The MIF receptor CD74 in diabetic podocyte injury

Abstract

Although metabolic derangement plays a central role in diabetic nephropathy, a better understanding of secondary mediators of injury may lead to new therapeutic strategies. Expression of macrophage migration inhibitory factor (MIF) is increased in experimental diabetic nephropathy, and increased tubulointerstitial mRNA expression of its receptor, CD74, has been observed in human diabetic nephropathy. Whether CD74 transduces MIF signals in podocytes, however, is unknown. Here, we found glomerular and tubulointerstitial CD74 mRNA expression to be increased in Pima Indians with type 2 diabetes and diabetic nephropathy. Immunohistochemistry confirmed the increased glomerular and tubular expression of CD74 in clinical and experimental diabetic nephropathy and localized glomerular CD74 to podocytes. In cultured human podocytes, CD74 was expressed at the cell surface, was upregulated by high concentrations of glucose and TNF-alpha, and was activated by MIF, leading to phosphorylation of extracellular signal-regulated kinase 1/2 and p38. High glucose also induced CD74 expression in a human proximal tubule cell line (HK2). In addition, MIF induced the expression of the inflammatory mediators TRAIL and monocyte chemoattractant protein 1 in podocytes and HK2 cells in a p38-dependent manner. These data suggest that CD74 acts as a receptor for MIF in podocytes and may play a role in the pathogenesis of diabetic nephropathy.

Figure 1.

Increased CD74 expression in human DN biopsies. (A) Transcriptomic analysis of CD74 mRNA in glomerular and tubulointerstitial compartments of living kidney donors (control, n = 6) and Pima Indians with diabetes (DN, n = 20) and MCD (n = 4). Data are means ± SD; ^#P < 0.0001 versus control. (B) Semiquantification of CD74 immunoreactivity within the glomerular and tubular compartments in the kidney biopsies from control subjects (n = 5) and patients with DN (n = 5) and MCD (n = 4). **P = 0.01 versus control. (C) Representative image of a control and a DN sample. A marked increase in CD74 staining in DN is visible in both glomerular and tubular cells. (D) Co-localization of CD74 and synaptopodin in podocytes in a glomerulus from DN biopsy.

Figure 2.

Expression of CD74 protein in experimental DN. (A) Western blot of whole kidney. Representative image. (B) Western blot quantification expressed as percentage increase over control. Data are means ± SD of 10 rats per group. *P < 0.05 versus control. (C) Immunohistochemistry, representative images of control kidney, DN, and a detail of DN in which podocyte staining for CD74 is observed (arrows). (D) Quantification of glomerular CD74 expression by immunohistochemistry. **P < 0.01 versus control. Magnification, ×200.

Figure 3.

Expression of CD74 by human cultured podocytes. (A) High glucose increases CD74 mRNA. Real-time reverse transcription–PCR (RT-PCR). Data are means ± SD of three independent experiments. **P < 0.01 versus control 48 h. (B) TNF increases CD74 mRNA. Real-time RT-PCR. Data are means ± SD of three independent experiments. **P < 0.01 versus control. (C) High glucose increases CD74 protein expression. Representative Western blot and quantification. Data are means ± SD of three independent experiments. **P < 0.01 versus control 48 h. (D) TNF increases CD74 protein. Data are means ± SD of three independent experiments. **P < 0.01 versus control 48 h. (E) Confocal microscopy localized CD74 to the perinuclear region and the cell membrane. Nephrin staining used the same secondary antibody and confirmed the differentiated status of podocytes. (F) Flow cytometry of nonpermeabilized cells showed that CD74 is expressed in the cell surface.

Figure 4.

MIF activates MAPK in cultured podocytes. (A through D) MIF stimulation results in ERK1/2 (A and B) and p38 (C and D) phosphorylation. Time course (A, **P < 0.01 versus control *P < 0.05 versus control; C, **P < 0.01 versus control *P < 0.05 versus control) and dose-response at 30 min (B, *P < 0.05 versus control; D, **P < 0.01 versus control). Representative Western blots and quantification (means ± SD) of three independent experiments.

Figure 5.

Downregulation of CD74 prevents MIF actions on podocytes. siRNA downregulation of CD74 protein (Western blot; A) prevents ERK1/2 (B) and p38 (C) phosphorylation induced by 50 ng/ml MIF for 30 min. Data are means ± SD of three independent experiments. (B) *P < 0.05 versus control; **P < 0.01 versus MIF alone. (C) *P < 0.05 versus control; **P < 0.01 versus MIF alone.

Figure 6.

MIF increases TRAIL and MCP-1 expression in podocytes. (A) Time course of TRAIL mRNA expression in podocytes treated with 10 ng/ml MIF. Real-time RT-PCR. Data are means ± SD of three independent experiments. *P < 0.05 versus control. (B and C) In cells stimulated with 10 ng/ml MIF for 24 h, pretreatment with inhibitors of ERK1/2 (20 μM PD98059) and p38 MAPK (5 μM SB203580) for 1 h prevented the induction of TRAIL mRNA (B, *P < 0.05 versus control, **P < 0.01 versus MIF alone) and MCP-1 mRNA (C, *P < 0.05 versus control, **P < 0.01 versus MIF alone). Data are means ± SD of three independent experiments.

Figure 7.

CD74 expression and MIF actions in cultured tubular cells. (A) High glucose increases CD74 mRNA expression at 24 h. Real-time RT-PCR. Data are means ± SD of three independent experiments. ^#P < 0.0001 versus control. (B) High glucose increases CD74 protein expression at 24 h. Representative Western blot and quantification. Data are means ± SD of three independent experiments. ^#P < 0.0001 versus low-glucose control. (C) Exposure to 10 ng/ml MIF for 24 h increases TRAIL expression in tubular cells. Representative Western blot and quantification. Data are means ± SD of three independent experiments. *P < 0.05 versus control. (D and E) Cells were pretreated with inhibitors of ERK1/2 (20 μM PD98059) and p38 MAPK (5 μM SB203580) for 1 h and stimulated with 10 ng/ml MIF for 24 h: Effect on TRAIL mRNA (D, *P < 0.05 versus control, **P < 0.01 versus MIF alone) and MCP-1 mRNA expression (E, *P < 0.05 versus control, **P < 0.01 versus MIF alone). Data are means ± SD of three independent experiments.

Figure 8.

TRAIL is expressed by podocytes in diabetic kidney injury and is lethal for cultured podocytes. (A) Representative TRAIL immunohistochemistry of a human glomerulus from a patient with DN. The arrow points a stained podocyte. De novo TRAIL expression in glomeruli was found in 13 of 17 biopsies from patients with DN by our group, whereas no glomerular staining for TRAIL was observed in control glomeruli. (B) Culture for 24 h in the presence of 100 ng/ml TRAIL increases the apoptosis rate in podocytes cultured in a high-glucose milieu. *P < 0.05 versus low glucose; **P < 0.01 versus high glucose. Data are means ± SD of three independent experiments.