Dent disease is a chronic kidney disorder that occurs almost exclusively in males. Genetics plays a strong role in urinary stone pathogenesis 14. Patients with Dent's disease often carry mutations in genes of CLCN5 or OCRL1 that are located on chromosome 2. A gene mutation can affect different genes the cell that influence physiological, morphological and pathological variation 15. Previous studies indicate that Dent's disease may be associated with aminoaciduria, phosphaturia, glycosuria, uricosuria, kaliuresis, and impaired urinary acidification, and is often complicated by rickets or osteomalacia 4, 16. The clinical diagnosis of Dent's disease is based on urinary LMWP, hypercalciuria, and at least one of the following: nephrocalcinosis, kidney stones, hematuria, hypophosphataemia, or renal insufficiency. The clinical diagnosis is confirmed by genetic tests of the identification of mutation in either CLCN5 or OCRL117,18. The CLC-5 chloride channel knock-out mouse was established as an animal model for Dent's disease 19, 20. This mouse model had elevated serum 1alpha,25-dihydroxyvitamin D3, alkaline phosphatase, osteocalcin, and urinary deoxypyridinoline that similar with the serum parameters of Dent disease patients 21, 22.
In this study, the original microarray analysis showed that hundreds of genes are expressed differentially in the proximal tubule samples of the Dent related CLCN5 knockout mice compared with the wild-type mice, 8-week-old23. A total of 500 DEGs were identified from dataset of GSE10162 by GEO2R analysis. These DEGs were then subjected to BP, CC and MF enrichment analysis. The up-regulated genes were mainly enriched in negative regulation of transcription from RNA polymerase II promoter (ontology: BP), dendrite (ontology: CC), and protein binding (ontology: MF), and the down-regulated genes were mainly enriched in oxidation-reduction process (ontology: BP), extracellular region (ontology: CC) and oxidoreductase activity (ontology: MF). KEGG pathway analysis showed the down-regulated genes were mainly enriched in metabolic pathways and fatty acid elongation, and the up-regulated genes were mainly enriched in neuroactive ligand-receptor interaction, nicotine addiction, and morphine addiction, TNF signaling pathway, calcium signaling pathway, and cAMP signaling pathway and oxidation-reduction process.
In addition to the pathway discussed above, the candidate hub genes were identified through PPI network construction and analysis, and ranking the hub genes by degree of connectivity. Our results indicated that 17 hub genes had significant correlations with Dent disease. The 17 hub genes enriched in lipid metabolic process, regulation of blood pressure, cellular response to hypoxia, positive regulation of angiogenesis, positive regulation of developmental growth, and positive regulation of cytosolic calcium ion concentration. KEGG pathway analysis showed that the 17 hub genes might participate in the TNF signaling pathway, fat digestion and absorption. The first hub genes are Apob and Lep, which serve as critical function in lipid metabolism. Apob gene encodes the main apolipoprotein of chylomicrons and low density lipoproteins 24. Lep gene encodes a protein that is secreted by white adipocytes into the circulation and plays a major role in the regulation of energy homeostasis, such as the regulation of energy balance and body weight control 25, 26. The gene C3, a Protein Coding gene, plays a central role in the activation of the complement system. There were some research indicated that C3 had relationship with acylation stimulating protein, which can stimulate triglyceride (TG) synthesis and glucose transport in adipocytes 27, 28. The Cxcl1 gene encoded protein is a secreted growth factor that signals through the G-protein coupled receptor and CXC receptor 2. The Acly gene encodes the primary enzyme (ATP citrate lyase), which responsible for the synthesis of cytosolic acetyl-CoA in many tissues. The Mmp9 gene is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Based on gene function description of the top five ranking hub genes, we could find them playing important roles in lipid metabolism.
These results suggested that these DEGs are involved in the lipid metabolism, calcium signaling pathway and. Dent disease has multiple defects attributed to proximal tubule malfunction including LMWP, hypercalciuria, aminoaciduria, phosphaturia, glycosuria, and nephrolithiasis29, but symptoms of Dent’s disease vary by patient. Some patients have typical symptoms such as kidney stones, nephrocalcinosis, hypercalciuria; and some patients exhibit other typical symptoms of nephrotic syndrome, including hypoproteinemia, hyperlipemia, or edema 3. Scheinman SJ’s study indicated that inactivation of CLCN5 can be found in the setting of hypercalciuria18. However, Dent's disease is often underdiagnoses since the mild clinical and biochemical signs, and unobvious of the X-linked inheritance30.
Some limitations exiting in our studies should be mentioned. There is only one available dataset from CLCN5 knockout mouse model in online GEO database. In this study, we conducted bioinformatics analysis with GO and KEGG pathway enrichment, and PPI network construction. All these analysis was based on the gene expression data from the comparison between 3 CLCN5 knockout mouse samples and 3 wild type mouse samples. In DEG identification, we identified 500DEGs with P value <0.01 and a |log2 fold change (FC)| ≥1 as the thresholds, and 63 DEGs with adjust P value <0.05 and a |log2 fold change (FC)| ≥1 as the thresholds. Comparing to these two methods, the 63 DEGs were also ranked in the top 63 list DEGs screened by p value. It is acceptable to use the p value instead adjust p value to identify the DEGs, since the small number of sample size. Multi-central and large sample studies on Dent disease expression profiles were needed for validation of our findings in the future. Integrating more omics data such as epigenetic or epidemiological data would help illustrate the genetic, epigenetic, and environmental factors for Dent disease. Moreover, the lack of experimental validation is another limitation of our research. And further experimental research is needed to verify these findings with a larger sample size.