TLR4 links podocytes with the innate immune system to mediate glomerular injury

Abstract

Toll-like receptors (TLR) classically recognize pathogen-associated danger signals but are also activated via endogenous ligands. For evaluation of their role in inflammatory kidney disease, the function of TLR was analyzed in two mouse models of cryoglobulinemic membranoproliferative glomerulonephritis (MPGN; mice transgenic for thymic stromal lymphopoietin [TSLP], with or without deletion of the Fcgamma receptor IIb). Expression of TLR1 through 9 and TLR11 mRNA was detectable in whole kidneys and in isolated glomeruli of wild-type mice, with TLR3 and TLR4 having the highest absolute levels of expression. TLR1, 2, and 4 were increased in TSLP transgenic mice and even higher in TSLP transgenic FcgammaRIIb-deficient mice. TLR5 through 9 and 11 were upregulated to similar degrees in TSLP transgenic and TSLP transgenic FcgammaRIIb-deficient mice. Immunohistochemical studies of nephritic glomeruli localized TLR4 protein to podocytes. Cultured podocytes also expressed TLR4, and stimulation with TLR4-specific ligands resulted in a marked induction of chemokines; this was reduced by specific knockdown of TLR4 with siRNA. Fibrinogen, a potential endogenous TLR4 ligand, was shown to induce a similar profile of chemokines. In conclusion, it was demonstrated that TLR4 is constitutively expressed by podocytes and is upregulated in MPGN, where it may mediate glomerular injury by modulating expression of chemokines; therefore, TLR4 may link podocytes with the innate immune system to mediate MPGN triggered by the deposition of immune complexes.

Figure 1.

TSLP transgenic mice as a model of MPGN. (A and B) WT mice with normal renal histology at the age of 120 d (A, silver stain; B, periodic acid-Schiff [PAS] stain). (C through E) TSLP transgenic mice at the age of 120 d show typical signs of MPGN as deposition of PAS-positive material (immune deposits including the containing cryoglobulins) in peripheral capillaries and the mesangium (D, PAS stain). The glomerular tuft shows prominent increase of silver staining mesangial matrix (C, silver stain). (E) Electron micrograph of a glomerulus depicting widespread deposition of electron-dense immune complexes in mesangial areas and in subendothelial portions of glomerular capillary walls. Podocytes show focal effacement of foot processes, but many of these are preserved (arrows). There are red blood cells in the capillary lumen. (M, mesangium; D, deposits; CL, capillary lumen). Magnification, ×100.

Figure 2.

Glomerular TLR4 expression and regulation during MPGN in vivo. Glomeruli were isolated by sieving from (WT), TSLP, and TSLP/FcγRIIb−, respectively, at the given time points. Expression of TLR4 mRNA was analyzed by real-time PCR. At least eight mice were investigated per experimental group and time point. Relative expression values are depicted as means ± SEM.

Figure 3.

Podocytes express TLR4 in vivo and in vitro. Immunohistochemical localization of TLR4 protein in glomeruli of WT mice (A) and TSLP/FcγRIIb− mice (B). (A and B) Double staining for TLR4 (brown signal) and podocytes (identified by staining for p27^kip1; blue signal). TLR4 knockout mice served as control and showed no staining for TLR4 (data not shown). (C and D) Cultured primary murine podocytes show a constitutive expression of TLR4 by Western blot analysis (C) and RT-PCR (D). Size marker (M), lane 1; podocyte total RNA (Podo), lane 2; RT− control, lane 3. Magnification, ×100.

Figure 4.

Time course of chemokine induction in podocytes upon activation of TLR4. Cultured podocytes were stimulated with LPS or Lipid A during the shown time range. Relative expression values were analyzed by real-time RT-PCR and normalized to the housekeeping gene cyclophilin. Significant differences are indicated for P < 0.05 (*) or P < 0.01 (**). At least three independent experiments were performed.

Figure 5.

Effects of TLR4-specific siRNA on cultured podocytes. (A) Reduction of TLR4 mRNA in podocytes after transfection with TLR4-specific siRNA for 12 or 24 h (real-time RT-PCR); TLR9-specific siRNA and a nonsilencing siRNA served as controls. (B) Presence of TLR4 protein on the surface of podocytes as detected by FACS analysis. (Left) Naive cells. (Right) Cells incubated with TLR4-specific siRNA for 48 h. (C) Effect of pretreatment with TLR4-specific siRNA on chemokine induction by LPS and Lipid A (real-time RT-PCR). In A and C, significant differences are indicated for P < 0.05 (*) or P < 0.01 (**).

Figure 6.

Fibrinogen activates podocytes via TLR4. (A) Fibrinogen stimulation of cultured podocytes led to an induction of chemokines that was inhibitable by transfection with TLR4-specific siRNA. Significant differences are indicated for P < 0.05 (*) or P < 0.01 (**). (B) Deposition of fibrinogen (brown signal) in glomeruli of MPGN mice shown by immunohistochemistry. Magnification, ×1000.