RAGE in the renal tubules recognizes DNA and activates immune cells

Lupus nephritis (LN) occurs in up to 60% of patients with systemic lupus erythematosus (SLE). Despite of current development of immunosuppressant agents, LN still impairs the survival and quality of life in SLE patients. Receptor for advanced glycation endproducts (RAGE) is a multi-ligand receptor that belongs to the immunoglobulin superfamily, which is associated with innate immune system. While nucleosome released from dead cells was shown to bind to RAGE on immune cells that derives SLE, the pathological role of kidney tubules RAGE in LN remains unknown. In the present study, we investigated whether and how RAGE in proximal tubular cells (PTCs) is involved in the progression of LN. Furthermore, we explored the therapeutic impact of DNA-aptamer directed against RAGE (RAGE-apt), a next-generation RAGE antagonist, in the context of LN.

[Protocol 1] LN was induced by peritoneally injecting pristane into wild-type and RAGE globally knockout mice. Cell-free DNA was measured in MRL/lpr, SLE prone mice and pristane-induced SLE mice. [Protocol 2] Primary PTCs isolated from RAGE knockouts and wild-type mice were co-incubated with or without CpG oligodeoxynucleotide (CpG-ODN), single-stranded synthetic DNA molecules. The lysates were comprehensively analysed by RNA-seq. [Protocol 3] PTCs-specific RAGE deletion mice were constructed by mating RAGE floxed mice with PaX8-rtTA-LC1 mice (RAGEDPT). CpG-ODN were administrated into RAGEDPT mice, followed by isolating the kidneys. [Protocol 4] MRL/lpr mice were subcutaneously administrated with RAGE-apt or control-aptamer (Ctrl-apt) for 10 weeks.

The concentration of cell-free DNA in serum and urine was increased in MRL/lpr mice. Globally knocking out of RAGE attenuated kidney dysfunction, albuminuria, kidney fibrosis, and increased T lymphocytes and macrophages infiltration with reduction in systolic blood pressure. In vitro RNA-seq results demonstrated that wild-type PTCs, but not RAGE KO PTCs, upregulated the genes of MHC class I in response to the co-incubation with CpG-ODN. Pro-inflammatory cytokines, including interleukin (IL)-6, IL-18, and tissue necrosis factor (TNF)-a were upregulated in wild-type PTCs when co-incubated with CpG-ODN, all of which were reduced in RAGE KO PTCs. To confirm the pathological role of PTC RAGE responding to the increased concentration of cell-free DNA in SLE, we administrated CpG-ODN into RAGEDPT and RAGE floxed mice, and identified that the number of T lymphocytes in the kidneys and KIM-1+ injured tubules were reduced in RAGEDPT. This finding suggests that PTC RAGE may be associated with T lymphocytes activation through MHC class I. We constructed RAGE-apt by SELEX method and confirmed the binding of RAGE-apt to V1-domain of RAGE in vitro binding assay. When we administrated RAGE-apt or Crtl-apt into MRL/lpr mice, serum and urine concentration of cell-free DNA was reduced by the treatment with RAGE-apt. Moreover, RAGE-apt attenuated systolic blood pressure, kidney dysfunction, mesangial expansion, crescent formation, and T lymphocytes infiltration with the reduction in gene expression of IL-6, IL-18 and TNF-a. 

Our findings indicate that the engagement of nucleosomes to PTCs RAGE may be implicated in T lymphocytes activation via MHC class I, leading to the progression of LN. PTCs RAGE can be a therapeutic target for the progression of LN, and RAGE-apt might be one of the promising therapeutic options in the context of LN.