Rf-2 Gene Modulates Proteinuria and Albuminuria Independently of Changes in Glomerular Permeability in the Fawn-Hooded Hypertensive Rat
Authors: Artur Rangel-Filho, Mukut Sharma, Yvonne H. Datta, Carol Moreno, Richard J. Roman, Yoshiki Iwamoto, Abraham P. Provoost, Jozef Lazar, Howard J. Jacob
Journal: Journal of the American Society of Nephrology (2005)

We report that Rab38, a gene within the Rf-2 locus appears to influence the development of proteinuria (UPV) and albuminuria (UAV) in fawn-hooded hypertensive rats (FHH). Using congenic animals, we narrowed the region to eight genes; however, only one gene had a sequence variant. Rab38 has a mutation in the start codon, resulting in a natural knockout in the FHH strain. Despite no differences in glomerular albumin permeability, congenic animals carrying the wild-type Brown Norway (BN) allele of Rab38 on the FHH background exhibited, on average, 40% and 60% less UPV and UAV, respectively, than FHH. These findings suggest that Rab38 may modulate the tubular processing of filtered proteins without affecting the glomerular filtration barrier. This is the first gene reported for an animal model of hypertension-associated renal failure. This gene resides on human chromosome 11, which has been linked to renal disease. The genetic dissection of quantitative traits, such as renal failure, has proven a challenging task in humans because of their polygenic nature and interactions with the environment (19,27). One solution is to use animal models to study the genetic basis of ESRD (17,18). The first direct genetic evidence for hypertension-associated renal disease came from the FHH strain, in which five genomic regions or quantitative trait loci (QTL) (Rf-1 through Rf-5) have been linked to the development of UPV, UAV, and focal glomerulosclerosis (1,2,31). Since then, several groups have found the homologous regions in humans to be also linked to renal failure (10,13,9,37). The Rf-2 locus, located on rat chromosome 1, showed a recessive mode of inheritance with significant linkage to UPV (logarithm of the odds ratio score 5.39) and UAV (logarithm of the odds ratio score 6.50) (31). This locus accounts for approximately 30 to 40% of the urinary protein excretion (1). Studies in other rat models of renal failure have confirmed a role for the Rf-2 region in the development of UPV and UAV (33,28,38,29). In addition, Winn et al. (37) have reported linkage to a familial form of focal segmental glomerulosclerosis (FGS) in a region of human chromosome 11 syntenic to Rf-2 in rat. In this study, we report that a natural knockout of the Rab38 gene is likely the Rf-2 gene. We investigated the effects of restoring Rab38 protein expression on UPV, UAV, and the permeability of isolated glomeruli to albumin. Finally, through comparative genomics we constructed a map of the synteny between the rat and human QTLs at the gene level of resolution. Materials and Methods Generation of Congenic Animals and Sequencing of Candidate Genes All experiments were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Congenic animals were developed by marker-assisted selective breeding of FHH and BN rats as reported previously (25). Sequencing of positional candidate genes was performed using genomic DNA and cDNA on an ABI3730 capillary sequencer according to the manufacturer's suggested protocol. Urinary Protein/Albumin Excretion and Assessment of Glomerular Permeability Urine from 12-wk-old animals, fed standard rat chow, was collected in two consecutive 24 h periods and analyzed for total protein by the Weichselbaum's Biuret method (36). Albumin excretion was measured using the AB580 assay (16). Results are reported as the average of the two collection days. Glomerular permeability was determined using an in vitro functional assay as described previously (30). Blood Pressure Measurement BP was measured directly, in conscious rats, by cannulation of the right femoral artery as reported before (34). Western Immunoblotting Proteins from an SDS-polycrylamide gel electrophoresis of whole kidney homogenate of 12-wk-old FHH, BN, and FHH.BN-Rab38 congenic rats were transferred to a polyvinylidene difluoride (PVDF) membrane and probed with a polyclonal mouse anti-rat Rab38 antibody (24). Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as a control. Statistical Analyses All data are presented as mean and SEM. All groups were compared by single factor ANOVA and all pairwise multiple comparisons were performed by the Student-Newman-Keuls method using the SigmaStat package v.2.03. Results Genetic Dissection of Proteinuria and Albuminuria Using Congenic Animals To investigate the role of the Rf-2 region in the pathogenesis of renal disease, we developed congenic animals carrying distinct segments of the renal failure–resistant BN genome on the renal disease–susceptible FHH background. We identified a congenic line, designated as FHH.BN-Rab38, which carries an approximately 1.5 Mb (Chr1: 143.4 to 144.9 Mb) region of the BN chromosome 1 containing the Rab38 gene (Figure 1) (11). Replacement of this portion of the Rf-2 locus had a significant impact on UPV and UAV (Figure 2A). Despite having genomes over 99.99% identical, 3-month-old FHH.BN-Rab38 congenics developed significantly less UPV and UAV than FHH. Proteinuria was on average 40% lower in congenics when compared with FHH (97 ± 4 mg/d versus 163 ± 15 mg/d, respectively, P < 0.001). The effect on UAV was even more pronounced, with FHH.BN-Rab38 excreting 60% less albumin than FHH (23 ± 2 mg/d versus 57 ± 8 mg/d, respectively, P < 0.001). The renoprotective effect of replacing the Rab38 region appears to be independent of BP, which averaged 119 ± 2 mmHg and 118 ± 2 mmHg in congenics and FHH, respectively. Although UPV and UAV in the congenic rats were significantly improved, they were still higher in the FHH.BN-Rab38 compared with BN. This incomplete phenotypic rescue was expected as only one of the five susceptibility loci was replaced in the congenic strain. To investigate possible effects on the glomerular filtration barrier, we compared albumin permeability (Palb) in isolated glomeruli from BN, FHH.BN-Rab38, and FHH (Figure 2B). The Palb in the FHH.BN-Rab38 congenic was virtually indistinguishable from that of FHH and both were significantly higher than the BN control (Palb 0.63 ± 0.03, 0.62 ± 0.03, and 0.22 ± 0.04 in FHH.BN-Rab38, FHH, and BN, respectively, P < 0.001), suggesting that Rab38 does not modify Palb. Sequence Analysis of Candidate Genes in the FHH.BN-Rab38 Congenic and Detection of Gene Product The FHH.BN-Rab38 congenic carries only 5 known genes and 3 predicted open reading frames from the BN strain. Sequencing of the Rab38 gene revealed a protein null mutation in the FHH strain (GeneBank accession number AY907524). This mutation has also been previously reported in other fawn-hooded strains derived from the Long Evans rat (21). The FHH has a G→A mutation that causes a substitution in the start codon (Met→Ile). Sequencing of the coding regions of the remaining 4 known genes revealed no polymorphic variants. The mutation in the start codon would be expected to prevent translation of the Rab38 protein. Western blot analysis unveiled an immunoreactive band at the apparent molecular weight of the Rab38 protein (27 kD) in kidney extracts of BN and FHH.BN-Rab38, but not in FHH rats (Figure 3), indicating that the transfer of the Rab38 gene from BN to FHH in the FHH.BN-Rab38 congenic restored protein expression. Comparative Genomics of the Human and Rat Syntenic Regions Linked to Renal Failure Winn et al. (37) reported linkage of focal segmental glomerulosclerosis to a region on human chromosome 11 homologous to Rf-2 (Figure 1). The linkage of the renal disease phenotypes to the same genomic region across species suggests that similar genes may influence the trait. The human locus is confined between the genetic markers D11S2002 and D11S1986, a region comprising approximately 33Mb. We identified two large blocks of conservation between the species. The majority of the FGS locus (21 Mb) is syntenic to rat chromosome 8. Approximately 12 Mb of that locus is homologous to the rat Rf-2 region and contains all of the genes carried by the FHH.BN-Rab38 congenic. The orientation (p to q) of this segment is inverted in humans relative to rats, but the genes maintain the same position relative to each other, demonstrating that the overall structure within the region has remained unchanged despite evolutionary rearrangements at the chromosomal level. Discussion In this study we demonstrated that the introgression of the BN allele of Rab38 on the FHH background significantly reduced UPV and UAV. This segment represents less than 0.01% of the rat genome and carries only 5 known and 3 predicted genes. Sequencing of the exons of the known genes in the congenic region revealed a G→A substitution in the start codon of Rab38 that would be expected to prevent protein translation. Western blotting confirmed that the Rab38 protein is not expressed in the kidneys of FHH rats and that the transfer of this gene from BN to FHH in the FHH.BN-Rab38 congenic rescued Rab38 protein expression, which was associated with improvement in UPV and UAV. Although we cannot formally exclude a contribution by other genes in the region, none of them seem likely functional candidates and sequence analysis revealed no variants in their coding regions. Rab38 is a member of the Rab family of small GTPases that regulate intracellular vesicle formation and trafficking (23). The specific functions of Rab38 remain to be determined. However, Rab38 has been shown to be responsible for the partial oculocutaneous albinism and platelet δ-granule storage pool bleeding disorder in FHH and other rat strains (5,21,20). Here we report the first evidence linking the mutation in Rab38 to the development of proteinuria in the FHH rat. Proteinuria and albuminuria were 40% and 60% lower in the FHH.BN-Rab38 congenic compared with FHH. Interestingly, this significant improvement in UPV and UAV occurred in the absence of any reduction in the glomerular albumin permeability, which remained markedly elevated in the FHH.BN-Rab38 and FHH relative to the levels seen in BN controls. This observation, together with the disproportionately higher protection from UAV in our congenic animals, suggests that the Rab38 gene might affect the reabsorption and degradation of filtered proteins by proximal tubular cells. Indeed, the mechanism of tubular reuptake and protein metabolism involves extensive trafficking of vesicles carrying protein receptors and their ligands from the apical membrane to the lysosomes (12,22,26). An increasing body of evidence supports the concept of aberrant trafficking in tubular cells as a mechanism for the development of several renal diseases characterized by tubular proteinuria, including Dent's disease, Lowe syndrome, and some forms of cystinosis (3,4,6–8,15,32,35). Moreover, Christiansen et al. (4) have demonstrated that the CLC-5 KO mouse model of Dent's disease exhibits proteinuria as a result of defective trafficking of the receptors cubilin and megalin to the apical membrane, and at least two Rab proteins (Rab5 and Rab7) are known to be involved in this process (4). Finally, the Long Evans Cinnamon rat, which carries the same Rab38 allele as the FHH, also has been reported to have a defect in the excretion of phenolsulfonphthalein as a result of altered vesicular trafficking in the proximal tubule (14). These results support our view that the mutation in Rab38 may contribute to the development of proteinuria in FHH by altering tubular reuptake and processing of filtered protein. There are several mechanisms by which the lack of Rab38 protein might increase UPV and UAV. We have previously shown that the FHH platelets have impaired function of lysosome-related organelles that is restored in the FHH.BN-Rab38 congenic (5). It is conceivable that dysfunction of the lysosomes in the proximal tubular cells may lead to decreased breakdown of the filtered load of proteins which would appear as measurable fragments in the urine. Another possibility is that aberrant vesicular trafficking may lead to redistribution of receptors such as megalin and cubilin, which would, in turn, lead to a decreased protein uptake by the proximal tubule. Further studies will be necessary to elucidate the exact mechanisms by which the absence of the Rab38 gene product in the kidney alters urinary protein excretion. In summary, we identified a mutation in the Rab38 gene that prevents translation of protein in the FHH rat. This mutation appears to contribute to the development of proteinuria and albuminuria in this strain at three months of age. The replacement of the defective Rab38 resulted in a significant improvement in both UPV and UAV without reducing the elevated Palb seen in FHH rats. These studies suggest that Rab38 may affect tubular reuptake and processing of filtered proteins. Overall, this is the first report of a gene responsible for a QTL linked to proteinuria in any model of hypertension-associated ESRD. Although Rab38 seems to influence proteinuria independently of glomerular damage in our model, the region in the human genome homologous to the rat Rf-2 has been linked to the development of proteinuria and FGS. Thus, Rab38 emerges as a positional candidate gene that might play a role in some forms of human FGS. Further investigation will be required to verify this interpretation of the data.Figure 1: Genetic makeup of the parentals and FHH.BN-Rab38 congenic. Left, Rf-1 and Rf-2 quantitative trait loci (QTL) on rat chromosome 1 and reference genetic markers. Right, Localization of the human QTL for focal segmental glomerulosclerosis (FGS) on chromosome 11. The human chromosome is displayed in q to p orientation for clarity. Solid bars indicate Brown Norway (BN) genome and open bars indicate fawn-hooded hypertensive (FHH) genome. * indicates bacterial artificial chromosome (BAC) from CHORI-230 library, from which custom simple sequence length polymorphism (SSLP) markers were designed. Double- and single-headed arrows show the known and predicted genes in the congenic region. Patterns show human/rat homology as noted in the figure legend. The congenic animal carries an approximately 1.5 Mb region of the BN genome on the FHH background. This region is syntenic to a small portion of the human FGS locus on chromosome 11. ○, Rab38 gene. A total genome scan with 96 markers at approximately 10 to 20 cM resolution and a thorough screen of chromosome 1 with 15 markers at a density of approximately 1 marker per 15Mb were performed to ensure that the animals do not carry any portion of the BN genome in addition to the congenic region.Figure 2: A, □ indicates proteinuria, ▪ indicates albuminuria. Number of animals used per group was 6, 11, and 10 for BN, FHH.BN-Rab38, and FHH, respectively. Data shown as mean and SEM. Means of all measurements were significantly different between the groups of animals (P < 0.001). B, Glomeruli were isolated in a 5% isotonic bovine serum albumin (BSA) solution which was changed to 1% BSA. Movement of fluid into the capillaries led to an increase in glomerular size that was recorded by videomicroscopy. The albumin reflection coefficient (σalb) was calculated by dividing the fractional change in volume of experimental rats by that seen in normal Sprague Dawley rats, which were assumed to have a reflection coefficient of 1. Convectional permeability (Palb) was calculated as Palb = 1 − σalb, as described previously (30). Glomerular permeability is expressed in arbitrary Palb units. Albumin permeability is significantly different between BN and the other strains (P < 0.001). There was no difference between FHH.BN-Rab38 and FHH. Data shown as mean ± SEM. Five glomeruli were studied per animal (n indicates the total number of glomeruli analyzed).Figure 3: Representative Western blot analysis of whole kidney homogenate. Top, A band at the apparent molecular weight of Rab38 (27KD) was detected in the wild-type BN, but not in the FHH strain. Replacement of the mutant allele (FHH) with the normal (BN) restores Rab38 protein expression in the FHH.BN-Rab38 congenic. Bottom, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein detection is shown as a control for equal loading of the lanes. Rab38 mRNA was expressed in the kidney of all strains as determined by reverse transcription followed by PCR. Amplification of the Rab38 cDNA in total kidney cDNA of all strains failed to identify any splice variants. The FHH mutation found in the genomic DNA was confirmed in the cDNA sequence.Acknowledgments We thank the University of Texas Southwestern in Program for Genomics Applications for the anti-rat Rab38 antibody. This study was performed with support from the National Institutes of Health (NIH-R01-HL69321) to H.J.J., and from the American Heart Association and National Blood Foundation to Y.H.D.