Telomere shortening reduces regenerative capacity after acute kidney injury

Abstract

Telomeres of most somatic cells progressively shorten, compromising the regenerative capacity of human tissues during aging and chronic diseases and after acute injury. Whether telomere shortening reduces renal regeneration after acute injury is unknown. Here, renal ischemia-reperfusion injury led to greater impairment of renal function and increased acute and chronic histopathologic damage in fourth-generation telomerase-deficient mice compared with both wild-type and first-generation telomerase-deficient mice. Critically short telomeres, increased expression of the cell-cycle inhibitor p21, and more apoptotic renal cells accompanied the pronounced damage in fourth-generation telomerase-deficient mice. These mice also demonstrated significantly reduced proliferative capacity in tubular, glomerular, and interstitial cells. These data suggest that critical telomere shortening in the kidney leads to increased senescence and apoptosis, thereby limiting regenerative capacity in response to injury.

Figure 1.

Histopathology after renal IRI is shown. (A) Acute tubular damage, shown as percentage of damage to total tubular area, was quantified 1, 3, 7, and 30 d after 30 min of IRI of the left kidney in Terc^+/+, G1 Terc^−/−, and G4 Terc^−/− kidneys. (B) Representative periodic acid-Schiff stainings 3 d after IRI showing more acute tubular damage in G4 Terc^−/− compared with G1 Terc^−/− and Terc^+/+. * areas of acute tubular damage. (C) Chronic tubular deterioration and (D) interstitial fibrosis—both reflecting chronic kidney damage—were quantified 1, 3, 7, and 30 d after IRI in Terc^+/+, G1 Terc^−/−, and G4 Terc^−/−. (E) Representative Masson Trichrome stainings showing increased interstitial fibrosis (blue staining, indicated by black arrowhead) in G4 Terc^−/− on day 30 after IRI. □, Terc^+/+; , G1 Terc^−/−; ■, G4 Terc^−/−. Data are means ± SEM; significances are indicated. Magnification, ×200.

Figure 2.

Renal Ctgf expression after IRI is shown. (A) Analysis of Ctgf protein expression given as fold expression on the basis of the expression of Terc^+/+ nonoperated controls. (B and C) Western blots showing Ctgf at 37 kD and β-actin as a loading control at 42 kD for day 7 (B) and day 30 (C). □, Terc^+/+; , G1 Terc^−/−; ■, G4 Terc^−/−. Data are means ± SEM; significances are indicated.

Figure 3.

Telomere Q-FISH and renal gene expression analysis are shown. (A) Representative pictures for Q-FISH analysis showing tubular cross-sections from nonoperated Terc^+/+ and G4 Terc^−/− controls and from Terc^+/+ and G4 Terc^−/− kidneys 30 d after IRI. (B) Distribution of TFI in kidneys showing that critically shortened telomeres, defined as TFI ≤40, are rarely found in nonoperated Terc^+/+ and G4 Terc^−/− controls, even though there is a significant difference in mean TFI between the two groups; however, kidneys from G4 Terc^−/− mice 30 d after IRI show a significantly higher number of critically shortened telomeres when compared with Terc^+/+ 30 d after IRI. (C) p21 expression was significantly higher in G4 Terc^−/− compared with Terc^+/+ and G1 Terc^−/− for days 3, 7, and 30 after IRI as well as for sham-operated controls. A similar tendency was seen for nonoperated controls. *Sham-operated controls (P < 0.005 for Terc^+/+, P < 0.001 for G1 Terc^−/−, and P < 0.05 for G4 Terc^−/−) as well as **nonoperated controls (P < 0.005 for Terc^+/+, P < 0.001 for G1 Terc^−/−, and P < 0.05 for G4 Terc^−/−) showed significantly lower p21 expression levels compared with days 1, 3, 7, and 30 after IRI. (D) Cell-cycle inhibitor p16^INK4a showed a continuous increase after IRI in Terc^+/+, G1 Terc^−/−, and G4 Terc^−/− kidneys with significantly higher levels on day 30 compared with sham-operated (***P < 0.05 for Terc^+/+, P < 0.001 for G1 Terc^−/−, and P < 0.001 for G4 Terc^−/−) and nonoperated controls (****P < 0.01 for Terc^+/+, P < 0.001 for G1 Terc^−/−, and P < 0.001 for G4 Terc^−/−). Apart from day 30, significantly higher values of p16^INK4a were seen in G4 Terc^−/− kidneys when compared with Terc^+/+ (day 3), G1 Terc^−/− (day 7), or both (day 1). □, Terc^+/+; , G1 Terc^−/−; ■, G4 Terc^−/−. Data are means ± SEM; significances are indicated. Figure 3 is also provided in color as Supplemental Figure 3.

Figure 4.

Proliferation and apoptosis after renal IRI are shown. (A through C) Quantification of Ki-67–positive tubular, glomerular, and interstitial nuclei in Terc^+/+, G1 Terc^−/−, and G4 Terc^−/− animals 3 (A) and 30 d (B) after IRI and in sham-operated controls (C). Lower proliferation rates were seen in tubular and interstitial cells of G4 Terc^−/− kidneys on day 3 and in tubular, glomerular, and interstitial cells of G4 Terc^−/− kidneys on day 30 compared with Terc^+/+ and G1 Terc^−/−. Very low proliferation rates were detected in renal cells from sham-operated controls with no significant differences among the three groups. (D) Representative Ki-67 immunostainings on day 30 after IRI showing reduced numbers of positively stained nuclei in G4 Terc^−/−. (E) Representative TUNEL stainings on day 30 after IRI showing increased numbers of apoptotic cells in G4 Terc^−/−. TUNEL-positive cells are marked by arrowheads. (F through H) Quantification of TUNEL-positive tubular, glomerular, and interstitial nuclei in Terc^+/+, G1 Terc^−/−, and G4 Terc^−/− animals 3 (F) and 30 d (G) after IRI and in sham-operated controls (H). For both time points after IRI, G4 Terc^−/− kidneys showed a significantly higher number of TUNEL-positive tubular and interstitial cells compared with Terc^+/+. The same was true for the comparison of G4 Terc^−/− with G1 Terc^−/− kidneys, with the exception of interstitial cells 3 d after IRI. Very low numbers of apoptotic tubular and interstitial cells and no apoptotic glomerular cells were detected in sham-operated controls, with no statistical differences between groups. □, Terc^+/+; , G1 Terc^−/−; ■, G4 Terc^−/−. Data are means ± SEM; significances are indicated. Magnification, ×200.