Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia

Abstract

Thrombospondin 1 (TSP-1) is a matricellular protein that inhibits angiogenesis and causes apoptosis in vivo and in vitro in several cancerous cells and tissues. Here we identify TSP-1 as the molecule with the highest induction level at 3 hours of IR injury in rat and mouse kidneys subjected to ischemia/reperfusion (IR) injury using the DNA microarray approach. Northern hybridizations demonstrated that TSP-1 expression was undetectable at baseline, induced at 3 and 12 hours, and returned to baseline levels at 48 hours of reperfusion. Immunocytochemical staining identified the injured proximal tubules as the predominant sites of expression of TSP-1 in IR injury and showed colocalization of TSP-1 with activated caspase-3. Addition of purified TSP-1 to normal kidney proximal tubule cells or cells subjected to ATP depletion in vitro induced injury as demonstrated by cytochrome c immunocytochemical staining and caspase-3 activity. The deleterious role of TSP-1 in ischemic kidney injury was demonstrated directly in TSP-1 null mice, which showed significant protection against IR injury-induced renal failure and tubular damage. We propose that TSP-1 is a novel regulator of ischemic damage in the kidney and may play an important role in the pathophysiology of ischemic kidney failure.

Figure 1

DNA microarray results. Two separate filters, GF301 (A) and GF300 (B), were probed with the ³³P-labeled cDNA, which was prepared using poly(A+) RNA from sham-operated kidneys and kidneys with IR injury (IRI). Aldo. synthase, aldosterone synthase; cyt. P-450, cytochrome p450; b[0], y intercept; b[1], slope; r², correlation coefficient.

Figure 2

Examination of the time course of expression of TSP-1 in kidney. (A) TSP-1 expression: Northern hybridization. Northern blot analyses were performed (30 μg/well of total RNA) to determine the time course of expression of TSP-1 in IR injury. The expression of TSP-1 was induced at 3 hours of reperfusion, remained elevated at 12 hours of reperfusion, and decreased to undetectable baseline levels at 24 and 48 hours of reperfusion. Equal loading was confirmed by the examination of 18S rRNA bands. (B) TSP-1 expression: immunoblot analysis. Left panel: Western blot analysis was performed as described in Methods. Right panel: quantitation of the results. (C) Expression of CD36 in mouse kidney cortex. Left panel: RT-PCR of CD36. Right panel: Northern hybridization of CD36 in kidney IR injury. (D) Expression of p53 in kidney IR injury. The time course of expression of p53 was examined in IR injury. As indicated, the expression of p53 remained unchanged at 3 hours of reperfusion, increased moderately at 12 hours of reperfusion, and peaked at 24 hours of reperfusion. The expression of p53 decreased significantly at 48 hours of reperfusion. Equal loading was confirmed by the examination of 28S rRNA bands.

Figure 3

Immunofluorescence labeling of TSP-1 in kidney IR injury. Paraffin-embedded kidney sections were examined for the expression of TSP-1 by immunofluorescent microscopy in control (sham operated) and at 3 hours after reperfusion. G, location of glomerulus. Magnification, ×400.

Figure 4

Double immunofluorescence labeling of TSP-1 and activated caspase-3 in kidney IR injury. Shown are 2 separate paraffin-embedded kidney sections from 3-hour reperfusion, examined for the expression of TSP-1 (red) and activated caspase-3 (green) by immunofluorescent microscopy. The merged images (center) indicate the colocalization of the 2 proteins in the same cells. Magnification, ×400.

Figure 5

Effect of TSP-1 on kidney proximal tubule cells in vitro. (A–D) Cytochrome c release at 24 hours. As indicated, immunocytochemical staining was performed for cytochrome c on paraffin-fixed cells. (A and B) Vehicle-treated cells showing fine punctate/granular staining for cytochrome c within the cytoplasm (A) and double labeling with nuclear staining (B). (C and D) Same staining procedure performed on cells after incubation with 1 ng/ml of TSP-1. Magnification, ×40. (E–H) Cytochrome c release at 48 hours. (E and F) Vehicle-treated cells. (G and H) TSP-1–treated cells. Other parameters, including magnification and acquisition of the images were similar to those for A–D. (I) Caspase-3 activity assay. Caspase-3 enzymatic activity was measured colorimetrically as described in Methods. As indicated, caspase-3 activity increased significantly in TSP-1–treated cells compared with vehicle-treated cells.

Figure 6

Effect of TSP-1 gene deletion on kidney function and structure in kidney IR injury. (A) Kidney function. As demonstrated, mice deficient in TSP-1 showed significant protection against acute renal failure in IR injury, as assessed by serum creatinine measurement at 24 hours after reperfusion compared with WT animals. (B) Histological analysis of kidneys. Top panel: 24-hour IR injury in WT and TSP-1 null mice. Extensive brush border loss and vacuolization (arrows) were observed in WT animals. Bottom panel: Sham-operated WT and TSP-1 null mice. a, epithelial vacuolization; b, loss of brush border/tubular dilatation. Magnification ×40. (C) Histological analysis of kidneys (low magnification). Top panels: 24-hour IR injury in WT. Bottom panels: TSP-1 null mice. Extensive brush border loss (light green arrows), tubular dilation (dark blue arrows), vacuolization (light blue arrows), cast formation (black arrows), and necrosis (area surrounded by dark green arrows) were observed in WT animals. Magnification ×10.