The association of HLA-G polymorphisms and the synergistic effect of sMICA and sHLA-G with chronic kidney disease and allograft acceptance

Abstract

The HLA-G and MICA genes are stimulated under inflammatory conditions and code for soluble (sMICA and sHLA-G) or membrane-bound molecules that exhibit immunomodulatory properties. It is still unclear whether they would have a synergistic or antagonistic effect on the immunomodulation of the inflammatory response, such as in chronic kidney disease (CKD), contributing to a better prognosis after the kidney transplantation. In this study, we went from genetic to plasma analysis, first evaluating the polymorphism of MICA, NKG2D and HLA-G in a cohort from Southern Brazil, subdivided in a control group of individuals (n = 75), patients with CKD (n = 94), and kidney-transplant (KT) patients (n = 64). MICA, NKG2D and HLA-G genotyping was performed by polymerase chain reaction with specific oligonucleotide probes, Taqman and Sanger sequencing, respectively. Levels of soluble forms of MICA and HLA-G were measured in plasma with ELISA. Case-control analysis showed that the individuals with haplotype HLA-G*01:01/UTR-4 have a lower susceptibility to develop chronic kidney disease (OR = 0.480; p = 0.032). Concerning the group of kidney-transplant patients, the HLA-G genotypes +3010 GC (rs1710) and +3142 GC (rs1063320) were associated with higher risk for allograft rejection (OR = 5.357; p = 0.013 and OR = 5.357, p = 0.013, respectively). Nevertheless, the genotype +3010 GG (OR = 0.136; p = 0.041) was associated with kidney allograft acceptance, suggesting that it is a protection factor for rejection. In addition, the phenotypic analysis revealed higher levels of sHLA-G (p = 0.003) and sMICA (p < 0.001) in plasma were associated with the development of CKD. For patients who were already under chronic pathological stress and underwent a kidney transplant, a high sMICA (p = 0.001) in pre-transplant proved to favor immunomodulation and allograft acceptance. Even so, the association of genetic factors with differential levels of soluble molecules were not evidenced, we displayed a synergistic effect of sMICA and sHLA-G in response to inflammation. This increase was observed in CKD patients, that when undergo transplantation, had this previous amount of immunoregulatory molecules as a positive factor for the allograft acceptance.

Fig 1. The three steps (a, b and c) of haplotype inference, HLA-G allele frequencies, and haplotype frequencies found in all samples (n = 169).

Haplotype inferences: MICA-129 Val/Met and MICA A5.1/Wt (a), between 17 variations found in 3’-UTR of the HLA-G gene (b), and HLA-G UTRs and alleles (c). New UTRs described and their inferred origin (d). Similarity of frequencies of HLA-G haplotypes with published data from a population in São Paulo [18] (e). Wt: wild type, which does not show MICA A5.1 variation. CDS: coding DNA sequence (exon 2, 3 and 4 of the HLA-G gene). Del: +2960 or 14-bp deletion and Ins: +2960 or 14-bp insertion. NC: new composition.

Fig 2. Case-control analysis of sMICA and sHLA-G production.

Analyses of sMICA and sHLA-G were performed for control (Ct) versus chronic kidney disease (CKD) (a-b), and after kidney transplant (KT) in patients with (KTR) and without (KTN) episodes of rejection (c-d). The production of regulatory molecules, sHLA-G and sMICA, is stimulated in a pathological condition, such as in patients with chronic kidney disease (CKD), but generally not in individuals in homeostasis, as observed in Ct. Once the CKD is established, the regulatory molecules in the pre-transplant can act in the post-transplant period as enhancers of immunoregulation, leading to allograft acceptance (e). Mann-Whitney test¹. Chi-square test². SD: Standard deviation.