Endotoxemia alters tight junction gene and protein expression in the kidney

Abstract

Intact tight junctional (TJ) proteins are required for tubular ion transport and waste excretion. Disruption of TJs may contribute to a decreased glomerular filtration rate in acute kidney injury (AKI) via tubular backleak. The effect of LPS-mediated AKI on murine TJs has not been studied extensively. We hypothesized LPS endotoxin administration to mice would disrupt tubular TJ proteins including zonula occludens-1 (ZO-1), occludin, and claudins. ZO-1 and occludin immunofluorescence 24 h post-LPS revealed a marked change in localization from the usual circumferential fencework pattern to one with substantial fragmentation. Renal ZO-1 expression was significantly reduced 24 h after LPS (decrease of 56.1 ± 7.4%, P < 0.001), with subsequent recovery. ZO-1 mRNA expression was increased 24 h post-LPS (4.34 ± 0.87-fold, P = 0.0019), suggesting disruption of ZO-1 protein is not mediated by transcriptional regulation, but rather by degradation or changes in translation. Similarly, claudin-4 protein expression was decreased despite elevated mRNA. LPS administration resulted in dephosphorylation of occludin and fragmented tubular redistribution. Protein expression of claudin-1, and -3 was increased after LPS. ZO-1, occludin, and claudin-1, -3, and -4 gene expression were increased 48 h after LPS, suggesting a renal response to strengthen TJs following injury. Interestingly, reduced mRNA expression was found only for claudin-8. This study provides further support that LPS-induced AKI is associated with structural injury and is not merely due to hemodynamic changes.

Fig. 1.

Effect of LPS on light microscopic pathology. Periodic acid-Schiff-stained light microscopy of mouse kidney tissue is shown. A: normal mouse kidney. B: mouse kidney 24 h post-LPS. Widespread vacuolization and occasional denuded cells (arrow) are seen in tubules. Magnification: ×60. Bar = 20 μm.

Fig. 2.

Effect of LPS on renal tight junction (TJ) protein expression. Mice were injected with LPS (10 mg/kg) and euthanized 6, 24, and 48 h after injection. Representative immunoblots are shown. Each lane represents a different mouse. A: zonula occludens-1 (ZO-1) expression is decreased 24 h following LPS administration. B: occludin is heavily phosphorylated, revealing multiple bands from 79 to 65 kDa. Total occludin expression is unchanged; however, a shift from higher molecular weight bands to the lower molecular weight 65-kDa band occurs following LPS administration. C: claudin-1 expression increases over time after LPS administration. D: claudin-3 expression increases over time after LPS administration. E: claudin-4 expression decreases at all time points after LPS administration. F: claudin-8 expression was not significantly changed at any time point after LPS administration.

Fig. 3.

TJ protein densitometry. Protein levels of ZO-1, occludin, and claudin-1, -3, -4, and -8 were determined at each time point. Band densities are expressed as a ratio to actin. Protein expression in control mice was normalized to a value of 1. A: total protein expression in whole mouse kidney for all 6 TJ proteins. B: occludin densitometry. Total occludin did not differ significantly between baseline and post-LPS mice. Highly phosphorylated occludin band (those >70 kDa in size) intensity decreased while the lower molecular weight 65-kDa occludin increased. The ratio of bands >70 kDa to the 65-kDa band decreased 24–48 h (T) after LPS administration. *Significant comparison with baseline with adjusted P < 0.05. +Significant comparisons to T = 6 with P < 0.05. ^Significant comparisons to T = 24 with P < 0.05.

Fig. 4.

TJ staining in mouse kidney. Frozen sections of kidney tissue were stained via immunofluorescence for TJ proteins in control mice and mice 24 h post-LPS. A: ZO-1 control stain. Tubules are shown with circumferential “fencework” cellular staining (arrowhead). B: ZO-1 24 h post-LPS. Tubules lack this usual circumferential pattern. Instead, there were significant fragmentation and nonlinear, diffuse staining of ZO-1. C: occludin control stain. Circumferential cellular staining is denoted by the arrowhead. D: occludin 24 h post-LPS. Tubules reveal decreased circumferential staining, with increased apical and intratubular distribution (arrow). E: claudin-1 control stain. Bowman's capsule and occasional tubular staining are observed. F: claudin-1 staining 24 h post-LPS. No discernible changes were noted compared with control. G: claudin-3 control stain. Circumferential tubular staining is shown. H: claudin-3 staining 24 h post-LPS. Tubules revealed increased total staining after LPS, but much of the staining was less specific to the cellular membrane, giving a more diffuse, cytoplasmic pattern. I: claudin-4 control stain. Staining appears to be mainly cytoplasmic, which may represent fixation artifact. J: claudin-4 staining 24 h post-LPS. Intensity of staining is decreased. Circumferential tubular cell staining showed occasional areas of fragmentation; circumferential staining was not appreciated. K: claudin-8 control stain. Bright linear tubular staining is noted with some circumferential staining. Occasional artifact nuclear staining was noted as well. L: claudin-8 staining 24 h post-LPS. Circumferential cellular staining is noted (arrowheads). Magnification: ×60. Bar = 20 μm.

Fig. 5.

Effect of LPS on renal TJ mRNA expression. mRNA expression by real-time PCR is provided as a ratio to 18S. RNA expression in control mice was normalized to 1. ZO-1 mRNA expression was increased 4.3 ± 0.9-fold at 24 h vs. baseline. Occludin mRNA expression was increased 1.5 ± 0.2-fold at 24 h vs. 6 h. Claudin-1 mRNA expression was increased 7.4 ± 1.1-fold at 48 h. Claudin-3 mRNA expression was increased 5.3 ± 0.8-fold at 48 h vs. baseline. Claudin-4 mRNA expression was increased 28.6 ± 9.9-fold at 48 h. Claudin-8 mRNA expression was decreased 5.7 ± 0.3-fold. *Significant comparison with control with adjusted P < 0.05. +Significant comparisons to T = 6 with P < 0.05; n = 4 for all groups except 24-h group, where n = 7.

Fig. 6.

Localization of ZO-1 after LPS administration. Frozen sections of control mice and mice 24 h post-LPS were stained with tubular markers of nephron segments. A: control section labeled with ZO-1 (blue). ZO-1 demonstrates expression in most tubules. B: control section labeled with megalin (green, proximal tubule). C: control section labeled with sodium-chloride cotransporter (NCC; red, distal convoluted tubule). D: merged image of A–C indicating ZO-1 is found distributed in both proximal and distal tubules. E: section 24 h post-LPS labeled with ZO-1 (blue). There is patchy, irregularly decreased ZO-1 expression after LPS administration. F: LPS section labeled with megalin (green, proximal tubule). G: LPS section labeled with NCC (red, distal convoluted tubule). H: Merged image of E–G indicating ZO-1 is found distributed in both proximal and distal tubules. Note that ZO-1 expression in proximal tubules may be either decreased (purple asterisk) or preserved (purple arrow) after LPS. ZO-1 expression in distal tubules may be either decreased (yellow asterisk) or preserved (yellow arrow). Magnification: ×20. Bar = 60 μm.

Fig. 7.

Administration of lower dose LPS. A: no statistical difference in blood urea nitrogen (BUN) was observed between control mice and mice receiving 0.5 mg/kg LPS. B: intact circumferential staining of ZO-1 was found in many tubules following low-dose LPS. C: real-time PCR revealed elevated gene expression of chemokine macrophage inflammatory protein (MIP)-2. In contrast, ZO-1 gene expression was unaltered by low-dose LPS.