involved in cell cycle regulation. Proliferation of tubular cells is a characteristic feature in obstructed kidneys of unilateral ureteral obstruction. Comparing Skp2(+/+) mice with Skp2(-/-) mice, we investigated the involvement of Skp2, a component of SCF-Skp2 ubiquitin ligase for p27, in the progression of renal lesions in unilateral ureteral obstructed kidneys. mRNA expression of Skp2 was markedly increased in the obstructed kidneys from Skp2(+/+) mice and peaked 3 days after unilateral ureteral obstruction. Renal atrophy, tubular dilatation, tubulointerstitial fibrosis, and increases in alpha-smooth muscle actin expression, the number of tubular cells, and proliferating tubular cells positive for Ki67 were observed in the obstructed kidneys from Skp2(+/+) mice; however, these findings were significantly attenuated in Skp2(-/-) mice. The p27 protein level was increased in the obstructed kidneys but was significantly greater in Skp2(-/-) mice. The number of Ki67-positive p27-negative cells was lower in obstructed kidneys from Skp2(-/-) mice than Skp2(+/+) mice, whereas that of Ki67-negative p27-positive cells was greater in Skp2(-/-) mice. These findings suggest that p27 accumulation, which results from SCF-Skp2 ubiquitin ligase deficiency in Skp2(-/-) mice, is involved in the amelioration of renal damage induced by obstructive nephropathy.

Figure 1

Representative pictures of UUO kidney sections (A; immunostaining for Ki67; magnification, ×300), percentage of dilated renal tubules (B), the number of Ki67-positive tubular (C) and interstitial cells (D), and the number of tubular epithelial cells (E) and interstitial cells (F) from Skp2^+/+ mice at the experimental time points after UUO. Many tubules were dilated in obstructed kidneys from day 3 after UUO (A and B). Numbers of Ki67-positive tubular cells and interstitial cells were increased in obstructed kidneys on day 3 after UUO (C and D). Tubular epithelial cells per tubule (E) and interstitial cells (F) were also increased in obstructed kidneys from day 3. The interstitial area was enlarged widely in obstructed kidneys (A). *P < 0.05 (C); P < 0.005 (B, E, and F); **P < 0.0005, ***P < 0.0001 versus sham-operated kidneys.

Figure 2

Level of Skp2 mRNA measured by quantitative RT-PCR in the obstructed kidneys from Skp2^+/+ wild-type mice at the experimental time points after UUO. In the obstructed kidneys from Skp2^+/+ mice, mRNA expression of Skp2 markedly increased and peaked 3 days after UUO. *P < 0.05, **P < 0.005 versus sham-operated kidneys.

Figure 3

Levels of Skp2 and GAPDH mRNA expression (A) and the representative macroscopic (B) and microscopic (C; periodic acid-Schiff, magnification, ×300) findings of the obstructed (UUO) and nonobstructed contralateral (CLK) kidneys of Skp2^+/+ (left) and Skp2^−/− mice (right) 7 days after UUO. No detectable expression of Skp2 mRNA was observed in Skp2^−/− mice (A). Although remarkable renal atrophy was noted in the obstructed kidneys from Skp2^+/+ mice, it was markedly less in Skp2^−/− mice (B). Tubular dilatation and atrophy and interstitial cell infiltration were observed in obstructed kidneys from Skp2^+/+ mice. However, the severity of these lesions was markedly less in obstructed kidneys from Skp2^−/− mice (C).

Figure 4

Representative pictures of immunostaining for Ki67 (A; magnification, ×600), percentage of dilated renal tubules (B), number of tubular epithelial cells per tubule (C), Ki67-positive tubular cells (D), and apoptosis (E) in the obstructed (UUO) and nonobstructed contralateral (CLK) kidneys of Skp2^+/+ and Skp2^−/− mice 7 days after UUO. Many tubules were dilated (A and B), with increased numbers of tubular epithelial cells (C), and their tubules had Ki67-positive tubular epithelial cells (A and D) in obstructed kidney from Skp2^+/+ mice. However, tubular dilatation, increases of tubular cells, Ki67-positive cells, and apoptosis were markedly less in obstructed kidneys from Skp2^−/− mice. *P < 0.0001; Skp2^+/+ CLK versus Skp2^+/+ UUO, #P < 0.0001; Skp2^+/+ UUO versus Skp2^−/− UUO in B, C, and D. *P = 0.0001; Skp2^−/− CLK versus Skp2^−/− UUO, **P < 0.0001; Skp2^+/+ CLK versus Skp2^+/+ UUO, ^#P < 0.0001; Skp2^+/+ UUO versus Skp2^−/− UUO in E.

Figure 5

Levels of p27 protein detected by Western blot analysis (A) and the representative pictures of immunostaining for Ki67 and p27 in consecutive renal sections (B; magnification, ×300) from the obstructed (UUO) kidneys of Skp2^+/+ (left) and Skp2^−/− mice (right) 7 days after UUO and the population of proliferating and quiescent tubular epithelial cells (C). p27 protein accumulated in obstructed kidneys from Skp2^−/− mice (A and B). Black arrows show each Ki67-positive cell in top panels or p27-positive cell in bottom panels. White arrows show each Ki67-negative cell in top panels or p27 negative cell in bottom panels. Proliferating cells, Ki67-positive/p27-negative (C, white bar), decreased and quiescent cells, Ki67-negative/p27-positive (C, black bar), increased in obstructed kidneys from Skp2^−/− mice.

Figure 6

The number of interstitial cells (A), Ki67-positive (B), and apoptotic interstitial cells (C) in the obstructed (UUO) and nonobstructed contralateral (CLK) kidneys of Skp2^+/+ and Skp2^−/− mice 7 days after UUO. The number of interstitial cells and apoptosis decreased in obstructed kidneys from Skp2^−/− mice. *P < 0.05; Skp2^−/− CLK versus Skp2^−/− UUO, **P < 0.0001; Skp2^+/+ CLK versus Skp2^+/+ UUO, ^#P < 0.005; Skp2^+/+ UUO versus Skp2^−/− UUO in A. *P < 0.05; Skp2^+/+ CLK versus Skp2^+/+ UUO in B. *P < 0.001; Skp2^−/− CLK versus Skp2^−/− UUO, **P < 0.0001; Skp2^+/+ CLK versus Skp2^+/+ UUO, ^#P < 0.05; Skp2^+/+ UUO versus Skp2^−/− UUO in C.

Figure 7

Representative pictures of the CLK and UUO kidney sections from Skp2^+/+ mice (A, left) and Skp2^−/− mice (A, right) and the semiquantification of staining intensity (B) for Masson’s trichrome 7 days after obstruction. The interstitial area was increased, and fibrosis progressed in obstructed kidneys from Skp2^+/+ mice. However, it was moderated in obstructed kidneys from Skp2^−/− mice. The grading of intensities shown in B was as follows: G0 (grade 0, 0%, white bars): G1 (grade 1, ≤20% in interstitial area colored green, yellow bars); G2 (grade 2, 21 ≈ 50%, red bars); G3 (grade 3, 51 ≈ 80%, blue bars); and G4 (grade 4, ≥81%, black bars). P < 0.0001; Skp2^+/+ UUO versus Skp2^−/− UUO.

Figure 8

Representative pictures of the CLK and UUO kidney sections from Skp2^+/+ mice (A, left) and Skp2^−/− mice (A, right) and the semiquantification of immunostaining intensity (B) for α-SMA 7 days after obstruction. Significant increases of interstitial myofibroblastic cells positive for α-SMA were observed in obstructed kidneys from Skp2^+/+ mice on day 7, but the levels were markedly attenuated in obstructed kidneys from Skp2^−/− mice (A, magnification, ×300). The grading of immunostaining intensities for α-SMA shown in B was as follows: G0 (grade 0, positive area 0%); G1 (grade 1, ≤20% in interstitial area were positive); G2 (grade 2, 21 ≈ 50% positive); G3 (grade 3, 51 ≈ 80% positive); and G4 (grade 4, ≥81% positive). P < 0.0001; Skp2^+/+ UUO versus Skp2^−/− UUO.