IDENTIFICATION OF GENES THAT PROTECT FATTY RATS FROM URINARY TRACT INFECTION AND KIDNEY DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1008912
• Project Start Date: Dec 16, 2015
• Project End Date: Jun 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Neurobiology, Physiology and Behavior

Non Technical Summary
Obese people are more likely than lean people to have diseases such as high cholesterol, high blood pressure, type 2 diabetes, kidney disease and many other diseases. Obesity does not directly causes these diseases but it increases the risk - the percent of people who have each disease. This means that some obese people do not have any of these diseases. Thus, understanding why some obese people develop diseases, and why some do not, could be useful for preventing disease. Risk of disease can be influenced by gender, age and diet, but these alone do not explain all of the correlation between obesity and its co-morbidities. Individual genetic factors also influence the risk that an obese person will develop a co-morbidity. This project is specifically focused on how obesity increases the risk for devloping kidney disease, because kidney disease is one of the major causes of death in obese people. Kidney disease can be caused by urinary tract infections - bacterial infections in the ureters that pass urine from kidneys to the outside, because these urinary tract infections can move upstream and infect the kidneys. Bacterial infections of the kidneys reduce functioning of the kidneys, and may cause kidney failure, by causing a disease called glomerulonephritis.We have found a rat model where obese fatty Zucker rats develop kidney disease and are also likely to have urinary tract infections. Urinary tract infections were determined by taking samples from the kidney at sacrifice and then growing (or not) bacteria from those samples. Congenic rats are identical to a donor strain except for a part of one chromosome from a donor strain. We have made a congenic strain with Brown Norway rat genes on chromosome 1 and all other 20 chromosomes from the Zucker strain. This congenic is as fat as fatty Zucker, but kidney disease is much less severe and it is also much less likely to develop urinary tract infections. We have identified differences in the proteins present in urine of Zucker and congenic rats. Some of the proteins that are present in different amounts, such as lactoperoxidase, have antibacterial properties.Thus, our project is to confirm that these antibacterial proteins are really present in different amounts in Zucker and congenic animals. We will confirm that the lactoperoxidase in urine is an active protein and not just a fragment. We will test males and females, lean and obese rats. These studies will identify proteins that may have antibacterial effects, and will identify proteins that differ between lean and obese rats. Thus, we hope to identify the genetic factors that can make congenic rats resistant to urinary tract infections and subsequent kidney disease. Future translational work could then be designed to test the hypothesis that amounts of these proteins in human urine predicts likelihood of urinary tract infection and likelihood of developing more severe kidney disease.This project uses biological samples collected for a project where Judy Stern (Nutrition) is PI and Craig Warden is co-PI. These samples are collected using an IACUC protocol approved for Dr. Stern's project. This IACUC protocol, including several parts written by Dr. Warden, includes all procedures that are described in this project.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3840	1020	34%
311	4010	1020	33%
723	4010	1020	33%

Knowledge Area
311 - Animal Diseases; 305 - Animal Physiological Processes; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
3840 - Laboratory animals; 4010 - Bacteria;

Field Of Science
1020 - Physiology;

Keywords

zucker rat model

obesity and kidney disease

kidney disease and urinary tract infection

genetic susceptibility to urinary tract infection

Goals / Objectives
Urinary tract infections can cause kidney disease by causing glomerulonephritis. Kidney failure is one of the main causes of death for obese humans. I have been studying with my long time collaborator Judy Stern a fatty Zucker rat model where obesity is correlated with type 2 diabetes and kidney disease. The focus of Judy's research has been on the relationship between obesity and type 2 diabetes. This project will focus on the relationship of obesity and kidney disease in the Zucker rat model. Samples will be taken from tissues and urine stored for Dr. Stern's continuing project funded by Nora Eccles (which has a type 2 diabetes focus). Thus no additional animals will need to be maintained or used for this project.Studies of identical and dizygotic twins have shown that heritability for infections can be high. Heritability for mutans streptococci colonization in preschool twins was estimated as 52% (Corby, Bretz et al. 2005). Studies of monozygotic twins raised apart for H. pylori infection lead to heritability estimates of 57% (Malaty, Engstrand et al. 1994).There are no papers locating chromosomal locations for quantitative trait loci (QTLs) for bacterial infection in rats and only one paper has been published about QTLs for kidney infection in mice. Studies in mouse models have identified UTIs caused by several gene knockout models.Preliminary data for this project was produced by a study of Zucker fatty rats. Male and female Zucker rats were either lean (homozygous LeprSte) or fatty (homozygous LeprStefa,) on chromosome 4 and either homozygous for Brown Norway (ZUC.BN-Chr1) or Zucker alleles at the distal chromosome 1 congenic region.We performed aerobic bacteria culture from the renal pelvis of 117 chow fed non-barrier rats. Bacteria (one or more species) were cultured from 50 samples. The remaining 67 samples had no bacteria cultured. Three most common: Lactobacillus (12 times), E. coli (11 times), and Staphylococcus (7 times). We observed equal likelihood of infection from any species of bacteria in males and females, and in lean and fatty Zucker rats. 12 out of 45 congenics (27%) were infected while 38 out of 72 (52%) of Zuckers were infected (p=0.008). Contingency table analysis revealed a significant effect of any infection to increase urine volume and ACR in fatty animals homozygous for LeprfaSte. For the proteomics experiment urine was taken at 9 weeks from male fatty congenic and Zucker strain animals. All animals were checked for kidney infection by aerobic culture at sacrifice. Included animals were negative for bacterial growth We used 9 week old animals to minimize the effects of albumin on the proteomics analysis, and also to focus attention on proteins that may have causal early influences on the development of kidney disease. We have identified significant genotype effects on urine proteins in the proteomics experiment. There is a substantial enrichment of chromosome 1 congenic donor region genes in the proteins differing between congenic and Zucker strain rats.Table 1. Urine proteins in the Brown Norway Chromosome 1 congenic donor region with statistically significant differences between congenic and Zucker strain animals.Gene name, p-value Congenic versus Zucker , Fold Congenic/Zucker, RNAseq data, FunctionalinformationAnpep, p=0.0023, 2.8-fold, mRNA 1.14-fold higher congenic kidney p=0.008 at 15 weeks, Reduces basolateral Na+ -K+ -ATPase in proximal tubule cells. Has a role in coronavirus infections.Tpp1, p = 0.029, 2.3-fold, Not differential mRNA. Missense mutation congenic Cys97Arg. Likely functional, Lysosomal serine protease with tripeptidyl-peptidase I activity.Folr1, p=0.01, 2.6-fold, RNAseq 1.08 fold increase congenic p=0.018 at 15 weeks. Folate receptor. Binds folic acid and its reduced derivatives, and transports 5-methyltetrahydrofolate into cells.Gp2, p=0.019, 4-fold,, Congenic mRNA 1.4-fold higher congenic kidney 9 weeks p=0.027. Missense Glu116Gly in congenic, GP2 binds Escherichia coli Type 1 fimbriae.Lhpp, p=0.032, 3.9-fold, mRNA 1.1-fold higher congenic liver at 15 weeks p=0.0004Phosphatase that hydrolyzes imidodiphosphate, 3-phosphohistidine and 6-phospholysine.Psbpc1,p=0.00036, 8.1-fold, No mRNA data, Major secretory glycoprotein of the rat ventral prostate gland, binds steroids.tear acid-lipase-like protein-like, p=0.032, 2.4-fold, mRNA 1.03-fold higher liver congenic p=0.021 at 15 weeks, Secreted protein that lacks detectable lipase activity.Lactoperoxidase (chromosome 10), p=0.014, 2.8-fold, mRNA not differential in Kidney, Liver, Gonadal adipose. General antibacterial. Peroxidase.Methods for proteomicsCRITERIA FOR PROTEIN IDENTIFICATION-- Scaffold (version Scaffold_4.4.5, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem. 2003;75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Proteins were annotated for functional information with GO terms from NCBI (downloaded Sep 2, 2015). (Ashburner, M et al Nat. Genet. 2000;25(1):25-9).Functional information for each protein is from NCBI. P-values were by T-test. Statistical significance was adjusted for false discovery rate by Hochberg-Benjamini.The RNAseq data is unpublished. We used kidney, liver and gonadal white adipose tissue of male fatty Zucker and congenic animals. We have performed quantitative PCR to confirm the RNAseq data, but have not completed analysis. Sequence information was from the RNAseq data where both our Zucker and our congenic strain were compared with the Brown Norway strain that is the reference sequence for rats. The Brown Norway strain used for our congenic is not identical to the reference sequence Brown Norway.Specific Goals/objectives1. Repeat the proteomics analysis with urine samples from 9 week old females.2. Repeat proteomics analysis for samples infected with the most common bacteria, so we can have adequate statistical power to determine if there are bacterial specific effects. These are: (1) Lactobacillus, (2) E. coli, and (3) Staphylococcus.3. Repeat proteomics using urine samples from lean Zucker rats to identify effects of obesity.4. Use Western blots to confirm that proteins identified as differential by proteomics are differential and of intact functional size at 9, 15 and 28 weeks of age.5. Perform enzymatic assays to confirm that urinary lactoperoxidase is active.6. Perform histology to assess kidney damage.7. Perform quantitative PCR to determine if mRNA levels are also differential for proteins that are differential in urine.ReferencesCorby, P. M. A., W. A. Bretz, T. C. Hart, M. M. Filho, B. Oliveira and M. Vanyukov (2005). "Mutans streptococci in preschool twins." Archives of Oral Biology 50(3): 347-351.Malaty, H. M., L. Engstrand, N. L. Pedersen and D. Y. Graham (1994). "Helicobacter pylori Infection: Genetic and Environmental Influences: A Study of Twins." Annals of Internal Medicine 120(12): 982-986.

Project Methods
Proteomics analysis will be performed as before. The proteomics was performed by the UC Davis Proteomics core. Analysis by Scaffold was preformed by Dr. Warden. Western blots will be performed as described in previous publications of Dr. Warden. Quantitation will be by non-radioactive chemiluminescence methods.Commercial spectrophotometric kits will be purchased to quantitate lactoperoxidase activity.Histology will be preformed by Steve Griffey of the UC Davis Comparative Pathology lab, using methods that have already been published for previous studies of the fatty Zucker and congenic rats.Quantitative PCR will be performed using UC Davis Genome Center cores.Analysis and writing papers will be performed by Dr Warden. All animal tissues used in this project were collected for another project with an approved animal care protocolStatistical analyses will use the UC Davis licensed JMPPro. Minimum sample sizes will be N=6 and preferred sample size will be N=8 to match existing data.

Progress 12/16/15 to 06/30/19

Outputs
Target Audience:I attended and gave a presentation at the "Tohoku University - Texas A&M University Nutrition Symposium" (November 2-3, 2018, Hilton Hotel, College Station, TX). The audience included Professors from Tohoku University in Sendai Japan and Texas A&M and undergraduate and graduate students from Texas A&M. My 35 minute talk was titled "Diet lessons from studies in mice". This talk continues as part of a many years collaboration with the faculty of the Graduate School of Agricultural Science, Tohoku University, Japan. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Warden regularly attends national and international meetings to present our data. He has attended The Obesity Society and the Complex Trait Consortium several times in the last few years. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Progress on Aim 1 includes three parts. First, we have been working on diet studies for several years. We had two goals - (1) find a diet that produces type 2 diabetes in both males and females; and (2) find a diet that magnifies the congenic effect - that is that makes the differences between congenic and control strain larger (bigger fold effect). The first goal failed - we could not find a diet that made males and females have equally severe type 2 diabetes. However, we also found that a high sucrose diet D12450B greatly increases the differences between congenic and ZUC control strain in males for fasted plasma glucose and plasma albumin. We have used this information Second, we have characterized two subcongenic strains that contain non-overlapping BN chromosome 1 donor regions on a ZUC background. Subcongenic 1A is ZUC strain alleles on chromosome 1 until the two most distal microsatellite markers: D1Rat309 and D1Rat90 which are BN alleles. Subcongenic 1B is BN alleles from D1Rat183 until D1Rat71, then has ZUC strain alleles for the last three microsatellite markers. Thus, the two subcongenic strains do not overlap. Data from subcongenics demonstrate that the 1A strain has type 2 diabetes and kidney function phenotypes the same as ZUC background and the 1B subcongenic has phenotypes the same as the full congenic. This means that the causal genes for the effects seen in the full congenic cannot be in 1A BN donor region and must be located in the BN donor region of the 1B subcongenic. This info eliminates many otherwise plausible candidate genes that are located in the 1A donor region and will make choice of gene to knockout with CRISPR better. Third, we have gathered urine volume data of chow fed fatty male and female animals recombinant for the 1B subcongenic region. These data indicate that we do not need to include females in future recombinant studies. We have next used the diet results from the first part of Aim 1 to guide us towards feeding the recombinants the high sucrose D12450B diet. We now have data on fed and fasted glucose, urine glucose and albumin to creatinine data. The results indicate that genes for type 2 diabetes and kidney disease appear to be in the proximal part of the 1B subcongenic. We plan to confirm this by breeding and phenotyping additional animals. Progress towards Aim 2 Eleven genes are being evaluated because they meet criteria as candidate genes to cause reductions of kidney disease or type 2 diabetes in our Zucker rat model. Criteria for inclusion are: 1. Chromosomal location in the 1B subcongenic which ranges from 137 to 240 Mbp on Chromosome 1. 2. Differential amounts of ZUC vs BN in any assay - RNAseq, qPCR, or urine proteomics. 3. Preference was also given to genes with missense mutations. 4. BioGPS was used to examine tissue distribution for mRNA in mice. Data from mice was used because the rat data is inadequate. Data were examined to find genes that are either specific to kidney or where Kidney expression is one of the 2-3 highest tissues. The main reason for this is that our first Crispr modified rat is likely to be a global KO, so we will get the most specific information from kidney specific genes. 5. Finally, Umod is included because it is kidney specific and known to cause kidney disease or diabetes. Immunohistochemistry, but not Western blots, have been completed for all 11 genes. The results reveal genotype effects (congenic versus background ZUC in males for the protein Gp2 with congenics with the BN alleles having more Gp2 than the ZUC background. Diet effects Genotype and gender effects of four different diets has been gathered. Sample sizes were 6-8 animals for all values. All animals were fatty (Leprfa/fa). Urine Volume per day was measured at 9 and 15 weeks. It was normalized to body weight. Percent brown adipose tissue is brown adipose tissue weight as a percent of total body weight, while percent liver is liver weight as a percent ot total body weight. The data show that the largest genotype effects (BN full congenic vs ZUC strain control) were for animals fed the D12450B high sucrose diet in males for urine volume. These effects were highly statistically significant (p<0.0001). Both males and females showed highly significant diet dependent genotype effects on liver weight when fed the high sucrose D12450B diet. The results suggest that whatever gene(s) are causal for the congenic genotype effects that they are influenced greatly by the high sucrose diet. This does not mean that the effect of sucrose is direct but could mean that something about a sucrose diet has different effects on the BN and ZUC alleles.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: How Can We Define "Optimal Microbiota?": A Comparative Review of Structure and Functions of Microbiota of Animals, Fish, and Plants in Agriculture. Ikeda-Ohtsubo W, Brugman S, Warden CH, Rebel JMJ, Folkerts G, Pieterse CMJ. Front Nutr. 2018 Oct 2;5:90. doi: 10.3389/fnut.2018.00090. eCollection 2018. Review. Erratum in: Front Nutr. 2018 Nov 28;5:113. PMID: 30333981

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:I attended and gave a presentation at the "Tohoku University - Texas A&M University Nutrition Symposium" (November 2-3, 2018, Hilton Hotel, College Station, TX). The audience included Professors from Tohoku University in Sendai Japan and Texas A&M and undergraduate and graduate students from Texas A&M. My 35 minute talk was titled "Diet lessons from studies in mice". This talk continues as part of a many years collaboration with the faculty of the Graduate School of Agricultural Science, Tohoku University, Japan. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We have had to hire a new SRA to manage the rat colonies and perform all the assays. There has been a long learning curve, but the new SRA is doing well and we should have more publishable data in this coming year. What do you plan to do during the next reporting period to accomplish the goals?5. Plans for the current grant year Specific Aims The specific aims for the current grant year are the same as the previous year detailed in section 4. Aim 1: This aim continues work of the present grant. Phenotype fatty animals with recombinations in the Subcongenic 1B BN donor region, with varying and even smaller BN donor regions. Aim 2: We will use antibodies to characterize specific protein locations and quantities in rat kidneys. We will determine if there are differences in protein amounts of candidate genes. Aim 3: This is a new aim, enabled by a revolution in genetic manipulation of rat models that has occurred during the current grant period. We will use CRISPR/Cas9 to genetically modify Zucker rats. We are working mostly on aims 1 and 2. Work on Aim 3 may start later this year, but is contingent on results of aims 1 and 2 - i.e., can we identify a good candidate for genetic modification in Zucker fatty rats? We have not completed phenotyping of animals for the diet study and for our study of the subcongenic 1A and 1B strains. We will need data on fed and fasted plasma glucose, urine glucose, urinary albumin excretion, and urinary creatinine. These are all routine assays that we have performed for many years, so we anticipate no problems completing the work. The subcongenic strain data is likely to be publishable since it will include and exclude chromosome 1 positional candidate genes. The diet data will be used to determine if the D12450B diet is indeed ideal for studies of type 2 diabetes genotype effects. Preliminary analysis of fed plasma glucose levels suggest highly significant BN vs ZUC genotype effects in animals fed the high sucrose D12450B diet. Phenotyping of recombinant animals for Aim 1 will focus on fatty animals fed the high sucrose D12450B diet since it increases the genotype effect for urine volume and liver weight and appears to also increase genotype effects for fed plasma glucose. Use of the high sucrose diet for recombinant study animals will improve ability to map the causal genes by increasing statistical power. We expect to have about 100 animals either completed or under study by the end of this project year. This is essential for us so that we can progress to picking genes for knockout by CRISPR in Aim 3. We will complete Aim 2 this year. This includes Immunohistochemistry of all target genes and quantitative Western blots for those genes that appear to have genotype effects on protein expression. This would include Gp2. Rat kidney tissues (cortex and medulla) will be fixed and embedded in wax. Tissues will be sectioned (5 µm) followed by rehydration, antigen retrieval and immunohistochemical staining. Optimal conditions, such as antigen retrieval and primary or secondary antibody concentration for each antibody that reacts to the target proteins will be first determined. The immunoreactivity will be developed in tissue sections using VECTASTAIN elite ABC and HRP substrate ImmPACT DAB kits which produced brown deposits in cells. For Western blot assay, rat kidney tissues will be lysed in M-PER buffer containing proteinase inhibitors. Protein concentrations will be determined using a BCA protein assay. Proteins will be separated in Criterion precast gels and transferred onto PDVF membranes. Membranes will be blocked and probed with primary antibodies against target proteins. Secondary antibodies will be applied to visualize protein bands using a chemiluminescent substrate on a ChemiDoc XRS+ System. Our top priority genes for Aim 3 will be in the right place (where there is a BN congenic genotype effect of recombinant animals in Aim 1) and will have differential amounts or localization of protein in Aim 2.

Impacts
What was accomplished under these goals? Progress on Aim 1 includes three parts. First, we have been working on diet studies for several years. We had two goals - (1) find a diet that produces type 2 diabetes in both males and females; and (2) find a diet that magnifies the congenic effect - that is that makes the differences between congenic and control strain larger (bigger fold effect). The first goal failed - we could not find a diet that made males and females have equally severe type 2 diabetes. However, we also found that a high sucrose diet D12450B greatly increases the differences between congenic and ZUC control strain in males for fasted plasma glucose and plasma albumin. Second, we have characterized two subcongenic strains that contain non-overlapping BN chromosome 1 donor regions on a ZUC background. Subcongenic 1A is ZUC strain alleles on chromosome 1 until the two most distal microsatellite markers: D1Rat309 and D1Rat90 which are BN alleles. Subcongenic 1B is BN alleles from D1Rat183 until D1Rat71, then has ZUC strain alleles for the last three microsatellite markers. Thus, the two subcongenic strains do not overlap. We present results for animals fed chow. These results demonstrate that the 1A subcongenic has type 2 diabetes and kidney function phenotypes the same as ZUC background and the 1B subcongenic has phenotypes the same as the full congenic. This means that the causal genes for the effects seen in the full congenic cannot be in 1A BN donor region and must be located in the BN donor region of the 1B subcongenic. This info eliminates many otherwise plausible candidate genes that are located in the 1A donor region and will make choice of gene to knockout with CRISPR better. Third, we have gathered urine volume data of chow fed fatty male and female animals recombinant for the 1B subcongenic region. These data indicate that we do not need to include females in future recombinant studies. We will soon have data on fasted glucose, urine glucose and albumin to creatinine data so that we can evaluate type 2 diabetes and kidney function using several phenotypes. How this data will influence our work in the current year. We have incorporated the diet data into our plans and all future phenotyping of recombinants will be restricted to fatty males fed high-sucrose, D12450B. This will greatly increase power to detect genotype effects. There are hints (not statistically significant) that the disease protection genes are located nearer the distal part of the 1B donor region than to the proximal part of the BN donor region. We will give preference to choosing breeders that provide information about the distal part of the donor region (D1Rat291). Progress towards Aim 2 Eleven genes are being evaluated because they meet criteria as candidate genes to cause reductions of kidney disease or type 2 diabetes in our Zucker rat model. Criteria for inclusion are: 1. Chromosomal location in the 1B subcongenic which ranges from 137 to 240 Mbp on Chromosome 1. 2. Differential amounts of ZUC vs BN in any assay - RNAseq, qPCR, or urine proteomics. 3. Preference was also given to genes with missense mutations. 4. BioGPS was used to examine tissue distribution for mRNA in mice. Data from mice was used because the rat data is inadequate. Data were examined to find genes that are either specific to kidney or where Kidney expression is one of the 2-3 highest tissues. The main reason for this is that our first Crispr modified rat is likely to be a global KO, so we will get the most specific information from kidney specific genes. 5. Finally, Umod is included because it is kidney specific and known to cause kidney disease or diabetes. Immunohistochemistry, but not Western blots, have been completed for Acsm5, Folr1, and Gp2. The results reveal genotype effects (congenic versus background ZUC in males for the protein Gp2 with congenics with the BN alleles having more Gp2 than the ZUC background. Diet effects Genotype and gender effects of four different diets has been gathered. Sample sizes were 6-8 animals for all values. All animals were fatty (Leprfa/fa). Urine Volume per day was measured at 9 and 15 weeks. It was normalized to body weight. Percent brown adipose tissue is brown adipose tissue weight as a percent of total body weight, while percent liver is liver weight as a percent ot total body weight. The data show that the largest genotype effects (BN full congenic vs ZUC strain control) were for animals fed the D12450B high sucrose diet in males for urine volume. These effects were highly statistically significant (p<0.0001). Both males and females showed highly significant diet dependent genotype effects on liver weight when fed the high sucrose D12450B diet. The results suggest that whatever gene(s) are causal for the congenic genotype effects that they are influenced greatly by the high sucrose diet. This does not mean that the effect of sucrose is direct but could mean that something about a sucrose diet has different effects on the BN and ZUC alleles.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Warden CH, Bettaieb A, Min E, Fisler JS, Haj FG, Stern JS. PLoS One. 2017 Dec 6;12(12):e0188175. doi: 10.1371/journal.pone.0188175. eCollection 2017. PMID: 29211750
• Type: Journal Articles Status: Published Year Published: 2018 Citation: How Can We Define "Optimal Microbiota?": A Comparative Review of Structure and Functions of Microbiota of Animals, Fish, and Plants in Agriculture. Ikeda-Ohtsubo W, Brugman S, Warden CH, Rebel JMJ, Folkerts G, Pieterse CMJ. Front Nutr. 2018 Oct 2;5:90. doi: 10.3389/fnut.2018.00090. eCollection 2018. Review. Erratum in: Front Nutr. 2018 Nov 28;5:113. PMID: 30333981

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?I provided a talk on "Obesity, diet, exercise and genetics" to the Davis Rotary club on Feb 3, 2017. What do you plan to do during the next reporting period to accomplish the goals?I plan to attend the next international meeting of the Center for Food and Agricultural Immunity (CFAI) on Nov 2-3, 2018 at Texas A&M. I will present a talk on Food and Health. Other participants will be from Tohoku University in Sendai Japan and Texas A&M. Research on the Zucker fatty rat and kidney disease or type 2 diabetes gene susceptibility continues to be funded by an Extramural grant from the Nora Eccles Foundation with Judy Stern as PI and Dr Warden as co-PI. We are focusing on the final steps of finding a few candidate genes that will be tested by using Cas/CRISPR to make genetically modified Zucker fatty rats. We also continue to make and phenotype sugcongenic strain animals. ZUC strain fatty females do not normally develop type 2 diabetes. We are attempting to find diets that will exacerbate type 2 diabetes in females, so that these animals will provide a better model of human disease. These studies continue to the present day. Finally, we have published a paper on kidney disease and type 2 diabetes in Female ZUC.BN-Chrom1 congenic animals in PLoSONE in Dec 2017 that will be detailed in the next annual report.

Impacts
What was accomplished under these goals? We submitted and revised a paper describing kidney disease and type 2 diabetes in female ZUC.BN-Chrom1 congenic strain animals. This paper was accepted and published just after the end of this reporting period.

Publications

• Type: Book Chapters Status: Published Year Published: 2017 Citation: CRAIG H. WARDEN, JANIS S. FISLER, Genetics of Nonsyndromic Human Obesity, With Suggestions for New Studies From Work in Mouse Models. In Ann M. Coulston, Carol J. Boushey, Mario G. Ferruzzi and Linda M. Delahanty, editors: Coulston - NUTRITION PREV & TREAT DISEASE, Oxford: Academic Press , 2017 , pp. 453 - 476.

Progress 12/16/15 to 09/30/16

Outputs
Target Audience:I presented a poster at the 13th annual International Congress on Obesity in Vancouver British Columbia on May 1, 2016. Title was "Effects of body and fat depot weights on quantitative levels of kidney expressed kidney disease genes in lean and obese, male and female Zucker rats." Authors were: Craig Warden, Ahmed Bettaieb, Esther Min, Fawaz Haj and Judith Stern. I also attended the Lorentz Center workshop in Leiden The Netherlands on "Innate Immunity of crops, livestock and Fish." The conference was Sept 19-23, 2016. I gave a Special Lecture on "Finding genes for bacterial resistance in rodents." I attended as an invited guest of the Center for Food and Agricultural Immunity (CFAI) of Tohoku University, Sendai Japan where I had been a visiting Professor in the Summer of 2015 while on sabbatical. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?These are provided in the target audience. Attendance at the 13th International Congress on obesity in Vancouver, BC was educational. Also attendance at the Lorentz Center conference (Leiden The Netherlands) on Innate Immunity of Crops, livestock and fish. Topics covered at the Lorentz center conference were: (1) Self-Defense in Livestock, Fish, Shellfish and Crops; (2) Comparing Innate Immune Systems among Diverse Organisms; (3) Microbiome and Immunity in Animals and Plants; (4) Environment and Food Safety in Agricultural Immunology; and (5) Feasibility of Drug-Independent Food Production How have the results been disseminated to communities of interest?See above. We have presented research results at international meetings. What do you plan to do during the next reporting period to accomplish the goals?Research on the Zucker fatty rat and kidney disease or type 2 diabetes gene susceptibility continues to be funded by an Extramural grant from the Nora Eccles Foundation with Judy Stern as PI and Dr Warden as co-PI. We are focussing on the final steps of finding a few candidate genes that will be tested by using Cas/CRISPR to make genetically modified Zucker fatty rats. We also continue to make and phenotype sugcongenic strain animals. ZUC strain fatty females do not normally develop type 2 diabetes. We are attempting to find diets that will exacerbate type 2 diabetes in females, so that these animals will provide a better model of human disease. These studies continue to the present day.

Impacts
What was accomplished under these goals? Research designed to find the genes that prevent development of type 2 diabetes and kidney disease in fatty Zucker rats continues. We previously reported development of a rat model congenic for Brown Norway chromosome 1 on a Zucker fatty rat background. We named this the ZUC.BN-(D1Rat183-D1Rat90) congenic. D1Rat183 and D1Rat90 are microsatellite markers that define the proximal and distal borders of the congenic. We had previously reported that male congenic strain animals had reduced kidney disease and milder glucose intolerance than the fatty ZUC strain animals. Our major effort for this year was to compare phenotypes of female congenic strain animals to female background strain ZUC. The results of this work were published in Dec 2017. For this grant period we also began to produce subcongenic strain mice with smaller BN donor regions. We also observed that rats of urinary tract infections are much lower in the congenic than in the background strain. We have not yet published this observation, but we continue to study this effect.

Publications