Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia
Charuhas V. Thakar, … , Hamid Rabb, Manoocher Soleimani
Charuhas V. Thakar, … , Hamid Rabb, Manoocher Soleimani
Published December 1, 2005
Citation Information: J Clin Invest. 2005;115(12):3451-3459. https://doi.org/10.1172/JCI25461.
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Research Article Cell biology

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.

Authors

Charuhas V. Thakar, Kamyar Zahedi, Monica P. Revelo, Zhaohui Wang, Charles E. Burnham, Sharon Barone, Shannon Bevans, Alex B. Lentsch, Hamid Rabb, Manoocher Soleimani

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Examination of the time course of expression of TSP-1 in kidney. (A) TSP...
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.