Invariant Natural Killer T Cell Agonist Modulates Experimental Focal and Segmental Glomerulosclerosis

A growing body of evidence demonstrates a correlation between Th2 cytokines and the development of focal and segmental glomerulosclerosis (FSGS). Therefore, we hypothesized that GSL-1, a monoglycosylceramide from Sphingomonas ssp. with pro-Th1 activity on invariant Natural Killer T (iNKT) lymphocytes, could counterbalance the Th2 profile and modulate glomerulosclerosis. Using an adriamycin(ADM)-based model of FSGS, we found that BALB/c mice presented albuminuria and glomerular degeneration in association with a Th2-like pro-fibrogenic profile; these mice also expressed a combination of inflammatory cytokines, such as IL-4, IL-1α, IL-1β, IL-17, TNF-α, and chemokines, such as RANTES and eotaxin. In addition, we observed a decrease in the mRNA levels of GD3 synthase, the enzyme responsible for GD3 metabolism, a glycolipid associated with podocyte physiology. GSL-1 treatment inhibited ADM-induced renal dysfunction and preserved kidney architecture, a phenomenon associated with the induction of a Th1-like response, increased levels of GD3 synthase transcripts and inhibition of pro-fibrotic transcripts and inflammatory cytokines. TGF-β analysis revealed increased levels of circulating protein and tissue transcripts in both ADM- and GSL-1-treated mice, suggesting that TGF-β could be associated with both FSGS pathology and iNKT-mediated immunosuppression; therefore, we analyzed the kidney expression of phosphorylated SMAD2/3 and SMAD7 proteins, molecules associated with the deleterious and protective effects of TGF-β, respectively. We found high levels of phosphoSMAD2/3 in ADM mice in contrast to the GSL-1 treated group in which SMAD7 expression increased. These data suggest that GSL-1 treatment modulates the downstream signaling of TGF-β through a renoprotective pathway. Finally, GSL-1 treatment at day 4, a period when proteinuria was already established, was still able to improve renal function, preserve renal structure and inhibit fibrogenic transcripts. In conclusion, our work demonstrates that the iNKT agonist GSL-1 modulates the pathogenesis of ADM-induced glomerulosclerosis and may provide an alternative approach to disease management

Primer sequences for mRNA quantification.

<p>Primer sequences for mRNA quantification.</p

GSL-1 treatment induces transcripts for GM3 and GD3 synthases and the anti-apoptotic protein Bcl-2.

<p>BALB/c mice were injected at day 0 with 10 mg/kg of adriamycin (ADM) or treated concomitantly with ADM and 5 µg/mouse GSL-1 (ADM+GSL-1). (A) The ADM group showed decreased levels of ST8Sia1, the enzyme responsible for the generation of GD3 gangliosides, molecules that have been associated with podocyte physiology. In contrast, GSL-1 treatment increased the levels of this transcript compared with both control and ADM mice. (B) The levels of mRNA for GM3 synthase (ST3Gal5) were not altered in the ADM group; however, they were significantly augmented after GSL-1 treatment. Because GM3 gangliosides were associated with resistance to ADM-cytotoxicity due to Bcl-2 induction, we measured the mRNA levels of this protein in our system. (C) Bcl-2 transcripts were not altered by ADM but increased in ADM+GSL-1 mice, indicating a relationship between increased ST3Galt5 and Bcl-2 transcripts with renal protection from effects of ADM. * p<0.05 vs. control; # p<0.05 vs. ADM.</p

GSL-1 treatment modulates TGF-β signaling.

<p>BALB/c mice were injected at day 0 with 10 mg/kg of adriamycin (ADM) or treated concomitantly with ADM and 5 µg/mouse GSL-1 (ADM+GSL-1). (A) Total TGF-β protein increased in the serum of both the ADM and ADM+GSL-1 groups compared with the control animals. (B) The increase in TGF-β transcripts in kidney tissue reflected the serum findings showing that the ADM and ADM+GSL-1 groups had higher levels of this cytokine compared with the control mice. GSL-1 treatment induced a more significant increase in serum TGFβ and mRNA transcripts compared with the ADM group; however, only ADM+GSL-1 mice presented transcripts to the TGFBI/BIGH3 protein (C), indicating that TGF-β production had a different effect in this group. This idea is supported by the presence of phosphorylated SMAD2/3 in ADM mice, whereas in the ADM+GSL-1 group, the expression of SMAD7 protein increased (D). These data indicate that GSL-1 treatment modulated the downstream cascade of TGF-β signaling. * p<0.05 vs. control; # p<0.05 vs. ADM.</p

GSL-1 treatment inhibits the ADM-induced expression of fibrogenic mRNAs.

<p>BALB/c mice were injected at day 0 with 10 mg/kg of adriamycin (ADM) or treated concomitantly with ADM and 5 µg/mouse GSL-1 (ADM+GSL-1). (A) ADM mice presented increased levels of vimentin compared with control mice; vimentin was diminished in GSL-1-treated group. (B) The levels of PAI-1 transcripts significantly increased in ADM mice, in contrast with ADM+GSL-1 animals, which had levels comparable to that of the control mice. (C) TIMP-1 mRNA increased in the ADM group compared with the control and ADM+GSL-1 mice (D) MMP9 transcripts were equally increased in the ADM and ADM+GSL-1 groups compared with the control mice. These data indicate the development of ADM-induced FSGS with renal remodeling. * p<0.05 vs. control; # p<0.05 vs. ADM.</p

GSL-1 treatment inhibits the inflammatory context induced by ADM administration.

<p>BALB/c mice were injected at day 0 with 10 mg/kg of adriamycin (ADM) or treated concomitantly with ADM and 5 µg/mouse GSL-1 (ADM+GSL-1). ADM mice had increased levels of the inflammatory cytokines IL-4, IL-1α, IL-1β, TNF-α, IL-12p40 and IL-17 compared with both the control and GSL-1-treated groups (A to F, respectively). The administration of ADM also induced high levels of inflammatory chemokines, such as RANTES and eotaxin, which were inhibited by GSL-1 treatment (G and H, respectively). These data indicate an inhibitory effect for GSL-1 treatment, supporting its renoprotective effect. * p<0.05 vs. control; # p<0.05 vs. ADM.</p

GSL-1 treatment inhibits ADM-induced renal failure due to podocyte injury in an iNKT-dependent manner.

<p>BALB/c WT or iNKT-deficient (Jalpha18^−/−) mice were injected at day 0 with 10 mg/kg of adriamycin (ADM) or treated concomitantly with ADM and 5 µg/mouse GSL-1 (ADM+GSL-1). (A) As a result of ADM cytotoxicity, the mice lost body weight in a time-dependent fashion, in contrast to the control, untreated mice, and the GSL-1-treated group. (B) The increased proteinuria/creatininuria found in the ADM group at day 7 post-ADM injection reflects impaired renal function compared with the control and ADM+GSL-1 animals. These data indicate a renoprotective effect of GSL-1 treatment. (C) The albuminuria levels found in ADM animals reflect the podocyte injury due to ADM cytotoxicity. In contrast, the albuminuria/creatininuria ratio in the ADM+GSL-1 group was comparable to that in the control animals, indicating podocyte conservation. The GSL-1 treatment failed to protect Jalpha18^−/− mice from ADM-induced renal injury, showing that its renoprotective effect is iNKT-dependent (D and E). * p<0.05 vs. control; # p<0.05 vs. ADM.</p

GSL-1 treatment inhibits renal injury due to ADM cytotoxicity.

<p>BALB/c mice were injected at day 0 with 10 mg/kg of adriamycin (ADM) or treated concomitantly with ADM and 5 µg/mouse GSL-1 (ADM+GSL-1). (A) Representative image of the renal alterations induced by ADM administration (black arrow). Figures B to D show graphic representation of the mesangial hypercellularity, signs of glomerular sclerosis and tubular degeneration induced by ADM administration. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032454#pone-0032454-g002" target="_blank">Figure 2</a> corroborates the renoprotective effect of GSL-1 treatment depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032454#pone-0032454-g001" target="_blank">Figure 1</a>. * p<0.05 vs. control; # p<0.05 vs. ADM.</p

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. the bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and alpha-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling. Kidney International (2011) 79, 1217-1227; doi:10.1038/ki.2011.14; published online 16 March 2011Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT Complex FluidsUniv São Paulo, Inst Biomed Sci 4, Dept Immunol, Lab Imunobiol Transplante, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, Dept Med, Lab Imunol Clin & Expt, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, Dept Med, Mol Biol Lab, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Patol, BR-04023900 São Paulo, BrazilUniv Paris 07, INSERM, U699, Paris, FranceUniv São Paulo, Dept Patol, BR-05508900 São Paulo, BrazilHosp Israelita Albert Einstein, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, Dept Med, Lab Imunol Clin & Expt, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Nefrol, Dept Med, Mol Biol Lab, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Patol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilFAPESP: 04/08226-9FAPESP: 07/07139-3FAPESP: 2007/07120FAPESP: 08/55125-4CNPq: 501848/2009-6Web of Scienc