Podocyte NF-κB is dispensable for the pathogenesis of renal ischemia-reperfusion injury

Abstract

Podocytes play a central role in the formation of the glomerular filtration barrier in the kidney, and their dysfunction has been shown to result in multiple proteinuric kidney diseases. In this study, we sought to determine whether NF-κB, a proinflammatory signaling, within podocytes was involved in renal ischemia-reperfusion (I/R) injury. Podocyte-specific IκBΔN transgenic (Pod-IκBΔN) mice, in which NF-κB was inhibited specifically in podocytes, were generated by the Cre-loxP technology, and their phenotype was compared with control mice after bilateral renal ischemia. The effect of systemic administration of a NF-κB inhibitor, pyrrolidinedithiocarbamate (PDTC), on renal I/R injury was also examined. Pod-IκBΔN mice were phenotypically normal before surgery. Following renal I/R injury, serum concentrations of urea nitrogen and creatinine were elevated in both Pod-IκBΔN and control mice to a similar extent, whereas PDTC treatment attenuated the elevation of these parameters. Renal histological damage in I/R-injured Pod-IκBΔN mice was also similar to I/R-injured control mice, although it was improved by PDTC treatment. Moreover, I/R induced accumulation of inflammatory cells, such as neutrophils and macrophages, was reduced by PDTC treatment, but not by podocyte-specific NF-κB inhibition. These results provide evidence that the NF-κB activity in podocytes does not contribute to the pathogenesis of renal I/R injury.

Keywords: Acute kidney injury; NF‐κB; ischemia‐reperfusion; podocytes.

Figure 1

The NF‐κB inhibitor, pyrrolidinedithiocarbamate (PDTC), but not podocyte‐specific NF‐κB inhibition, attenuated renal ischemia‐reperfusion (I/R) injury. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. Serum levels of urea nitrogen (A) and creatinine (B) were measured 24 h after reperfusion. n = 5–8 per each group. *P < 0.05 compared with sham‐operated mice. #P < 0.05 compared with I/R‐injured control mice.

Figure 2

Pyrrolidinedithiocarbamate (PDTC), but not podocyte‐specific NF‐κB inhibition, improved renal histological damage following ischemia‐reperfusion (I/R) injury. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. Renal histology was examined 24 h after reperfusion. (A) Representative pictures of hematoxylin–eosin staining are shown. Bar: 100 μm. Arrows indicate intratubular casts. Arrowheads indicate tubular necrosis. B, C: Levels of the formation of proteinaceous casts (B) and tubular necrosis (C) were scored semiquantitatively. n = 5–8 per each group. *P < 0.05 compared with sham‐operated mice.

Figure 3

Tumor necrosis factor (TNF) induced p65 translocation from the cytoplasm to the nucleus in cultured podocytes derived from control mice, but not from Pod‐IκBΔN mice. Primary cultures of podocytes derived from Pod‐IκBΔN and control mice, respectively, were treated with TNF for 24 h. Expression of p65 (Red) as well as podocin (Green) was examined by immunofluorescence studies. Nuclear staining was performed with 4′,6‐diamidino‐2‐phenylindole (DAPI; Blue). Bar: 100 μm.

Figure 4

Pyrrolidinedithiocarbamate (PDTC), but not podocyte‐specific NF‐κB inhibition, attenuated ischemia‐reperfusion (I/R) injury induced increases in NGAL expression. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. A duration of 24 h after reperfusion, renal NGAL expression was examined by real‐time RT‐PCR. n = 5–8 per each group. *P < 0.05 compared with sham‐operated mice.

Figure 5

Ischemia‐reperfusion (I/R) induced accumulation of inflammatory cells was reduced by pyrrolidinedithiocarbamate (PDTC), but not by podocyte‐specific NF‐κB inhibition. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg PDTC intraperitoneally 3 h before I/R. Accumulation of neutrophils (A and C) and macrophages (B and D) was examined 24 h after reperfusion. n = 5–7 per each group. A and B: Representative pictures of immunohistochemical staining for neutrophils (A) and macrophages (B) are shown. Neutrophils (A) and macrophages (B) were visualized by diaminobenzidine, and sections were counterstained with hematoxylin. Bars: 100 μm. C and D: The numbers of neutrophils (C) and macrophages (D) per five random fields in the kidneys were quantified. *P < 0.05 compared with sham‐operated mice. #P < 0.05 compared with I/R‐injured control mice.

Figure 6

Renal ischemia‐reperfusion (I/R) injury was modest at later stage of AKI. Pod‐IκBΔN and control mice were subjected to bilateral renal ischemia for 35 min (I/R) or sham‐operation. A subset of mice were treated with 200 mg/kg pyrrolidinedithiocarbamate (PDTC) intraperitoneally 3 h before I/R. Serum levels of urea nitrogen (A) and creatinine (B) were measured 72 h after reperfusion. n = 4 per each group.