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Rf-2 Modulates Proteinuria and Albuminuria Independently of Changes in Glomerular Permeability in the Fawn-Hooded Hypertensive Rat

Artur Rangel-Filho,*† Mukut Sharma,‡ Yvonne H. Datta,†† Carol Moreno,*† Richard J. ʈ Roman,† Yoshiki Iwamoto,* Abraham P. Provoost,** Jozef Lazar,*¶ Howard J. Jacob*†§ *Human and Molecular Genetics Center, †Department of Physiology, ‡Division of Nephrology, Department of ʈ Medicine, §Department of Pediatrics, Department of Urology, ¶Department of Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin; **Department of Pediatric Surgery, Erasmus Medical Center, Rotterdam, The Netherlands; ††Department of Hematology and Oncology, Winship Cancer Institute, Emory University, Atlanta, GA J Am Soc Nephrol 16: 852–856, 2005. doi: 10.1681/ASN.2005010029

e report that Rab38, a gene within the Rf-2 locus models of renal failure have confirmed a role for the Rf-2 region appears to influence the development of - in the development of UPV and UAV (33,28,38,29). In addition, W uria (UPV) and albuminuria (UAV) in fawn- Winn et al. (37) have reported linkage to a familial form of focal hooded hypertensive rats (FHH). Using congenic animals, we segmental glomerulosclerosis (FGS) in a region of human chro- narrowed the region to eight ; however, only one gene mosome 11 syntenic to Rf-2 in rat. In this study, we report that had a sequence variant. Rab38 has a mutation in the start codon, a natural knockout of the Rab38 gene is likely the Rf-2 gene. We resulting in a natural knockout in the FHH strain. Despite no investigated the effects of restoring Rab38 protein expression differences in glomerular albumin permeability, congenic ani- on UPV, UAV, and the permeability of isolated glomeruli to mals carrying the wild-type Brown Norway (BN) allele of albumin. Finally, through comparative genomics we con- Rab38 on the FHH background exhibited, on average, 40% and structed a map of the synteny between the rat and human QTLs 60% less UPV and UAV, respectively, than FHH. These find- at the gene level of resolution. ings suggest that Rab38 may modulate the tubular processing of filtered without affecting the glomerular filtration bar- Materials and Methods rier. This is the first gene reported for an animal model of Generation of Congenic Animals and Sequencing of hypertension-associated renal failure. This gene resides on hu- Candidate Genes man 11, which has been linked to renal disease. All experiments were performed in compliance with the National The genetic dissection of quantitative traits, such as renal Institutes of Health Guide for the Care and Use of Laboratory Animals. failure, has proven a challenging task in humans because of Congenic animals were developed by marker-assisted selective breed- ing of FHH and BN rats as reported previously (25). Sequencing of their polygenic nature and interactions with the environment positional candidate genes was performed using genomic DNA and (19,27). One solution is to use animal models to study the cDNA on an ABI3730 capillary sequencer according to the manufac- genetic basis of ESRD (17,18). The first direct genetic evidence turer’s suggested protocol. for hypertension-associated renal disease came from the FHH strain, in which five genomic regions or quantitative trait loci Urinary Protein/Albumin Excretion and Assessment of (QTL) (Rf-1 through Rf-5) have been linked to the development Glomerular Permeability of UPV, UAV, and focal glomerulosclerosis (1,2,31). Since then, Urine from 12-wk-old animals, fed standard rat chow, was collected several groups have found the homologous regions in humans in two consecutive 24 h periods and analyzed for total protein by the to be also linked to renal failure (10,13,9,37). The Rf-2 locus, Weichselbaum’s Biuret method (36). Albumin excretion was measured located on rat chromosome 1, showed a recessive mode of using the AB580 assay (16). Results are reported as the average of the inheritance with significant linkage to UPV (logarithm of the two collection days. odds ratio score 5.39) and UAV (logarithm of the odds ratio Glomerular permeability was determined using an in vitro functional score 6.50) (31). This locus accounts for approximately 30 to assay as described previously (30). 40% of the urinary protein excretion (1). Studies in other rat Blood Pressure Measurement BP was measured directly, in conscious rats, by cannulation of the right femoral artery as reported before (34). Published online ahead of print. Publication date available at www.jasn.org.

Address correspondence to: Dr. Howard J. Jacob, Human and Molecular Genetics Western Immunoblotting Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Proteins from an SDS-polycrylamide gel electrophoresis of whole WI, 53226. Phone: 414-456-4887 Fax: 414-456-6516; E-mail: [email protected] kidney homogenate of 12-wk-old FHH, BN, and FHH.BN-Rab38 con-

Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1604-0852 J Am Soc Nephrol 16: 852–856, 2005 Effects of the Rf-2 Locus on Proteinuria 853 genic rats were transferred to a polyvinylidene difluoride (PVDF) with FHH (97 Ϯ 4 mg/d versus 163 Ϯ 15 mg/d, respectively, membrane and probed with a polyclonal mouse anti-rat Rab38 anti- P Ͻ 0.001). The effect on UAV was even more pronounced, with body (24). Detection of glyceraldehyde-3-phosphate dehydrogenase FHH.BN-Rab38 excreting 60% less albumin than FHH (23 Ϯ 2 (GAPDH) was performed as a control. mg/d versus 57 Ϯ 8 mg/d, respectively, P Ͻ 0.001). The reno- protective effect of replacing the Rab38 region appears to be Statistical Analyses independent of BP, which averaged 119 Ϯ 2 mmHg and 118 Ϯ All data are presented as mean and SEM. All groups were compared 2 mmHg in congenics and FHH, respectively. by single factor ANOVA and all pairwise multiple comparisons were Although UPV and UAV in the congenic rats were signifi- performed by the Student-Newman-Keuls method using the SigmaStat package v.2.03. cantly improved, they were still higher in the FHH.BN-Rab38 compared with BN. This incomplete phenotypic rescue was Results expected as only one of the five susceptibility loci was replaced Genetic Dissection of Proteinuria and Albuminuria Using in the congenic strain. Congenic Animals To investigate possible effects on the glomerular filtration To investigate the role of the Rf-2 region in the pathogenesis barrier, we compared albumin permeability (Palb) in isolated of renal disease, we developed congenic animals carrying dis- glomeruli from BN, FHH.BN-Rab38, and FHH (Figure 2B). The tinct segments of the renal failure–resistant BN genome on the Palb in the FHH.BN-Rab38 congenic was virtually indistinguish- renal disease–susceptible FHH background. We identified a able from that of FHH and both were significantly higher than Ϯ Ϯ Ϯ congenic line, designated as FHH.BN-Rab38, which carries an the BN control (Palb 0.63 0.03, 0.62 0.03, and 0.22 0.04 in Ͻ approximately 1.5 Mb (Chr1: 143.4 to 144.9 Mb) region of the FHH.BN-Rab38, FHH, and BN, respectively, P 0.001), sug- BN chromosome 1 containing the Rab38 gene (Figure 1) (11). gesting that Rab38 does not modify Palb. Replacement of this portion of the Rf-2 locus had a significant impact on UPV and UAV (Figure 2A). Despite having genomes Sequence Analysis of Candidate Genes in the FHH.BN- over 99.99% identical, 3-month-old FHH.BN-Rab38 congenics Rab38 Congenic and Detection of Gene Product developed significantly less UPV and UAV than FHH. Protein- The FHH.BN-Rab38 congenic carries only 5 known genes and uria was on average 40% lower in congenics when compared 3 predicted open reading frames from the BN strain. Sequenc-

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 . 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. E, 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. 854 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 852–856, 2005

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 dehydro- genase (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.

Figure 2. A, ᮀ indicates proteinuria, Ⅵ 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 Comparative Genomics of the Human and Rat Syntenic and SEM. Means of all measurements were significantly differ- Regions Linked to Renal Failure ent between the groups of animals (P Ͻ 0.001). B, Glomeruli Winn et al. (37) reported linkage of focal segmental glomer- were isolated in a 5% isotonic bovine serum albumin (BSA) ulosclerosis to a region on human chromosome 11 homologous solution which was changed to 1% BSA. Movement of fluid into to Rf-2 (Figure 1). The linkage of the renal disease phenotypes the capillaries led to an increase in glomerular size that was to the same genomic region across species suggests that similar recorded by videomicroscopy. The albumin reflection coeffi- genes may influence the trait. The human locus is confined ␴ cient ( alb) was calculated by dividing the fractional change in between the genetic markers D11S2002 and D11S1986, a region volume of experimental rats by that seen in normal Sprague comprising approximately 33Mb. Dawley rats, which were assumed to have a reflection coeffi- We identified two large blocks of conservation between the cient of 1. Convectional permeability (P ) was calculated as alb species. The majority of the FGS locus (21 Mb) is syntenic to rat P ϭ 1 Ϫ ␴ , as described previously (30). Glomerular per- alb alb chromosome 8. Approximately 12 Mb of that locus is homolo- meability is expressed in arbitrary Palb units. Albumin perme- ability is significantly different between BN and the other gous to the rat Rf-2 region and contains all of the genes carried strains (P Ͻ 0.001). There was no difference between FHH.BN- by the FHH.BN-Rab38 congenic. The orientation (p to q) of this Rab38 and FHH. Data shown as mean Ϯ SEM. Five glomeruli segment is inverted in humans relative to rats, but the genes were studied per animal (n indicates the total number of glo- maintain the same position relative to each other, demonstrat- meruli analyzed). ing that the overall structure within the region has remained unchanged despite evolutionary rearrangements at the chro- ing of the Rab38 gene revealed a protein null mutation in the mosomal level. FHH strain (GeneBank accession number AY907524). This mu- tation has also been previously reported in other fawn-hooded Discussion strains derived from the Long Evans rat (21). The FHH has a In this study we demonstrated that the introgression of the G3A mutation that causes a substitution in the start codon BN allele of Rab38 on the FHH background significantly re- (Met3Ile). Sequencing of the coding regions of the remaining duced UPV and UAV. This segment represents less than 0.01% 4 known genes revealed no polymorphic variants. of the rat genome and carries only 5 known and 3 predicted The mutation in the start codon would be expected to pre- genes. Sequencing of the exons of the known genes in the vent translation of the Rab38 protein. Western blot analysis congenic region revealed a G3A substitution in the start codon unveiled an immunoreactive band at the apparent molecular of Rab38 that would be expected to prevent protein translation. weight of the Rab38 protein (27 kD) in kidney extracts of BN Western blotting confirmed that the Rab38 protein is not ex- and FHH.BN-Rab38, but not in FHH rats (Figure 3), indicating pressed in the kidneys of FHH rats and that the transfer of this that the transfer of the Rab38 gene from BN to FHH in the gene from BN to FHH in the FHH.BN-Rab38 congenic rescued FHH.BN-Rab38 congenic restored protein expression. Rab38 protein expression, which was associated with improve- J Am Soc Nephrol 16: 852–856, 2005 Effects of the Rf-2 Locus on Proteinuria 855 ment in UPV and UAV. Although we cannot formally exclude prevents translation of protein in the FHH rat. This mutation a contribution by other genes in the region, none of them seem appears to contribute to the development of proteinuria and likely functional candidates and sequence analysis revealed no albuminuria in this strain at three months of age. The replace- variants in their coding regions. ment of the defective Rab38 resulted in a significant improve-

Rab38 is a member of the Rab family of small GTPases that ment in both UPV and UAV without reducing the elevated Palb regulate intracellular vesicle formation and trafficking (23). The seen in FHH rats. These studies suggest that Rab38 may affect specific functions of Rab38 remain to be determined. However, tubular reuptake and processing of filtered proteins. Rab38 has been shown to be responsible for the partial oculo- Overall, this is the first report of a gene responsible for a QTL cutaneous albinism and platelet ␦-granule storage pool bleed- linked to proteinuria in any model of hypertension-associated ing disorder in FHH and other rat strains (5,21,20). Here we ESRD. Although Rab38 seems to influence proteinuria indepen- report the first evidence linking the mutation in Rab38 to the dently of glomerular damage in our model, the region in the development of proteinuria in the FHH rat. homologous to the rat Rf-2 has been linked to Proteinuria and albuminuria were 40% and 60% lower in the the development of proteinuria and FGS. Thus, Rab38 emerges FHH.BN-Rab38 congenic compared with FHH. Interestingly, as a positional candidate gene that might play a role in some this significant improvement in UPV and UAV occurred in the forms of human FGS. Further investigation will be required to absence of any reduction in the glomerular albumin permeabil- verify this interpretation of the data. ity, which remained markedly elevated in the FHH.BN-Rab38 and FHH relative to the levels seen in BN controls. This obser- Acknowledgments vation, together with the disproportionately higher protection We thank the University of Texas Southwestern in Program for from UAV in our congenic animals, suggests that the Rab38 Genomics Applications for the anti-rat Rab38 antibody. This study was gene might affect the reabsorption and degradation of filtered performed with support from the National Institutes of Health (NIH- proteins by proximal tubular cells. Indeed, the mechanism of R01-HL69321) to H.J.J., and from the American Heart Association and tubular reuptake and protein metabolism involves extensive National Blood Foundation to Y.H.D. trafficking of vesicles carrying protein receptors and their li- gands from the apical membrane to the lysosomes (12,22,26). An increasing body of evidence supports the concept of References aberrant trafficking in tubular cells as a mechanism for the 1. Brown DM, Provoost AP, Daly MJ, Lander ES, Jacob HJ: development of several renal diseases characterized by tubular Renal disease susceptibility and hypertension are under independent genetic control in the fawn-hooded rat. Nat proteinuria, including Dent’s disease, Lowe syndrome, and Genet 12: 44–51, 1996 some forms of cystinosis (3,4,6–8,15,32,35). Moreover, Chris- 2. Brown DM, Van Dokkum RP, Korte MR, McLauglin MG, tiansen et al. (4) have demonstrated that the CLC-5 KO mouse Shiozawa M, Jacob HJ, Provoost AP: Genetic control of model of Dent’s disease exhibits proteinuria as a result of susceptibility for renal damage in hypertensive fawn- defective trafficking of the receptors cubilin and megalin to the hooded rats. Ren Fail 20: 407–411, 1998 apical membrane, and at least two Rab proteins (Rab5 and 3. Carr G, Simmons N, Sayer J: A role for CBS domain 2 in Rab7) are known to be involved in this process (4). Finally, the trafficking of chloride channel CLC-5. Biochem Biophys Res Long Evans Cinnamon rat, which carries the same Rab38 allele Commun 310: 600–605, 2003 as the FHH, also has been reported to have a defect in the 4. Christensen EI, Devuyst O, Dom G, Nielsen R, Van der excretion of phenolsulfonphthalein as a result of altered vesic- Smissen P, Verroust P, Leruth M, Guggino WB, Courtoy PJ: ular trafficking in the proximal tubule (14). These results sup- Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney prox- port our view that the mutation in Rab38 may contribute to the imal tubules. Proc Natl Acad Sci U S A 100: 8472–8477, 2003 development of proteinuria in FHH by altering tubular re- 5. Datta YH, Wu FC, Dumas PC, Rangel-Filho A, Datta MW, uptake and processing of filtered protein. Ning G, Cooley BC, Majewski RR, Provoost AP, Jacob HJ: There are several mechanisms by which the lack of Rab38 Genetic mapping and characterization of the bleeding dis- protein might increase UPV and UAV. We have previously order in the fawn-hooded hypertensive rat. Thromb Hae- shown that the FHH platelets have impaired function of lyso- most 89: 1031–1042, 2003 some-related organelles that is restored in the FHH.BN-Rab38 6. De Camilli P, Emr SD, McPherson PS, Novick P: Phosphoi- congenic (5). It is conceivable that dysfunction of the lysosomes nositides as regulators in membrane traffic. Science 271: in the proximal tubular cells may lead to decreased breakdown 1533–1539, 1996 of the filtered load of proteins which would appear as measur- 7. Devonald MA, Karet FE: Renal epithelial traffic jams and able fragments in the urine. Another possibility is that aberrant one-way streets. J Am Soc Nephrol 15: 1370–1381, 2004 8. Faucherre A, Desbois P, Satre V, Lunardi J, Dorseuil O, vesicular trafficking may lead to redistribution of receptors Gacon G: Lowe syndrome protein OCRL1 interacts with such as megalin and cubilin, which would, in turn, lead to a Rac GTPase in the trans-Golgi network. Hum Mol Genet 12: decreased protein uptake by the proximal tubule. Further stud- 2449–2456, 2003 ies will be necessary to elucidate the exact mechanisms by 9. Freedman BI: End-stage renal failure in African Americans: which the absence of the Rab38 gene product in the kidney Insights in kidney disease susceptibility. Nephrol Dial alters urinary protein excretion. Transplant 17: 198–200, 2002 In summary, we identified a mutation in the Rab38 gene that 10. Freedman BI, Rich SS, Yu H, Roh BH, Bowden DW: Link- 856 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 852–856, 2005

age heterogeneity of end-stage renal disease on human 25. Provoost AP, Shiozawa M, Van Dokkum RP, Jacob HJ: chromosome 10. Kidney Int 62: 770–774, 2002 Transfer of the Rf-1 region from FHH onto the ACI back- 11. Genome Browser: Rat Genome Assembly Jun 2003. Genome ground increases susceptibility to renal impairment. Bioinformatics Group of UC Santa Cruz. Available online at Physiol Genomics 8: 123–129, 2002 http://genome.ucsc.edu/. Accessed November 3, 2004. 26. Russo LM, Osicka TM, Brammar GC, Candido R, Jerums 12. Gudehithlu KP, Pegoraro AA, Dunea G, Arruda JA, Singh G, Comper WD: Renal processing of albumin in diabetes AK: Degradation of albumin by the renal proximal tubule and hypertension in rats: Possible role of TGF-beta1. Am J cells and the subsequent fate of its fragments. Kidney Int 65: Nephrol 23: 61–70, 2003 2113–2122, 2004 27. Schork NJ: Genetics of complex disease: Approaches, prob- 13. Hunt SC, Hasstedt SJ, Coon H, Camp NJ, Cawthon RM, lems, and solutions. Am J Respir Crit Care Med 156: S103– Wu LL, Hopkins PN: Linkage of creatinine clearance to S109, 1997 chromosome 10 in Utah pedigrees replicates a locus for 28. Schulz A, Litfin A, Kossmehl P, Kreutz R: Genetic dissec- end-stage renal disease in humans and renal failure in the tion of increased urinary albumin excretion in the Munich fawn-hooded rat. Kidney Int 62: 1143–1148, 2002 Wistar Fromter rat. J Am Soc Nephrol 13: 2706–2714, 2002 14. Itagaki S, Shimamoto S, Hirano T, Iseki K, Sugawara M, 29. Schulz A, Standke D, Kovacevic L, Mostler M, Kossmehl P, Nishimura S, Fujimoto M, Kobayashi M, Miyazaki K: Com- Stoll M, Kreutz R: A major gene locus links early onset parison of urinary excretion of phenolsulfonphthalein in albuminuria with renal interstitial fibrosis in the MWF rat an animal model for Wilson’s disease (Long-Evans Cinna- with polygenetic albuminuria. J Am Soc Nephrol 14: 3081– mon rats) with that in normal Wistar rats: Involvement of 3089, 2003 primary active organic anion transporter. J Pharm Pharm 30. Sharma M, Sharma R, Reddy SR, McCarthy ET, Savin VJ: Sci 7: 227–234, 2004 Proteinuria after injection of human focal segmental glo- 15. Kalatzis V, Cohen-Solal L, Cordier B, Frishberg Y, Kemper merulosclerosis factor. Transplantation 73: 366–372, 2002 M, Nuutinen EM, Legrand E, Cochat P, Antignac C: Iden- 31. Shiozawa M, Provoost AP, van Dokkum RP, Majewski RR, tification of 14 novel CTNS mutations and characterization Jacob HJ: Evidence of gene-gene interactions in the genetic of seven splice site mutations associated with cystinosis. susceptibility to renal impairment after unilateral nephrec- Hum Mutat 20: 439–446, 2002 tomy. J Am Soc Nephrol 11: 2068–2078, 2000 16. Kessler MA, Meinitzer A, Wolfbeis OS: Albumin blue 580 32. Shotelersuk V, Larson D, Anikster Y, McDowell G, Lemons fluorescence assay for albumin. Anal Biochem 248: 180–182, R, Bernardini I, Guo J, Thoene J, Gahl WA: CTNS muta- 1997 tions in an American-based population of cystinosis pa- 17. Korstanje R, DiPetrillo K: Unraveling the genetics of tients. Am J Hum Genet 63: 1352–1362, 1998 chronic kidney disease using animal models. Am J Physiol 33. St. Lezin E, Griffin KA, Picken M, Churchill MC, Churchill Renal Physiol 287: F347–F352, 2004 PC, Kurtz TW, Liu W, Wang N, Kren V, Zidek V, Pravenec 18. Kwitek-Black AE, Jacob HJ: The use of designer rats in the M, Bidani AK: Genetic isolation of a chromosome 1 region genetic dissection of hypertension. Curr Hypertens Rep 3: affecting susceptibility to hypertension-induced renal 12–18, 2001 damage in the spontaneously hypertensive rat. Hyperten- 19. Lander ES, Schork NJ: Genetic dissection of complex traits. sion 34: 187–191, 1999 Science 265: 2037–2048, 1994 34. Stoll M, Cowley AW Jr, Tonellato PJ, Greene AS, Kaldunski 20. Loftus SK, Larson DM, Baxter LL, Antonellis A, Chen Y, ML, Roman RJ, Dumas P, Schork NJ, Wang Z, Jacob HJ: A Wu X, Jiang Y, Bittner M, Hammer JA 3rd, Pavan WJ: genomic-systems biology map for cardiovascular function. Mutation of melanosome protein RAB38 in chocolate mice. Science 294: 1723–1726, 2001 Proc Natl Acad Sci U S A 99: 4471–4476, 2002 35. Suchy SF, Olivos-Glander IM, Nussabaum RL: Lowe syn- 21. Oiso N, Riddle SR, Serikawa T, Kuramoto T, Spritz RA: drome, a deficiency of phosphatidylinositol 4,5-bisphos- The rat Ruby (R) locus is Rab38: Identical mutations in phate 5-phosphatase in the Golgi apparatus. Hum Mol fawn-hooded and Tester-Moriyama rats derived from an Genet 4: 2245–2250, 1995 ancestral Long Evans rat sub-strain. Mamm Genome 15: 36. Weichselbaum TE: A accurate and rapid method for the 307–314, 2004 determination of proteins in small amounts of blood serum 22. Osicka TM, Comper WD: Protein degradation during renal and plasma. Am J Clin Path 16: 40–49, 1946 passage in normal kidneys is inhibited in experimental 37. Winn MP, Conlon PJ, Lynn KL, Howell DN, Slotterbeck albuminuria. Clin Sci (Lond) 93: 65–72, 1997 BD, Smith AH, Graham FL, Bembe M, Quarles LD, Peri- 23. Pereira-Leal JB, Seabra MC: Evolution of the Rab family of cak-Vance MA, Vance JM: Linkage of a gene causing fa- small GTP-binding proteins. J Mol Biol 313: 889–901, 2001 milial focal segmental glomerulosclerosis to chromosome 24. University of Texas Southwestern Program for Genomics Ap- 11 and further evidence of genetic heterogeneity. Genomics plications: Genomics and Proteomics of Cell Injury and Inflam- 58: 113–1120, 1999 mation. National Heart, Lung, and Blood Institute Program for 38. Yagil C, Sapojnikov M, Katni G, Ilan Z, Zangen SW, Rosen- Genomic Applications (grant ID 5U01HL6688002), University of mann E, Yagil Y: Proteinuria and glomerulosclerosis in the Texas Southwestern Medical Center. Available online at http:// Sabra genetic rat model of salt susceptibility. Physiol pga.swmed.edu. Accessed October 15, 2004. Genomics 9: 167–178, 2002