Plasma ANGPTL8 Levels and Risk for Secondary Cardiovascular Events in Japanese Patients With Stable Coronary Artery Disease Receiving Statin Therapy.

BACKGROUND: The ability to predict secondary cardiovascular events could improve health of patients undergoing statin treatment. Circulating ANGPTL8 (angiopoietin-like protein 8) levels, which positively correlate with proatherosclerotic lipid profiles, activate the pivotal proatherosclerotic factor ANGPTL3. Here, we assessed potential association between circulating ANGPTL8 levels and risk of secondary cardiovascular events in statin-treated patients. METHODS: We conducted a biomarker study with a case-cohort design, using samples from a 2018 randomized control trial known as randomized evaluation of high-dose (4 mg/day) or low-dose (1 mg/day) lipid-lowering therapy with pitavastatin in coronary artery disease (REAL-CAD [Randomized Evaluation of Aggressive or Moderate Lipid-Lowering Therapy With Pitavastatin in Coronary Artery Disease])." From that study's full analysis set (n=12 413), we selected 2250 patients with stable coronary artery disease (582 with the primary outcome, 1745 randomly chosen, and 77 overlapping subjects). A composite end point including cardiovascular-related death, nonfatal myocardial infarction, nonfatal ischemic stroke, or unstable angina requiring emergent admission was set as a primary end point. Circulating ANGPTL8 levels were measured at baseline and 6 months after randomization. RESULTS: Over a 6-month period, ANGPTL8 level changes significantly decreased in the high-dose pitavastatin group, which showed 19% risk reduction of secondary cardiovascular events compared with the low-dose group in the REAL-CAD [Randomized Evaluation of Aggressive or Moderate Lipid-Lowering Therapy With Pitavastatin in Coronary Artery Disease] study. In the highest quartiles, relative increases in ANGPTL8 levels were significantly associated with increased risk for secondary cardiovascular events, after adjustment for several cardiovascular disease risk factors and pitavastatin treatment (hazard ratio in Q4, 1.67 [95% CI, 1.17-2.39). Subgroup analyses showed relatively strong relationships between relative ANGPTL8 increases and secondary cardiovascular events in the high-dose pitavastatin group (hazard ratio in Q4, 2.07 [95% CI, 1.21-3.55]) and in the low ANGPTL8 group at baseline (166

Does the routine handling affect the phenotype of disease model mice?

The three different mouse handling methods, picking up by tails, tunnels, and open hands were performed using the ICGN glomerulonephritis mouse and the severity of symptoms was evaluated. The handling groups exhibited a tendency of more severe symptoms than the non-handling control group. Female mice handled by their tails showed significantly more severe symptoms than the control group. In addition, we subjected the normal laboratory mice, C57BL/6 and BALB/c mice to tail and tunnel handling to assess the stress conditions. The plasma corticosterone level in the tail-handled mice was higher than that in control mice. These results indicate that handling causes stress and may affect the phenotype of disease model mice.

Identification of multiple genetic loci in the mouse controlling immobility time in the tail suspension and forced swimming tests.

Depression is one of the most famous psychiatric disorders in humans in all over the countries and considered a complex neurobehavioral trait and difficult to identify causal genes. Tail suspension test (TST) and forced swimming test (FST) are widely used for assessing depression-like behavior and antidepressant activity in mice. A variety of antidepressant agents are known to reduce immobility time in both TST and FST. To identify genetic determinants of immobility duration in both tests, we analyzed 101 F2 mice from an intercross between C57BL/6 and DBA/2 strains. Quantitative trait locus (QTL) mapping using 106 microsatellite markers revealed three loci (two significant and one suggestive) and five suggestive loci controlling immobility time in the TST and FST, respectively. Results of QTL analysis suggest a broad description of the genetic architecture underlying depression, providing underpinnings for identifying novel molecular targets for antidepressants to clear the complex genetic mechanisms of depressive disorders.

Efficacy and safety in mice of repeated, lifelong administration of an ANGPTL3 vaccine.

Previously, we reported that an ANGPTL3 vaccine is a hopeful therapeutic option against dyslipidemia. In our current study, we assess durability and booster effects of that vaccine over a period representing a mouse's lifespan. The vaccine remained effective for over one year, and booster vaccination maintained suppression of circulating triglyceride levels thereafter without major adverse effects on lungs, kidneys, or liver, suggesting vaccine efficacy and safety.

The chicken 2'-5' oligoadenylate synthetase A inhibits the replication of West Nile virus.

West Nile virus (WNV) is a pathogen to cause West Nile encephalitis when the infection occurs in the brain. Previous studies in mice identified the 2'-5' oligoadenylate synthetase 1b (Oas1b) gene as a determining factor for resistance to WNV infection. In addition, it has been suggested that human OAS1 and OASL are associated with the resistance to the WNV infection. WNV is maintained in nature through a complex life cycle involving wildbirds and mosquitoes. Birds are not only susceptible to the WNV, but also act as reservoir hosts, thus participating in the spread of the disease. It has previously been reported that chicken OASL possesses the oligoadenylate synthetase activity. However, until now the antiviral activity of chicken OASL has not been determined. In this study, we investigated the putative antiviral activity of chicken OASL by ectopic expression of this enzyme in mammalian cells and then infecting these cells with WNV replicon. We demonstrate that chicken OASL has an antiviral activity against the WNV. This is the first report to show that chicken OASL is associated with the resistance to the WNV infection.

High-resolution linkage mapping of the rat hooded locus.

To identify a gene responsible for the hooded phenotype in the rat, high-resolution linkage mapping for the hooded locus was performed using IS (non-hooded) and LEA (hooded) rats. The map revealed that only Kit gene existed in the critical region, suggesting that the Kit is a strong candidate gene. However, mutation was not found in the coding region of the LEA rat Kit gene. Further, the expressions of Kit mRNA were not different in fetal neural tubes and both neonatal and adult skins between IS and LEA rats. Furthermore, Kit-positive cells, possibly melanocytes, were found in the non-pigmented hair follicles of hooded phenotype rats. Several hypotheses are conceivable to account for mechanisms in the appearance of hooded phenotype.

Stroma-derived ANGPTL2 establishes an anti-tumor microenvironment during intestinal tumorigenesis.

Previous studies show that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) functions as a tumor promoter in some cancer contexts. However, we recently reported that host ANGPTL2 also shows tumor suppressive activity by enhancing dendritic cell-mediated CD8+ T cell anti-tumor immune responses in mouse kidney cancer and murine syngeneic models. However, mechanisms underlying ANGPTL2-mediated tumor suppression are complex and not well known. Here, we investigated ANGPTL2 tumor suppressive function in chemically-induced intestinal tumorigenesis. ANGPTL2 deficiency enhanced intestinal tumor growth in an experimental mouse colitis-associated colon cancer (CAC) model. Angptl2-deficient mice also showed a decrease not only in CD8+ T cell responses but in CD4+ T cell responses during intestinal tumorigenesis. Furthermore, we show that stroma-derived ANGPTL2 can activate the myeloid immune response. Notably, ANGPTL2 drove generation of immunostimulatory macrophages via the NF-κB pathway, accelerating CD4+ T helper 1 (Th1) cell activation. These findings overall provide novel insight into the complex mechanisms underlying ANGPTL2 anti-tumor function in cancer pathology.

Genetic analysis of modifiers for the hooded phenotype in the rat.

The hooded phenotype is one of the coat color phenotype seen peculiarly in the rat. The hooded locus showing autosomal recessive inheritance is mapped to chromosome (Chr) 14 and that the hooded phenotype receives modification by hooded-modifier gene showing the linkage to the hooded locus. However, a gene responsible for either the hooded or hooded-modifier gene is not yet identified. To clarify genetic control of hooded phenotype, we carried out genetic linkage studies using BN and LEA rats. For determination of phenotypic variation, we measured ratio of pigmented coat area in parental and their F1 and F2 rats. We, then, conducted a genome-wide scan on 152 F2 rats for linkage with ratio of pigmented coat area for the dorsal, ventral, and total regions. A major quantitative trait locus (QTL), D14Got40, showing highly significant linkage contributing 70-90% of the variance for hooded phenotype was detected on Chr 14, which may be correspondent to the hooded locus. In addition, another QTL, D17Rat2, showing highly significant linkage was also detected on Chr 17 in dorsal region phenotype as well as a QTL showing suggestive linkage on Chr15 in ventral region phenotype. We, further, investigated a genome-wide scan for epistatic interactions and detected significant interactions between D14Got40 and D20Mit1, and between D14Got40 and D17Rat2 in the dorsal region phenotype. These results suggest that a major QTL in Chr 14, which is possibly correspondent to the hooded locus, mainly regulates the hooded phenotype with some modifier loci, two of which show epistatic interactions with the hooded locus.

Aging- and obesity-related peri-muscular adipose tissue accelerates muscle atrophy.

Sarcopenia due to loss of skeletal muscle mass and strength leads to physical inactivity and decreased quality of life. The number of individuals with sarcopenia is rapidly increasing as the number of older people increases worldwide, making this condition a medical and social problem. Some patients with sarcopenia exhibit accumulation of peri-muscular adipose tissue (PMAT) as ectopic fat deposition surrounding atrophied muscle. However, an association of PMAT with muscle atrophy has not been demonstrated. Here, we show that PMAT is associated with muscle atrophy in aged mice and that atrophy severity increases in parallel with cumulative doses of PMAT. We observed severe muscle atrophy in two different obese model mice harboring significant PMAT relative to respective control non-obese mice. We also report that denervation-induced muscle atrophy was accelerated in non-obese young mice transplanted around skeletal muscle with obese adipose tissue relative to controls transplanted with non-obese adipose tissue. Notably, transplantation of obese adipose tissue into peri-muscular regions increased nuclear translocation of FoxO transcription factors and upregulated expression FoxO targets associated with proteolysis (Atrogin1 and MuRF1) and cellular senescence (p19 and p21) in muscle. Conversely, in obese mice, PMAT removal attenuated denervation-induced muscle atrophy and suppressed upregulation of genes related to proteolysis and cellular senescence in muscle. We conclude that PMAT accumulation accelerates age- and obesity-induced muscle atrophy by increasing proteolysis and cellular senescence in muscle.

Null mutation of the endothelin receptor type B gene causes embryonic death in the GK rat.

The Hirschsprung disease (HSCR) is an inherited disease that is controlled by multiple genes and has a complicated genetic mechanism. HSCR patients suffer from various extents of constipation due to dysplasia of the enteric nervous system (ENS), which can be so severe as to cause complete intestinal obstruction. Many genes have been identified as playing causative roles in ENS dysplasia and HSCR, among them the endothelin receptor type B gene (Ednrb) has been identified to play an important role. Mutation of Ednrb causes a series of symptoms that include deafness, pigmentary abnormalities, and aganglionosis. In our previous studies of three rat models carrying the same spotting lethal (sl) mutation on Ednrb, the haplotype of a region on chromosome (Chr) 2 was found to be responsible for the differing severities of the HSCR-like symptoms. To confirm that the haplotype of the responsible region on Chr 2 modifies the severity of aganglionosis caused by Ednrb mutation and to recreate a rat model with severe symptoms, we selected the GK inbred strain, whose haplotype in the responsible region on Chr 2 resembles that of the rat strain in which severe symptoms accompany the Ednrbsl mutation. An Ednrb mutation was introduced into the GK rat by crossing with F344-Ednrbsl and by genome editing. The null mutation of Ednrb was found to cause embryonic death in F2 progeny possessing the GK haplotype in the responsible region on Chr 2. The results of this study are unexpected, and they provide new clues and animal models that promise to contribute to studies on the genetic regulatory network in the development of ENS and on embryogenesis.

Verification of genetic loci responsible for the resistance/susceptibility to the Sendai virus infection using congenic mice.

Sendai virus (SeV) is one of the most important pathogens in the specific-pathogen free rodents. It is known that there are some inbred mouse strains susceptible or resistant to SeV infection. The C57BL/6 (B6) and DBA/2 (D2) mice are representative of the resistant and susceptible strains, respectively. Previous study with the quantitative trait locus (QTL) analysis identified three QTLs responsible for resistance or susceptibility to SeV infection on different chromosomes and indicated that resistance or susceptibility to SeV infection was almost predicted by genotypes of these three QTLs. In this paper, to verify the above hypothesis, congenic lines were generated as follows; B6-congenic lines carrying one of the D2 alleles of three QTLs and combination of these three QTLs, and D2-congenic lines carrying single or combination of B6 alleles of three QTLs. All these congenic lines were then challenged with SeV infection. D2 congenic lines introgressed single or combination of B6 alleles of QTLs changed to resistance to SeV infection. Especially, a D2 triple-congenic line became resistant as similar level to B6-parental strain. However, B6-congenic lines introgressed single or combination of D2 alleles of QTLs all remained to be resistant to SeV infection. Both IL-6 and TNF-α in broncho-alveolar lavage fluid of D2 triple-congenic line were decreased to the similar level of B6 mice, suggesting that this is a part of factors that D2 triple-congenic line became resistant to the similar level of B6 mice. Data obtained from these congenic mice verified that three QTLs identified previously were indeed responsible for the resistance/susceptibility to SeV infection in B6 and D2 mice.

Analysis for genetic loci controlling protoscolex development in the Echinococcus multilocularis infection using congenic mice.

The resistance/susceptibility to Echinococcus multilocularis infection in mice is genetically controlled. However, genetic factors responsible for these differences remain unknown. Our previous study in genetic linkage analysis has revealed that there is a significant quantitative trait locus (QTL) for the establishment of cyst (Emcys1), and a highly significant QTL for the development of protoscolex of E. multilocularis larvae (Empsc1), on mouse chromosomes 6 and 1, respectively. The current study aimed to confirm these QTLs and narrow down the critical genetic region that controls resistance/susceptibility to E. multilocularis infection by establishing congenic and subcongenic lines from C57BL/6 (B6) and DBA/2 (D2) mice. For protoscolex development phenotype, two congenic lines, B6.D2-Empsc1 and D2.B6-Empsc1 were developed, where responsible QTL, Empsc1 was introgressed from D2 into B6 background and vice versa. For cyst establishment phenotype, two congenic lines, B6.D2-Emcys1 and D2.B6-Emcys1 were developed, where responsible QTL, Emcys1 was introgressed from D2 into B6 background and vice versa. Because there was no significant difference in cyst establishment between B6.D2-Emcys1 and D2.B6-Emcys1 mice after challenge with E. multilocularis, it is suggested that the Emcys1 does not solely control the cyst establishment in mouse liver. However, infection experiments with B6.D2-Empsc1 and D2.B6-Empsc1 mice showed a significant difference in protoscolex development in the cyst. It confirms that the Empsc1 controls phenotype of the protoscolex development in the cyst. Subsequently, two subcongenic lines, B6.D2-Empsc1.1 and B6.D2-Empsc1.2 from B6.D2-Emcys1 and one subcongenic line, D2.B6-Empsc1.1 from D2.B6-Empsc1 were developed to narrow down the critical region responsible for protoscolex development. From the results of infection experiments with E. multilocularis in these subcongenic mice, it is concluded that a gene responsible for protoscolex development is located between D1Mit290 (68.1 cM) and D1Mit511 (97.3 cM).

Analysis of the Relationship Between Enzymatic and Antiviral Activities of the Chicken Oligoadenylate Synthetase-Like.

The oligoadenylate synthetase (OAS) is well known as an antiviral factor against the flavivirus infection in mammals. It is known that the oligoadenylate synthetase-like (ChOAS-L) gene is only present in the chicken genome. It has been shown in the previous report that the ChOAS-L possesses enzymatic activity to convert ATP into 2'-5'-linked oligoadenylates and antiviral activity against West Nile virus (WNV) replicon. Therefore, this study aimed to investigate the relationship between enzymatic and antiviral activities of ChOAS-L. Eight mutated ChOAS-L proteins were generated using either the site-directed mutagenesis or standard polymerase chain reaction protocol. The wild-type and mutated proteins were ectopically expressed in 293FT cells to analyze the enzymatic activity and in BHK-21 and BALB/3T3 cells to analyze the antiviral activity using WNV replicon. The results revealed that all mutated proteins showed no enzymatic activity except for ChOAS-L-AΔUbL2. However, all mutated proteins showed antiviral activity to inhibit the replication of the WNV replicon except for ChOAS-L-AΔUbL1/UbL2, which showed a partial inhibition compared to the wild-type ChOAS-L-A or other mutated proteins. These results suggest that the ChOAS-L expresses the antiflavivirus activity in a manner independent of enzymatic activity. Our results propose reconsideration of the mechanism of antiviral activity against the flavivirus replication of ChOAS-L.

The role of mouse 2',5'-oligoadenylate synthetase 1 paralogs.

The interferon-induced oligoadenylate synthetase (OAS) family is one of the most important immune response proteins to the viral infection. The OAS protein binds dsRNA and is activated to produce 2',5'-oligoadenylates, which lead to the activation of latent form of RNase L, resulting in degradation of cellular and viral RNA and inhibition of viral replication. In mice, the Oas gene family locates on chromosome 5. The mouse Oas gene locus undergoes a recent series of duplication event, leading to the presence of eight paralogs of Oas1 genes (Oas1a through Oas1h) that forms Oas gene cluster with the Oas2, Oas3 and two OasL (OasL1 and OasL2) genes. Previous studies demonstrated that the mouse Oas1b gene conferred resistance to the flavivirus infection in mice; however, the antiviral activity of other mouse Oas1 gene family is still unknown. Therefore, in the present study, we have evaluated the mouse Oas1 paralogs regarding the enzymatic activity and antiviral activity against the two neurotropic flaviviruses, West Nile virus and tick-borne encephalitis virus. The mouse Oas1 genes were cloned from C57BL/6J (B6) as well as the Oas1b derived from feral mouse strain, MSM. The obtained results demonstrated that only Oas1a and Oas1g showed enzymatic activity. Although MSM-derived Oas1b showed antiviral activity to both viruses, all B6-derived OAS paralogs did not show antiviral activity. These results suggest that Oas1a and Oas1g play a role in potentiating viral RNA-induced interferon response in the cell, whereas the Oas1b works as a specific anti-flavivirus factor unless it is mutated. However, the role of other paralogs is unknown and should wait for further investigation.

Genetic variation in the GDNF promoter affects its expression and modifies the severity of Hirschsprung's disease (HSCR) in rats carrying Ednrb(sl) mutations.

Glial cell line-derived neurotrophic factor (GDNF) is necessary for the migration of neural crest stem cells in the gut. However, mutations in GDNF per se are deemed neither necessary nor sufficient to cause Hirschsprung's disease (HSCR). In a previous study, a modifier locus on chromosome 2 in rats carrying Ednrb(sl) mutations was identified, and several mutations in the putative regulatory region of the Gdnf gene in AGH-Ednrb(sl) rats were detected. Specifically, the mutation -232C>T has been shown to be strongly associated with the severity of HSCR. In the present study, the influence of genetic variations on the transcription of the Gdnf gene was tested using dual-luciferase assay. Results showed that the mutation -613C>T, located near the mutation -232C>T in AGH-Ednrb(sl) rats, decreased Gdnf transcription in an in vitro dual-luciferase expression assay. These data suggested an important role of -613C in Gdnf transcription. Expression levels of the Gdnf gene may modify the severity of HSCR in rats carrying Ednrb(sl) mutations.

QTL analysis of modifiers for pigmentary disorder in rats carrying Ednrb sl mutations.

Pigmentary variation in animals has been studied because of its application in genetics, evolution, and developmental biology. The large number of known color loci provides rich resource to elucidate the functional pigmentary system. Nonetheless, more color loci remain to be identified. In our previous study, we revealed that two different strains, namely, AGH rats and LEH rats, but which had the same null mutation of the Ednrb gene (Ednrb(sl)) showed markedly different pigmented coat ratio. This result strongly suggested that the severity of pigment abnormality was modified by genetic factor(s) in each strain. To elucidate the modifier locus of pigment disorder, we carried out whole-genome scanning for quantitative trait loci (QTLs) on 149 F2 (AGH-Ednrb(sl) × LEH-Ednrb(sl)) rats. A highly significant QTL, constituting 26% of the total pigmentation phenotype variance, was identified in a region around D7Got23 on chromosome (Chr) 7. In addition, investigation on epistatic interaction revealed significant interactions between D7Got23 and D3Rat78 and between D7Got23 and D14Mit4. Results suggested that a modified locus on Chr 7 was mainly responsible for the variance of pigmentary disorder between AGH-Ednrb(sl) rats and LEH-Ednrb(sl) rats, and two modifier loci showing epistatic interaction may, in part, influence pigment phenotype.

Identification of genetic loci affecting the severity of symptoms of Hirschsprung disease in rats carrying Ednrbsl mutations by quantitative trait locus analysis.

Hirschsprung's disease (HSCR) is a congenital disease in neonates characterized by the absence of the enteric ganglia in a variable length of the distal colon. This disease results from multiple genetic interactions that modulate the ability of enteric neural crest cells to populate developing gut. We previously reported that three rat strains with different backgrounds (susceptible AGH-Ednrbsl/sl, resistant F344-Ednrbsl/sl, and LEH-Ednrbsl/sl) but the same null mutation of Ednrb show varying severity degrees of aganglionosis. This finding suggests that strain-specific genetic factors affect the severity of HSCR. Consistent with this finding, a quantitative trait locus (QTL) for the severity of HSCR on chromosome (Chr) 2 was identified using an F2 intercross between AGH and F344 strains. In the present study, we performed QTL analysis using an F2 intercross between the susceptible AGH and resistant LEH strains to identify the modifier/resistant loci for HSCR in Ednrb-deficient rats. A significant locus affecting the severity of HSCR was also detected within the Chr 2 region. These findings strongly suggest that a modifier gene of aganglionosis exists on Chr 2. In addition, two potentially causative SNPs (or mutations) were detected upstream of a known HSCR susceptibility gene, Gdnf. These SNPs were possibly responsible for the varied length of gut affected by aganglionosis.

Genetic background-dependent diversity in renal failure caused by the tensin2 gene deficiency in the mouse.

Tensin2 (Tns2) is thought to be a component of the cytoskeletal structures linking actin filaments with focal adhesions and is known to play a role as an intracellular signal transduction mediator through integrin in podocytes, although the mechanism by which it functions remains unclear. A Tns2-null mutation (nph) leads to massive albuminuria following podocyte foot process effacement in the ICGN mice, the origin of the mutation, and the DBA/2J (D2) mice, but not in the C57BL/6J (B6) mice or 129(+Ter)/SvJcl (129T) mice. Elucidating the reasons for these differences in diverse genetic backgrounds could help in unraveling Tns2 function in podocytes. We produced congenic mice in which Tns2(nph) was introgressed into a FVB/NJ background (FVB-Tns2(nph)), and evaluated the progression of kidney disease. FVB-Tns2(nph) mice developed albuminuria, renal fibrosis and renal anemia as seen in ICGN mice. The FVB-Tns2(nph) mice demonstrated podocyte foot process alteration under an electron microscope by as early as 4 weeks of age. This revealed that FVB strain is susceptible to Tns2-deficiency.

Development of an ELISA using a recombinant P46-like lipoprotein for diagnosis of Mycoplasma pulmonis infection in rodents.

Mycoplasma pulmonis is one of the most prevalent bacterial pathogens that infects laboratory mice and rats. To develop an M. pulmonis-specific antigen for serological diagnosis, we cloned the cDNA of P46-like lipoprotein (P46L), an M. pulmonis putative periplasmic protein. P46L is a homolog of P46, an M. hyopneumoniae antigen. We produced recombinant P46L fused to glutathione S-transferase (GST) in Escherichia coli. Immunoblot analysis revealed that sera from Mycoplasma-infected mice and rats contained anti-P46L antibodies. We developed an ELISA using the recombinant P46L-GST protein as an antigen. Thirteen of the 14 samples from rats naturally infected with M. pulmonis were determined to be positive according to the commercial ELISA (MONILISA Myco) and positive by our ELISA. Furthermore, 18/19 samples from mice experimentally infected with M. pulmonis were positive using our P46L-GST ELISA. In contrast, only 8/19 samples from infected mice were positive by the commercial ELISA. Our results indicate that P46L-GST was an appropriate antigen for developing a serological test to determine M. pulmonis infection in laboratory mice and rats.

Quantitative trait Loci for resistance to the congenital nephropathy in tensin 2-deficient mice.

The ICR-derived glomerulonephritis (ICGN) mouse is a chronic kidney disease (CKD) model that is characterized histologically by glomerulosclerosis, vascular sclerosis and tubulointerstitial fibrosis, and clinically by proteinuria and anemia, which are common symptoms and pathological changes associated with a variety of kidney diseases. Previously, we performed a quantitative trait locus (QTL) analysis to identify the causative genes for proteinuria in ICGN mice, and found a deletion mutation of the tensin 2 gene (Tns2nph, MGI no: 2447990). Interestingly, the congenic strain carrying the Tns2nph mutation on a C57BL/6J (B6) genetic background exhibited milder phenotypes than did ICGN mice, indicating the presence of several modifier genes controlling the disease phenotype. In this study, to identify the modifier/resistant loci for CKD progression in Tns2-deficient mice, we performed QTL analysis using backcross progenies from susceptible ICGN and resistant B6 mice. We identified a significant locus on chromosome (Chr) 2 (LOD = 5.36; 31 cM) and two suggestive loci on Chrs 10 (LOD = 2.27; 64 cM) and X (LOD = 2.65; 67 cM) with linkage to the severity of tubulointerstitial injury. One significant locus on Chr 13 (LOD = 3.49; approximately 14 cM) and one suggestive locus on Chr 2 (LOD = 2.41; 51 cM) were identified as QTLs for the severity of glomerulosclerosis. Suggestive locus in BUN was also detected in the same Chr 2 region (LOD = 2.34; 51 cM). A locus on Chr 2 (36 cM) was significantly linked with HGB (LOD = 4.47) and HCT (LOD = 3.58). Four novel epistatic loci controlling either HGB or tubulointerstitial injury were discovered. Further genetic analysis should lead to identification of CKD modifier gene(s), aiding early diagnosis and providing novel approaches to the discovery of drugs for the treatment and possible prevention of kidney disease.