Differential Expression Of Genes In Adult Sporadic Refractory Nephrotic Syndrome

Abstract:Objective:By comparing the differential expression of genes between adult sporadic refractory nephrotic syndrome (RNS), steroid-sensitive nephrotic syndrome (SSNS), and healthy controls, we may identify some specific steroid-resistant genes in RNS,by which we will reveal a molecular basis for the pathogenesis of primary nephrotic syndrome (PNS).Methods:(1). Four adult RNS patients (R1-4) and 4 SSNS patients (S1-4),together with,2 age-matched healthy adults (H1-2), were randomly chosen. The total RNA was extracted from the blood. A RT-PCR differential display technology (DDRT-PCR) was used to determine the gene expression profiling.The RT-PCR products were illuminated by agarose gel electrophoresis, or were separated by high voltage-denaturing urea polyacrylamide gel electrophoresis (PAGE) with silver nitrate staining. The differentially displayed bands were compared between samples from RNS patients, SSNS patients, and healthy controls..The identified fragments were recovered and sent for sequencing by a commercial service. The sequence was compared to human genome by using the BLAST software. In order to ensure the RNA quality,a positive RT-PCR (GAPDH) reaction was evaluated in all samples.(2) Identification of the expression of the MAML2 gene by RT-PCR. In this study,14 adult RNS patients (R1'～14) and 26 SSNS patients (S1'-26) were chosen, in addition to 8 healthy adult controls (H1'-8).The total RNA was extracted from the blood. RT-PCR was used to specifically amplify a fragment of the MAML2 gene, in order to determine whether there is any difference in the expression of this gene between these groups. Results:(1)By careful analyzing the differentially displayed bands, we have identified two unique expression bands.The first one, namely R31,was identified from RNS patients, and second one, namely S34, was from SSNS patients.Via homology comparison with known genes in Genbank, the fragment R31 has a high degree of homology to a known gene with unidentified functions, and the fragment S34 has a high degree of homology to the MAML2 gene. RT-PCR has demonstrate that these identified fragments are consistently expressed in patients with the same diagnosis.(2) The expression of the MAML2 gene is not found in healthy control adults,but showed a higher level in RNS patients, and the highest level in SSNS patients. These differences are all significant (P<0.01).Conclusions:(1).Differential display of mRNAs is an easy and feasible means to screen gene expression. (2).With this technology, we have found that there are differences in the expression of genes between RNS patients, SSNS patients, and healthy adults, while our experiments have only identified a few differences so far. (3).The identified specific expression in SSNS and RNS patients was not previously reported in any publications.One identified fragment has a high homology to the MAML2 gene. (4).MAML2 may play a role in the steroid-resistant of RNS,since this gene could be activated by the use of the steroids, and could enhance the effect of the steroid by activating P300,so that SSNS patients are more sensitive to a steroid treatment.