Uncovering Modifier Genes of X-Linked Alport Syndrome Using a Novel Multiparent Mouse Model.

BACKGROUND: Mutations in COL4A5 are responsible for 80% of cases of X-linked Alport Syndrome (XLAS). Although genes that cause AS are well characterized, people with AS who have similar genetic mutations present with a wide variation in the extent of kidney impairment and age of onset, suggesting the activities of modifier genes. METHODS: We created a cohort of genetically diverse XLAS male and female mice using the Diversity Outbred mouse resource and measured albuminuria, GFR, and gene expression. Using a quantitative trait locus approach, we mapped modifier genes that can best explain the underlying phenotypic variation measured in our diverse population. RESULTS: Genetic analysis identified several loci associated with the variation in albuminuria and GFR, including a locus on the X chromosome associated with X inactivation and a locus on chromosome 2 containing Fmn1. Subsequent analysis of genetically reduced Fmn1 expression in Col4a5 knockout mice showed a decrease in albuminuria, podocyte effacement, and podocyte protrusions in the glomerular basement membrane, which support the candidacy of Fmn1 as a modifier gene for AS. CONCLUSION: With this novel approach, we emulated the variability in the severity of kidney phenotypes found in human patients with Alport Syndrome through albuminuria and GFR measurements. This approach can identify modifier genes in kidney disease that can be used as novel therapeutic targets.

FAR2 is associated with kidney disease in mice and humans.

Mesangial matrix expansion is an important process in the initiation of chronic kidney disease, yet the genetic factors driving its development are unknown. Our previous studies have implicated Far2 as a candidate gene associated with differences in mesangial matrix expansion between mouse inbred strains. Consistent with the hypothesis that increased expression of Far2 leads to mesangial matrix expansion through increased production of platelet-activating factor precursors, we show that FAR2 is capable of mediating de novo platelet-activating factor synthesis in vitro and driven by the transcription factor NKX3.2. We demonstrate that knockdown of Far2 in mice delays the progression of mesangial matrix expansion with at least six months (equivalent to ~15 yr in human). Furthermore, we show that increased FAR2 expression in human patients is associated with diabetic nephropathy, lupus nephritis, and IgA nephropathy. Taken together, these results highlight FAR2's role in the development of mesangial matrix expansion and chronic kidney disease.

Continuous Glucose Monitoring in Female NOD Mice Reveals Daily Rhythms and a Negative Correlation With Body Temperature.

Previous studies with continuous glucose monitoring in mice have been limited to several days or weeks, with the mouse's physical attachment to the equipment affecting behavior and measurements. In the current study, we measured blood glucose and body temperature at 10-second intervals for 12 weeks in a cohort of NOD/ShiLtJ female mice using wireless telemetry. This allowed us to obtain a high-resolution profile of the circadian rhythm of these two parameters and the onset of hyperglycemic development in real time. The most striking observations were the elevated nocturnal concentrations of glucose into the diabetic range days before elevations in diurnal glucose (when glucose concentrations are historically measured) and the strong, negative correlation between elevated blood glucose concentrations and body temperature with a steady decline of the body temperature with diabetes development. Taken together, this technological advancement provides improved resolution in the study of the disease trajectory of diabetes in mouse models, including relevant translatability to the current technologies of continuous glucose monitoring now regularly used in patients.

Early gene expression differences in inbred mouse strains with susceptibility to pulmonary adenomas.

Lung cancer is the most common cause of cancer-related deaths in both men and women, and effective preventatives are rare due to the difficulty of early detection. Specific gene expression signatures have been identified in individuals that already developed lung cancer. To identify if gene expression differences could be detected in individuals before the onset of the disease, we obtained lung tissues for microarray analysis from young, healthy mice of 9 inbred strains with known differences in their susceptibility to spontaneous pulmonary adenomas when aged. We found that the most common differentially expressed genes among all possible 36 strain comparisons showed significant associations with cancer- and inflammation-related processes. Significant expression differences between susceptible and resistant strains were detected for Aldh3a1, Cxcr1 and 7, Dpt, and Nptx1-genes with known cancer-related functions, and Cd209, Cxcr1 and 7, and Plag2g1b-genes with known inflammatory-related functions. Whereas Aldh3a1, Cd209, Dpt, and Pla2g1b had increased expression, Cxcr1 and 7, and Nptx1 had decreased expression in strains susceptible to pulmonary adenomas. Thus, our study shows that expression differences between susceptible and resistant strains can be detected in young and healthy mice without manifestation of pulmonary adenomas and, thus, may provide an opportunity of early detection. Finally, the identified genes have previously been reported for human non-small cell lung cancer suggesting that molecular pathways may be shared between these two cancer types.

A major X-linked locus affects kidney function in mice.

Chronic kidney disease is a common disease with increasing prevalence in the western population. One common reason for chronic kidney failure is diabetic nephropathy. Diabetic nephropathy and hyperglycemia are characteristics of the mouse inbred strain KK/HlJ, which is predominantly used as a model for metabolic syndrome due to its inherited glucose intolerance and insulin resistance. We used KK/HlJ, an albuminuria-sensitive strain, and C57BL/6J, an albuminuria-resistant strain, to perform a quantitative trait locus (QTL) cross to identify the genetic basis for chronic kidney failure. Albumin-creatinine ratio (ACR) was measured in 130 F2 male offspring. One significant QTL was identified on chromosome (Chr) X and four suggestive QTL were found on Chrs 6, 7, 12, and 13. Narrowing of the QTL region was focused on the X-linked QTL and performed by incorporating genotype and expression analyses for genes located in the region. From the 485 genes identified in the X-linked QTL region, a few candidate genes were identified using a combination of bioinformatic evidence based on genomic comparison of the parental strains and known function in urine homeostasis. Finally, this study demonstrates the significance of the X chromosome in the genetic determination of albuminuria.

Comparison of unrestrained plethysmography and forced oscillation for identifying genetic variability of airway responsiveness in inbred mice.

Lung function detection in mice is currently most accurately measured by invasive techniques, which are costly, labor intensive, and terminal. This limits their use for large-scale or longitudinal studies. Noninvasive assays are often used instead, but their accuracy for measuring lung function parameters such as resistance and elastance has been questioned in studies involving small numbers of mouse strains. Here we compared parameters detected by two different methods using 29 inbred mouse strains: enhanced pause (Penh), detected by unrestrained plethysmography, and central airway resistance and lung elastance, detected by a forced oscillation technique. We further tested whether the phenotypic variations were determined by the same genomic location in genome-wide association studies using a linear mixed model algorithm. Penh, resistance, and elastance were measured in nonexposed mice or mice exposed to saline and increasing doses of aerosolized methacholine. Because Penh differed from airway resistance in several strains and because the peak genetic associations found for Penh, resistance, or elastance were located at different genomic regions, we conclude that using Penh as an indicator for lung function changes in high-throughput genetic studies (i.e., genome-wide association studies or quantitative trait locus studies) measures something fundamentally different than airway resistance and lung elastance.