Inhibition of c-Maf and Chemokine receptors prevents the progression of Fabry Disease nephropathy

Fabry disease (FD) is an X-linked genetic disorder caused by deficiency of α-galactosidase A (GLA), a lysosomal hydrolytic enzyme; thus, globotriaosylceramide (Gb3) accumulates in lysosomes, causing visceral organ damage. Kidney injury occurs in approximately 55% of FD patients, which has an impact on mortality. Although enzyme replacement therapy (ERT) has been established in clinics, kidney injury progressed in some cases even after ERT reduced Gb3 accumulation. Furthermore, cardiac and kidney dysfunction appears in female FD patients despite little amount of Gb3 accumulation, suggesting that cytotoxic factors other than Gb3 accumulation might be involved in the progression of FD nephropathy. Moreover, considering the higher risk of end stage kidney disease in Japan, it was hypothesized the recent changes in lifestyle factors in Japan, including high-salt (HS) and high-fat diets (HFD) might be associated with the progression of FD nephropathy. 

Protocol 1: Kidneys were isolated from GLA globally knockout (GLA KO) mice and wild-type mice at the age of 16 weeks. α1,4-galactosyltransferase transgenic mice (TgG3S), highly producing Gb3 in every tissue with GLA KO mice to create symptomatic FD mouse model (TgG3S;GLAKO), the kidneys of which were extracted. Protocol 2: Proximal tubular cells (PTCs) isolated from GLA KO mice were co-cultured with or without lyso-Gb3. Furthermore, GLA was genetically reduced by RNA-silencing technique in murine distal tubular cells (MDCTs) and co-cultured with or without lyso-Gb3. Protocol 3: GLA KO mice were fed with water containing 1.0% NaCl and HFD and cultured PTCs or MDCTs were co-incubated with HS in the presence or absence of palmitic acid. Protocol 4: c-Maf or CCR2 inhibitors were administrated into GLA KO mice fed with HS+HFD or TgG3S/GLA KO mice for eight weeks.

GLA knockouts and TgG3S;GLAKO mice showed an increase in kidney fibrosis and immune cells infiltration with the upregulation of proinflammatory cytokines, such as interleukin (IL)-6 and IL-18 when compared to wild-type mice. Kidney expression levels of chemokines, such as CCL2, CCL7, CCL8, were elevated with increased chemokine receptors, CCR1, CCR2, and CCR4 in GLA KO mice and TgG3S;GLAKO mice. Defects of GLA in both PTCs and MDCTs upregulated CCL2, CCL7, CCL8 CCR1, CCR2, and CCR4 even in the absence of Gb3 accumulation, suggesting that the activation of chemokine-chemokine receptor axis in intrinsic kidney cells might promote the development of FD nephropathy independent of Gb3 accumulation. c-Maf, a transcriptional factor regulating CCL8, was upregulated in GLA KO, TgG3S/GLA KO mice, GLA KO PTCs, and GLA knockdown MDCTs when compared to each control. While GLA knockouts were asymptomatic, GLA knockouts fed with HS+HFD showed the increase in albuminuria, kidney fibrosis and tubular injury. Thus, CCR2 or c-Maf inhibitors were administrated into GLA KO mice fed with HS+HFD and TgG3S/GLA KO mice. We identified that both CCR2 and c-Maf inhibitors inhibited kidney fibrosis and immune cells infiltration with the reduction in proinflammatory cytokines expression in GLA KO fed with HS+HFD.

C-Maf and CCR -targeted therapies may have a potential to inhibit the progression of FD nephropathy independent of Gb3 accumulation.