PMC

Immunofluorescence staining of healthy murine renal tissue for Gb₃ and AQP-2. (A) High-power images of a medullary tubule stained with anti-Gb₃ and anti-AQP-2, and the merged image is shown, with overlap colored in yellow. The inset shows magnified view of the boxed region. (B) Low-power images of renal medulla stained with anti-Gb₃ and anti-AQP-2 and the merged image. Bars, 10 μm (A) and 100 μm (B).

Immunofluorescence staining of healthy murine renal tissue for Gb₃ and AQP-1. Cortical (A) and medullary (B) tubules stained with anti-Gb₃ and anti-AQP-1, and the merged image is shown, with overlap colored in yellow. Some cortical tubules express only AQP-1 (single white arrow), some express only Gb₃ throughout (two white arrows), while others express AQP-1 and Gb₃ in a punctuate pattern (large white arrowhead). Bar, 20 μm.

Gb₃ localization in untreated mouse renal tissue by immunohistochemistry. (A) Isotype control shows no renal staining. Sections from cortex (B), papilla (C), and medulla (D) demonstrate anti-Gb₃ staining of some population of tubules. No staining was observed in glomeruli. Asterisks in panel C are examples of Gb₃-positive tubules in this region. All images shown are representative of images from eight mice and are at a magnification of ×200.

Western immunoblots of activated p38 (phospho-38) and total p38 from differentiated human and murine glomerular cells over a 12-h time course. Cells were incubated in media alone as a control (Cont) or challenged with Stx2 (Stx), 1 μg/ml LPS, or Stx2 plus LPS (Stx LPS). For human cells, 1 pM Stx2 was employed, while 1 nM Stx2 was used for the murine cells.

Quantification of renal apoptosis, renal failure, urine osmolality, and weight loss in mice injected with Stx2 plus LPS alone or with the nonselective caspase inhibitor Q-VD-OPH (WVDOPH) or with the DMSO vehicle alone. Mice challenged with Stx2 plus LPS and treated with the caspase inhibitor demonstrated decreased numbers of apoptotic nuclei on TUNEL-stained renal tissue sections (A), increased urine osmolality (B), decreased BUN levels (C), and less weight loss (D) than mice challenged with Stx2 plus LPS alone. Values that were significantly different are indicated as follows: *, P < 0.05 compared to the values for the control; **, P < 0.05 compared to the values for mice challenged with Stx2 plus LPS alone.

Human glomerular cell caspase 3 activity and cell death inhibition by the nonselective caspase inhibitor Q-VD-OPH. Human glomerular endothelial cells (A) and human glomerular podocytes (B) were treated with Stx2, LPS, or Stx2 plus LPS for 12 h, and lysates were isolated. The viability of cells after incubation of human glomerular endothelial cells (C) and human glomerular podocytes (D) with Q-VD-OPH (QVDOPH) was compared to that of cells grown in media alone (control) or in media containing 0.5% DMSO (vehicle). Values that were significantly different are indicated as follows: *, P < 0.05 compared to the value for the control (not challenged with Stx2); **, P < 0.05 compared to the value for mice challenged with Stx2 alone (without Q-VD-OPH).

Quantification of renal apoptosis, renal failure, urine osmolality, and weight loss in mice injected with Stx2 plus LPS. (A) The total number of TUNEL-stained nuclei were counted per 16 fields (at a magnification of ×200) per tissue section and averaged. Renal apoptosis increased over the time course and became significant starting 60 h after injection. An example of TUNEL stain at 72 h after Stx2-plus-LPS injection is shown in the inset. The black arrowheads indicate TUNEL-positive nuclei. (B) Increased BUN levels occurred over a time course similar to that of tubular apoptosis. (C) Urine osmolality was decreased late in the time course, although these mice demonstrated an initial increase in urine solute concentration. (D) Mice lost substantial weight over the time course. Values that were significantly different (P < 0.05) from those of the control are indicated by an asterisk.

Human and murine glomerular cell expression of SV40 large T antigen and Gb₃. (A) Whole-cell lysates from human glomerular cells and murine glomerular cells grown at the permissive temperature (33°C) compared to human and murine glomerular cells grown at the nonpermissive temperature (37°C). Differentiated cells grown for 2 weeks at the nonpermissive temperature degraded the SV40 temperature-sensitive large T antigen (T-Ag). HUVEC human primary endothelial cells (ENDO) were used as a control. PODO, podocytes. (B) (Top) Thin-layer chromatography of total neutral lipids with Stx1B overlay to specifically detect Gb₃. (Bottom) Total neutral lipids visualized by cupric sulfate to demonstrate similar loads in the lanes. RPTEC are human proximal tubule cells, whereas TKPTS are murine proximal tubule cells. The glycosphingolipid standards included the following: ceramide monohexoside (glucosylceramide) (CMH), ceramide dihexoside (lactosylceramide) (CDH), globotriaoslyceramide (Gb₃), and globotetraosylceramide (Gb₄). Glom, glomerular; Endo, endothelial.