Wnt/β-catenin signaling is considered an important player when it comes to development but has also been found to reactivate during many different chronic kidney diseases. Being able to block this signal would make it where damage to the kidney, lesions, and increased proteinuria could be prevented. Recently, the renin-angiotensin system (RAS), which also has a role in many chronic kidney diseases, has been found to have a possible connection with the Wnt/β-catenin signaling with a key player in both being β-catenin.
The authors’ goal was to determine the exact connection between RAS and Wnt/β-catenin signaling, reinforce this idea, and also determine the further implications of their results by using an inhibitory of β-catenin (ICG-001).
Methods used include urine albumin being measured by using ELISA and then the urine creatinine also being determined. Using the amount of urine creatinine as a standard, urine albumin was represented as milligrams per milligram of urine creatinine. Histology and Immunohistochemical staining were used, and each section of the kidney was prepared and stained with hematoxylin-eosin and periodic acid–Schiff reagent.
Also, Masson’s Trichrome was used as a stain, which is specifically used to determine collagen structure and fibrotic lesions. Immunofluorescence staining was used next with confocal microscopy to observe the staining. Western Blot analysis was used to determine protein expression for several kidney-associated genes. Real-Time RT-PCR was used to determine the expression of mRNA in several genes in RAS and Wnt/β-catenin signaling, and a chromatin immunoprecipitation (ChIP) assay to analyze the interactions of β-catenin promoted putative T-cell factor (TCF)/lymphoid enhancer factor (LEF), and the binding sites for these factors in the promoters of human RAS genes.
Statistics were done using a one-way ANOVA and a Newman–Kuels test to compare different groups. In regards to critter ical analysis of this section, the method section was confusing and could have been written more clearly.
As for results, the bioinformatics analyses found that all RAS had putative binding sites in their promoter regions linking them to the Wnt/β-catenin signaling pathway. The authors then transfected a kidney proximal tubular epithelial cell to overexpress β-catenin, and the results showed an increase in RAS mRNA expression in all genres. The authors also found an increase inβ-catenin-induced binding sites of RAS genes. They also looked further into the pathway by using various Wnt vectors and found that they also simulate mRNA expression of all genes. Results suggest that Wnt/β-catepathwaysnin signaling directly controls RAS gene expression, which the authors prove over and over again through their experiments both in vitro and in vivo.
The experimenters also used the IGG-001 and add it to kidney proximal tubular epithelial cells showing that blocking β-catenin directly represses RAS genes in vitro. Two different protocols using IGG-001 were used on a mouse and human model, and both showed that they repressed the expression of RAS genes, fibrosis-related g,enes, and matrix genes involved in fibrotic lesions. Other components involved in the structure of the podocyte like nephrin and podocin had an increased mRNA expression. All this suggests that blockage of Wnt/β-catenin by ICG-001 can reverse the damage to the kidney and proteinuria.
The results all were found by accurate ways to test their hypothesis. The authors also supported their hypothesis multiple times and in multiple ways both in vitro and in vivo making their conclusions all the more reinforced. The method for statistics is a sound way of doing those calculations too. However, controls were not described in the article. The authors only mention that a normal control was used to compare with the experimental groups. One major criticism, which was already mentioned, is the way the methods section is written. More precision and more information could have been used to make the experiment an easier read. Most of the information for the method section was crammed into the results, and it would be difficult to replicate this experiment even if you were an expert in the field because they are not clear on what methods were used to find the results.