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Original paper

Modifier loci in non-mutant, female Wistar Kyoto rats influence cellular pathogenesis of nephronophthisis in Lewis polycystic kidney rats

Jada Pasquale Yengkopiong
,
Joseph Daniel Wani Lako

Pol J Pathol 2015; 66 (3): 276-287
Online publish date: 2015/10/23
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Introduction

Genetic modifier loci are sections of the chromosomes, which influence the inheritance and progression of genetic diseases. These chromosomal sections contain genetic elements that interact with other chromosomal loci, especially the disease loci, and cause variability in disease presentation and progression. Nephronophthisis (NPHP) is one of these genetic diseases whose cellular progression is influenced by genetic loci of the background strains. Pathologically, NPHP simply means damaged nephrons; it is a form of autosomal recessive polycystic kidney disease (ARPKD) [1, 2], which is a chronic interstitial nephropathy whose cellular pathogenesis involves multiple organ systems and causes cystic kidneys, which eventually deteriorate to end stage renal disease [3].
Using different murine models, the pathogenesis of NPHP has been linked to many causative genetic mutations, some of which have been mapped to genes that are involved in centrosome and cilia functions [4, 5]. However, the pathogenesis of the classical ARPKD is caused by mutations only in the Pkhd1 gene, which was mapped to human chromosome 6p21.1–p12 [6], and in murine models, the orthologs are intermittently located in different chromosomes [7, 8, 9]. Like NPHP, ARPKD is also characterized by multiple fluid-filled cystic kidneys, anemia, hypertension and eventually end stage renal disease [10].
Although the pathogenesis of NPHP and ARPKD results from mutations in different genetic loci [6, 11, 12, 13], both are monogenic renal cystic disorders, which share common cellular and macroscopic features of cyst development [2, 14, 15]. The disorders also have common clinical presentation, which include infantile incidence, renal failure, anemia, polyuria without hematuria, proteinuria and hypertension [10, 16]. It has, however, become clearer that the variability of disease phenotypes, especially the progression and extrarenal manifestation, is a feature common to both NPHP and ARPKD. This variability is attributed to individual genetic heterogeneity, but importantly to the genetic background of the parental strains [17]. Regardless of the different genetic mutations that lead to cellular pathogenesis of NPHP, it is now accepted that NPHP is a less common form of ARPKD [18, 19].
Recently, we carried out a rigorous study to elucidate the molecular pathogenesis and inheritance of NPHP in Lewis polycystic kidney (LPK) rats, and the quantitative trait locus (QTL) responsible for the disease phenotypes was mapped to chromosome 10q21–q26 [10]. The QTL contains genes that are important in signal transduction, cell growth, cell proliferation and cell differentiation [20, 21], and they were also associated with unregulated cell proliferation and cancer [22, 23, 24]. Fine mapping of the entire rat chromosome 10 using a dense set of markers identified a region within the regulator of chromosome condensation 1 (RCC1) in the never in mitosis A (NIMA)-related kinase 8 (Nek8) gene as the cause of molecular and cellular pathogenesis of the disease [25]. Previous studies established that mutations in the Nek8 genes caused pathogenesis and development of NPHP in human and other animal species [26, 27, 28]. This resulted to the consideration of NPHP as a less common form of ARPKD [29, 30].
The current work is a further extension of the study in which a genome-wide linkage scan analysis identified a mutation in the Nek8 gene as the cause of NPHP in LPK rats [10, 25, 31]. It is now reported that this mutation is influenced by genetic modifier loci in the non-mutant, female WKY rats. In this context, we document a clear influence of genetic background on cellular pathogenesis, severity and progression of the phenotypes in the backcross 1 (BC1) progeny. The BC1 progeny arose as a result of an intercross between distantly related mutant, male LPK and non-mutant, female Wistar Kyoto (WKY) rat strains.
Linkage analysis between phenotypes and genotypes of the BC1 progeny provided significant evidence for the presence of modifier genes on chromosomes 5q34–q36 and 7q11–q34. The association between phenotypes and genotypes of alleles amplified by markers that segregate with these genetic loci strongly suggests that cellular pathogenesis of NPHP, its severity and progression are modified by loci located in both chromosomes.

Material and methods

Background

A colony of Lewis (LEW/SsNArc–/–) rats spontaneously developed enlarged cystic kidneys at the Animal Resources Center of Western Australia. Mating experiments between male and female progeny with enlarged cystic kidneys produced 100% pups with bilaterally enlarged, fluid-filled cystic kidneys [10]. This colony is now called the Lewis polycystic kidney (LPK/SsNArc+/+) rats.

Mating experiments

Three mutant, male Lewis polycystic kidney (LPK/SsNArc+/+) rats were mated with seven non-mutant, female Wistar Kyoto (WKY/NArc–/–) rats to produce first filial (F1) generations. Fifteen female rats from the heterozygote F1 progeny were mated with 3 mutant LPK parent male rats and produced 139 pups in the BC1 (Fig. 1). All the animals were kept in a 12-hour light/dark cycle and they had free access to the same food and water. The experiments were performed in accordance with the guidelines of the Animal Ethic Committees of Murdoch University and the Animal Resources Center of Western Australia [18].

Phenotypic traits analysis

The BC1 progeny were earmarked and recorded and each was palpated once every week from immediately after birth until euthanasia to determine which rat had developed enlarged cystic kidneys. Enlarged kidneys were confirmed on euthanasia and the presence of cysts was established on histological examination of the kidneys. Systolic blood pressure was measured when the rats were 12 weeks old, by the tail-cuff method, using NIBP Controller (ADI Instruments, Castle Hill, NSW, Australia). Three measurements were taken and the average measurement was used in statistical analysis. However, telemetry will be used in future studies to measure systolic blood pressure [10].

Euthanasia

Euthanasia of the BC1 rats was carried out using a carbon dioxide and oxygen gas mixture, in the proportions of 80 : 20 respectively. After euthanasia, the rats were weighed and the mass recorded in grams. Blood was removed by cardiac puncture into lithium-heparin tubes. The rats were opened on the ventral side along the linear alba and kidneys and sections of liver and pancreas were removed. Each kidney was weighed and the mass was recorded and the kidneys and sections of the liver and pancreas were fixed in 4% formaldehyde solution [10].

Packed cell volume

The packed cell volume (PCV) was determined in 2 ml of blood samples at the Department of Pathology, Murdoch University, by microhematocrit technique using Haeraeus Biofuge hemo centrifuge, according to the manufacturer’s instructions (RANDOX Laboratories Ltd, London, UK), as previously described [10].

Blood chemistry

Plasma total solid protein (TSP) was estimated on ethylenediamine tetra-acetic acid (EDTA) using a Refractometer according to the manufacturer’s instructions (RANDOX Laboratories Ltd). To 20 µl of samples, blank and standard solution, 1000 µl of pyrogallol red was added. This was mixed and incubated at 37°C for 5 minutes. The absorbance of the samples (Asample) and the standard (Astandard) was measured against the reagent blank at 600 nm wavelength using a spectrophotometer. Protein concentration was calculated according to the instructions of the manufacturer (RANDOX Laboratories Ltd):
Protein concentration (g/l) = Asample × [standard]/Astandard, where [standard] is the concentration of the standard solution in g/l, Asample is the absorbance of the sample, and Astandard is the absorbance of the standard solution.
Plasma creatinine (PC) was determined as follows: To 20 µl of samples, sodium hydroxide and picric acid were added, as per the instructions provided by the manufacturer (RANDOX Instruments). Creatinine, in the presence of sodium hydroxide solution, reacts with picric acid to form a colored complex. The rate of formation of the complex was measured using the colorimetric method.
Plasma urea was measured on the Randox Daytona, the reaction catalyzed by urease enzyme (RANDOX Laboratories Ltd). Ammonia produced during the reaction combined with -oxoglutarate and hydrogenated nicotinamide adenine dinucleotide (NADH), in the presence of glutamate-dehydrogenase, to yield glutamate and NAD+. The NAD+ produced was measured in µmol/l using the ultraviolet method, as previously described [10].

Histological examination of tissues

Histological examination of the sections of kidney, liver and pancreas were carried out at the Department of Histology, Murdoch University. The tissues were paraffin embedded and sectioned (4 µm thickness) using a microtome. The sections were fixed on glass slides, stained with hematoxylin and eosin and viewed with a light microscope, and the images were digitalized using a camera (Olympus, Perth, WA, Australia), as previously described [10].

Genetic analysis

Simple sequence repeat markers

The simple sequence repeat (SSR) markers used for the mapping studies were chosen from the rat genome database: http://rgd.mcw.edu/. One SSR marker was taken from the extreme ends of each chromosome and two or more markers between. A total of 150 SSR markers distributed across the 20 rat autosomes were screened in the study, but only 96 were found informative, and therefore used in the mapping studies, as described in the previous study [10].

DNA extraction and PCR analysis

The extraction of deoxyribonucleic acid (DNA) from liver tissue was carried out according to the instructions provided in the Standard Tissue Kit Protocol (QIAamp DNA Mini Kit from Qiagen, Melbourne, Australia). The DNA was quantified using a NanoDrop, ND-1000 (BIOLAB, Wilmington, USA).
The polymerase chain reaction (PCR) was performed in a total volume of 10 µl containing 10% (w/v) Cresol Red solution; PCR buffer [6.7 mM Tris-HCl, pH 8.8, 1.66 mM (NH4)2SO4, 0.045% Triton X-100, 0.02 mg/ml gelatin]; 0.25 mM of total dNTPs; 10 pM each of forward and reverse primers; 1U Taq polymerase; 1.5 mM MgCl2, and 20 ng/µl genomic DNA template in the reaction mixture. The amplification of the DNA was performed using touchdown conditions, with initial denaturation temperature of 94°C for 3 minutes, followed by 8 cycles at 94°C for 30 seconds, 63°C for 30 seconds and 72°C for 30 seconds, and the temperature decreased to 55°C, one cycle/1°C. A further 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds were carried out. A final extension at 72°C for 5 minutes was allowed.
The PCR products were separated using 8% polyacrylamide (acrylamide/bis-acrylamide solutions, 40% w/v) gels and electrophoresis was carried out for 20 hours using 1X Tris-base-Boric acid-EDTA (TBE) buffer. The potential difference for electrophoresis was set at 4 volts/cm and the gels were stained with ethidium bromide solution from Sigma-Aldrich, Pty. Ltd. Sydney, Australia (E1510; 10 mg/ml). The image of the gel was visualized using ultraviolet light on a trans-illuminator.

Scoring of genotypes and linkage analyses

The alleles amplified by each SSR in all the BC1 progeny were independently scored by three people either as homozygote parent A or heterozygote parent H (Fig. 2). Where there was no agreement in the score because the alleles were not informative, the fragment was scored as a dash. Linkage analysis between the genotype and the phenotypes to identify the quantitative trait locus (QTL) that controlled the phenotypic trait variation in the LPK rats was carried out using Map Manager QTX20b [32]. A minimum log10 likelihood ratio (LOD) score of 3 was used to determine the presence of a QTL [33] and the additive effect for each QTL was calculated [32, 34].

Results and statistical analysis

Statistical analysis was carried out using Statistical Package for Social Sciences, SPSS16 (Chicago, Ill., USA). The analysis of variance and multiple comparison tests with post hoc analysis of the combined male and female data sets were carried out and significance was considered at p < 0.05, unless otherwise stated. The segregation ratio showed the inheritance of a recessive mutation in a single gene (Table I).
Cysts were only found in the kidneys of rats with the disease; see Fig. 2 (left) and Fig. 3 (above). On histological examination, the normal kidneys did not have any cysts (Fig. 4), but the enlarged kidneys had cysts, some coalescing to form very large cysts (Fig. 5). The blood chemistry (Table II) showed that the cysts in the kidneys led to the deterioration of kidney function. As a result, the kidneys were unable to regulate the concentrations of protein, urea and creatinine. The variation of the mean values of the phenotypic traits between the mutant and the non-mutant progeny suggests the presence of modifier loci somewhere along the chromosomes in the WKY rat strain.
Figure 6 shows the alleles in the BC1 rats. Table III shows the LOD scores on chromosome 10, and Table IV shows an epistatic interaction between modifier loci on chromosomes 5q34–q36, 7q11–q34 and the Nek8 gene on chromosome 10q25. These modifier loci significantly influenced the cellular pathogenesis of NPHP, its severity and progression to end stage renal disease (Fig. 7 and Fig. 8). The effect of the genetic mutation (Fig. 9) located on the Nek8 gene led to interconnected events, but influenced by modifier genes on chromosomes 5q34–q36 and 7q11–q34.

Discussion

We have established that cellular pathogenesis and expression of NPHP in the BC1 rats is modified by genetic loci on chromosomes 5q34–q36 and 7q11–q34 of the non-mutant, female WKY rats. Genetic markers D5Rat111 and D5Rat132, and D7Rat36 and D7Rat11 flank these chromosomal loci respectively. This finding was established by the use of a dense set of polymorphic simple sequence repeat markers that mapped the genetic loci. Previously, we identified mutation in the Nek8 gene, located on the RCC1 on chromosome 10q25, as the cause of pathogenesis of NPHP [10, 18]. Later, we observed that the BC1 progeny had much more variable phenotypes than the LPK parental strain. The disease severity and its progression were less severe in the BC1 progeny than the LPK parental strain. This finding suggested that at least one genetic modifier loci was introduced by the WKY background strain. By analyzing the association between phenotypic and genotypic trait variations in the BC1 populations, presumptive modifying loci on chromosomes 5q34–q36 and 7q11–q34 were identified. We now report that these genetic loci in the non-mutant, female WKY rats are important in the modification of cellular pathogenesis of NPHP in the LPK rats.
General inspection of the locus on chromosome 5q34–q36 identified many candidate genes, including the regulator of chromosome condensation 2 (RCC2), as possible modifier genes. The genes in this genetic locus are important in cellular regulation, but RCC2 in particular is a known regulatory element, which was previously reported to be required in the signaling pathways [35]. The RCC2 gene regulates directional cell migration and alignment of chromosomes on the spindle during specific stages of the cell cycle [36]. By localizing the modifier elements to this genetic locus, it means that the RCC2 gene and some of the regulatory elements found within the locus are important in regulating the cellular pathogenesis of NPHP, and therefore influence the severity and progression of the kidneys to end stage renal disease in the BC1 progeny. What have not been identified are the specific gene responsible for the modification and the pathway through which this modification occurs.
We also identified that the pathogenesis of NPHP in the mutant BC1 progeny was modified by loci on chromosome 7q11–q34 of the WKY rats. This section of the chromosome contains a number of candidate genes, but the most important is the Cyp11b1 (11-hydroxylase) gene. A previous study reported that mutation in this gene resulted in the development of hypertension in congenic Dahl rat strains [37]. By localizing the modifier locus to this genetic region, it strongly suggests that this genetic locus is essential in the modification of systolic blood pressure in the mutant BC1 progeny.
The LOD scores for the association between genotypes and phenotypes within both chromosomal intervals were greater than 3. LOD scores greater than 3 provide significant evidence for linkage [7, 38] and show that the loci associated with the phenotypic traits are responsible for cellular modification and progression of the disease phenotypes. Therefore, these modifier loci caused significant variations in the mean values of the phenotypic traits between the LPK and the BC1 rats with NPHP.
During mapping of the QTLs responsible for the trait variation, assigning the allele fragments to the BC1 progeny was not biased. The accuracy in data preparation for the analysis was considered more important than the ease of computation. In addition, we did not underestimate that variation in quantitative traits is also influenced by environmental factors. In the study, however, we controlled the environmental factors during the growth of the pups because these factors are important features for increasing the precision of mapping the QTLs.
Since the inheritance of NPHP follows Mendelian ratios for traits controlled by single recessive genes, we expected that when an informative marker was linked to the locus that modified the disease phenotype or when the segregation of the mean values of the phenotypic traits was dependant on the segregation of the informative marker, then the alleles would show at this locus an increasing frequency of homozygosity for the alleles inherited from LPK rats over the frequency of 50% [39]. However, this percentage frequency also depends on the disease penetrance and its expressivity and also on the distance of the marker from the modifier loci [40, 41, 42, 43].
In the phenotypic study, we established that only 48% of the BC1 progeny carried the LPK phenotype and this percentage varied for every marker used in genotyping. The variation in the percentage frequency of the marker genotypes is a result of the varying number of informative alleles for the markers used. However, the linkage of all phenotypic traits to the same QTLs strongly demonstrates the relationship that exists among loci during pathogenesis of diseases [34, 44].
This study has provided evidence that modifier loci on chromosomes 5q34q36 and 7q11q34 of the non-mutant, female WKY rats significantly influenced cellular progression of NPHP in the mutant BC1 progeny. Therefore, we propose that a new QTL mapping framework, where an interval test separates and isolates the effects of individual QTLs, be carried out. This framework will improve the precision of mapping and will isolate the QTLs that specifically modify the disease phenotypes. This mapping framework will be particularly necessary because NPHP in our model is highly heritable and the genetic variation of the background strains can be easily controlled.
Therefore, mapping studies and linkage analysis have provided invaluable evidence that cellular pathogenesis of NPHP in this rat model is modified by genetic loci on the non-mutant, female WKY rats located on chromosomes 5q34q36 and 7q11q34. These modifier loci caused the phenotypic trait variation between the LPK parental strain and the mutant BC1 rats. The LOD scores associated with the genetic loci are greater than 3 and therefore provide substantial evidence for the presence of modifier genes within both loci. The study has therefore presented an opportunity to identify the modifier genes and the pathways through which modification of NPHP phenotypes in the mutant BC1 rats occurs. Presently, work is in progress to establish this.

Acknowledgments

We would like to thank Ms. Dora Li of Murdoch University for her technical assistance. We would also like to thank Professor Rudi Appels (Murdoch University), in whose laboratory this research was carried out, Professor Jacqueline Phillips (Macquarie University) and Professor Grant Morahan (Harry Perkins Institute of Medical Research) for their unconditional technical advice in the study.

This study was supported by Murdoch University.
The authors declare no conflict of interest.

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Address for correspondence

Jada Pasquale Yengkopiong
John Garang Memorial University of Science and Technology
Faculty of Science and Technology
Bor, Jonglei State
Republic of South Sudan
tel. +211 955 621 320
e-mail: jadalojuka@yahoo.com.au
Copyright: © 2015 Polish Association of Pathologists and the Polish Branch of the International Academy of Pathology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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