Activation and partial inactivation of NF-?B signaling lead to hypertension and chronic kidney disease
Activation of NF-?B-signalling is key in the pathogenesis of chronic kidney diseases (CKD). However, a certain level of NF-?B activity is necessary to enable tissue repair and hence maintain a healthy kidney. To investigate the relationship between activated and inactivated NF-?B signaling on the pathogenesis of CKD, mouse models of NF-?B partial inactivation (mutating cysteine at position 59 of the sixth exon on the NF-?B gene into alanine) and activation (mutating cysteine at position 59 of the sixth exon on the NF-?B gene into serine) were used. Blood pressure, serum creatinine and urinary albumin-to-creatinine-ratio markedly increased in NF-?B activated and inactivated mice compared to controls. PAS-, Masson trichrome-, and Sirius-Red-staining, as well as a-SMA immunofluorescence staining in kidneys of NF-?BC59A, and NF-?BC59S mice demonstrated kidney fibrosis in both models. Transmission electron microscopy indicated that the glomerular basement membrane was thicker in both NF-?BC59A and NF-?BC59S mice compared to wild-type mice. KEGG enrichment analysis of RNA sequencing data revealed that the PPAR pathway was most markedly upregulated in both mouse models. However, there were also differences such as fatty acid degradation, peroxisome and glutathione metabolism, and downregulation of retinol metabolism in NF-?BC59A mice with decreased NF-?B signaling, whereas cell adhesion molecules, fat digestion, and absorption were upregulated in mice with activated NF-?B signaling (NF-?BC59S mice). In conclusion, using mice models with primarily activated and partially inactivated NF-?B pathways suggests that there is an apparently U-shaped relationship between kidney function as well as morphology and the activation of the NF-?B pathway. Overall design: For active and inactive transgenic NF-?B mice, knock-in mice of NF-?B1C59A and NF-?B1C59S (NF-?B activated and inactivated) were obtained from the Modern Animal Research Center of Nanjing University. Exon 6 of the NF-?B1 (p50) gene, codon TGT for Cys-59 was mutated to either GCT or TCA (encoding Ala) by site-directed mutagenesis, in which the substitution of p50 either reduces or increases DNA binding activity respectively. This means that the NF-?B signal is either inactivated or activated to some extent in the transgenic mice5-7. A PGK-neo cassette was inserted in an intron near the mutation point as a selective marker. Standard cloning techniques were used to construct targeting vectors. The fragment containing the 5?kb 5'arm, the mutation point, PGK-neo, and the 5?kb 3'arm. The targeting vector was linearized and transferred to the C57BL/6NTac-derived ES cell line. The target clone was screened by long-range polymerase chain reaction (PCR) and southern blot. ES cell clones carrying the expected NF-?B1 (p50) mutation were injected into E3.5 C57BL/6 blastocysts that were subsequently transferred into foster mothers. Knock-in mutation was confirmed by sequencing tail DNA samples from offspring mice. Multiplex PCR genotyping used four primers to detect the knock-in alleles (Primer 1, Primer 2, Primer 3, and Primer 4). The following conditions for PCR were used to detect wild-type,NF-?B1C59A and NF-?B1C59S alleles: 94°C, 5?min; 41 cycles of 94°C, 30?s; 58°C, 30?s; 72°C, 45?s; 72°C, 5?min. Primers were obtained from Sangon Biotech (Shanghai, China), and the sequences are listed in Supplementary Table 1. All of the offspring mice were maintained under a 12:12 light/dark cycle at a constant temperature of approximately 25°C and humidity between 35% and 75%. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Jinan University. All surgeries were performed under pentobarbital anesthesia, and all efforts were made to minimize mouse suffering.