Shiga toxin 1 causes direct renal injury in rats

Abstract

Infection with Shiga toxin (Stx)-producing Escherichia coli has been implicated to cause hemolytic uremic syndrome, which is characterized by histological abnormalities such as microvascular thrombi and tubular cell damage in the kidney. Although Stx is known to be the major virulence factor of the pathogen, it is still unclear whether Stx directly impairs renal cells in vivo to cause such histological changes and deterioration of renal function. To assess the consequence of the direct action of Stx on renal cells, left kidneys of rats were perfused with Stx1 from the renal artery through the renal vein and then revascularized. Kidneys of control animals were perfused with the vehicle alone. On day 1, apoptosis and induction of tumor necrosis factor alpha gene expression were noticed to occur in the medulla of the Stx1-perfused kidneys. On day 3, extensive tubular injuries were observed by light microscopy: aggregated platelets and monocytic infiltrates in both glomeruli and the medullary interstitium were detected by immunostaining. Tubular changes were more extensive on day 9, with areas of infarction seen in the cortex and medulla. These changes were not found to occur in the sham-operated kidneys. No obvious glomerular changes were detected by light microscopy at any time point. When nonperfused right kidneys were removed after the Stx1 perfusion of the left kidneys, the serum creatinine and blood urea nitrogen levels were increased from day 2, and acute renal failure followed on day 3. These results indicate that Stx1 caused glomerular platelet aggregation, tubular damage, and acute deterioration of renal function by acting directly on renal cells.

FIG. 1.

Typical histological changes in tubulointerstitium of Stx1-perfused and revascularized kidneys seen under light microscopy. Cortex (A and B), outer stripe (C and D), inner stripe (E and F), and inner medulla (G and H) of Stx-perfused kidney on day 3 (A, C, E, and G) and day 9 (B, D, F, and H) postperfusion are shown. Asterisks, tubular dilation; black arrows, cytoplasmic vacuolation; white arrows, desquamation; black arrowheads, leukocyte infiltration; white arrowheads, pyknosis. In the Stx1-perfused kidney, massive infiltration of inflammatory cells is seen on day 3. Severe tubular dilatation and degeneration are notable on day 9. Hematoxylin-eosin staining. Bar, 100 μm.

FIG. 2.

Semiquantitative analysis of tubulointerstitial injuries seen under light microscopy. Ranking system at left is described in Materials and Methods. Filled circles, Stx-perfused rats; open circles, sham-operated control rats. Each symbol represents one animal. The differences between the two groups on days 3 and 9 in all areas are statistically significant (P < 0.05).

FIG. 3.

TUNEL labeling of rat kidneys on day 1. Some tubular cells in the kidneys of Stx-perfused rats were positive for TUNEL staining, but those in the kidneys of the sham-operated rats were not.

FIG. 4.

Expression of TNF-α mRNA in renal cortex and medulla on day 1. Filled circles, Stx-perfused rats (S); open circles, sham-operated control rats (C). Data are expressed as the ratios of the levels of TNF-α mRNA to those of the internal control. Each symbol represents one animal. NS, not significant.

FIG. 5.

Monocyte/macrophage infiltration in glomeruli and interstitium on days 1 and 3. Filled circles, Stx-perfused rats (S); open circles, sham-operated control rats (C). Each symbol represents one animal. NS, not significant.

FIG. 6.

Glomerular platelet aggregation on day 3. (a) Immunostaining of platelets in glomeruli and (b) quantitative analysis of the platelet aggregation. Glomerular platelet aggregation is expressed as a percentage of the area of glomeruli in cross-section that is occupied by platelets. Filled circles, Stx-perfused rats; open circles, sham-operated control rats. Each symbol represents one animal.

FIG. 7.

Levels of serum creatinine and BUN in Stx-perfused rats with uninephrectomy. Filled circles, Stx-perfused rats; open circles, sham-operated control rats. Each symbol represents one animal. NS, not significant.