Development of Overt Proteinuria in the Munich Wistar Frömter Rat Is Suppressed by Replacement of Chromosome 6 in a Consomic Rat Strain
Angela Schulz*,
Judith Weiss*,
Maria Schlesener*,
Jonna Hänsch*,
Markus Wehland*,
Norbert Wendt*,
Peter Kossmehl*,
Anika Sietmann,
Daniela Grimm*,
Monika Stoll,
Jens Randel Nyengaard and
Reinhold Kreutz*
* Institut für Klinische Pharmakologie und Toxikologie, Campus Benjamin Franklin, Charité–Universitätsmedizin Berlin, Berlin, and Leibniz-Institut für Arterioskleroseforschung, Münster, Germany; and Stereology and Electron Microscopy Research Laboratory and MIND Center, University of Aarhus, Aarhus, Denmark
Address correspondence to: Dr. Reinhold Kreutz, Department of Clinical Pharmacology and Toxicology, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Phone: +49-30-8445-2280; Fax: +49-30-8445-4482; E-mail: reinhold.kreutz{at}charite.de
Received for publication March 7, 2006.
Accepted for publication October 23, 2006.
In a cross between the Munich Wistar Frömter (MWF) ratand spontaneously hypertensive rats (SHR), a major quantitativetrait locus (QTL) was identified on rat chromosome 6 (RNO6)that demonstrated the strongest linkage to albuminuria amongseveral QTL identified. The QTL represented the only locus thatis linked to both early-onset albuminuria and increased renalinterstitial fibrosis in adult animals. A consomic MWF-6SHRstrain in which chromosome 6 from SHR was introgressed intothe MWF background therefore was generated to test the relevanceof this QTL. Phenotype analysis at 8 wk of age revealed thatearly onset of albuminuria in MWF with a 55-fold elevation ofurinary albumin excretion compared with SHR (P < 0.0001)was completely abolished in MWF-6SHR. Time-course analysis untilweek 24 demonstrated only a moderate increase of urinary albuminexcretion in MWF-6SHR, whereas MWF reached levels in the nephroticrange (16.6 ± 3.5 versus 162.6 ± 16.0 mg/24 h;P < 0.0001). At this age, analysis of glomerulosclerosis,tubulointerstitial damage, renal interstitial fibrosis, andrenal collagen III mRNA expression revealed a significant improvementof all parameters in MWF-6SHR compared with MWF (P < 0.05).At 32 wk, MWF but not MWF-6SHR demonstrated overt proteinuria(354.6 ± 37.6 versus 48.8 ± 13.2; P < 0.0001),whereas serum urea, cholesterol, and triglyceride concentrationswere lower and creatinine clearance was higher in MWF-6SHR comparedwith MWF (P < 0.05). Therefore, although albuminuria in MWFis determined by a complex interplay of several QTL, our datademonstrate that genetic exchange of one locus on RNO6 leadsto marked suppression of early-onset albuminuria and renal damagein MWF.
An elevated urinary albumin excretion (UAE) rate is a predictorfor the development of chronic nephropathy, and a moderate increaseof UAE in the range of microalbuminuria represents an independentrisk factor for cardiovascular events in the general population,in arterial hypertension, and particularly in patients withdiabetes or documented cardiovascular disease (1–4). Albuminuriaand proteinuria are complex phenotypes that are influenced byboth environmental and genetic factors (5–7). ElevatedUAE rates represent a hallmark of diabetic nephropathy (7),and previous genetic segregation analysis of UAE in familieswith type 2 diabetes demonstrated that levels of UAE are determinedby mixture of probably recessive genes with large and smalleffect in individuals both with and without diabetes (8,9).More recently, genetic analyses in extended families confirmedthat similar genes contribute to the heritability of UAE infamily members with and without diabetes (7). Moreover, severalquantitative trait loci (QTL) have been identified by genome-widelinkage analyses for UAE in diabetes, hypertension, and thegeneral population (6,7,10).
Previous studies in hypertensive genetic rat models have shownthat elevated UAE levels are also influenced by several QTLin the Fawn-hooded hypertensive (FHH) rat (11,12), Dahl salt-sensitiverat (SS) (13,14), Munich Wistar Frömter rat (MWF) (5,15),and Sabra rat (16). Overall, it turned out that increased UAElevels that are observed in these models are influenced by multipleUAE QTL, whereas single-locus effects on UAE were modest andrequired the homozygous state of susceptibility alleles, asa result of a recessive mode of inheritance (15). Therefore,in experimental crosses with these strains, it could be shownthat a considerable increase in UAE levels required the synergisticinteraction among several UAE QTL (5,12–15). In our cross-breedingand linkage studies involving the MWF rat, we identified multiplepotential QTL with suggestive or significant linkage to UAE(5,15) and proposed that a QTL that was identified on rat chromosome(RNO) 6 could play a major role. This QTL was identified intwo independent crosses involving either Lewis rats (5) or spontaneouslyhypertensive rats (SHR) (15) as reference strains with low UAE.Moreover, in the mapping study using SHR as a contrasting model,the homozygous MWF genotype at this QTL on RNO6 demonstratedthe strongest linkage to UAE and was the only QTL linked toboth the onset of albuminuria in young animals and morphologicchanges in the kidney (e.g., increased renal interstitial fibrosis[RIF]), in adult animals (15). We therefore set out to testthe potential functional relevance of this QTL for early manifestationof elevated UAE and development of overt proteinuria in MWFby generating a consomic MWF-6SHR strain in which RNO6 fromSHR was introgressed into the isogenic background of MWF.
Animals
Male MWF/Rkb and SHR/Rkb rats were obtained from our colonies(laboratory code Rkb) at the Charité, Campus BenjaminFranklin, Germany. Rats were grouped under conditions of regular12-h diurnal cycles using an automated light-switching deviceand climate-controlled conditions at a room temperature of 22°C.The rats were fed a normal diet that contained 0.2% NaCl andhad free access to food and water.
Experimental Groups
To test the influence of chromosome 6 transfer from SHR on earlyonset of UAE and the subsequent development of proteinuria inMWF, we first performed in accordance with our previous linkagestudies (5,15) time-course analysis for UAE at 8, 14, and 24wk of age, comparing MWF (n = 18) with MWF-6SHR (n = 19) duringtime-course analysis. In addition, because the absolute amountof UAE (in the range of 1 mg/24 h) and variation over time arelow in the parental SHR donor strain, a somewhat smaller groupof SHR (n = 10) also were included for comparison. After completionof UAE analysis, systolic BP (SBP) was measured at 24 wk ofage and all rats subsequently were killed at the end of wk 24for further analysis.
In addition, to test whether the transfer of RNO6 from SHR intoMWF protects against the development of overt proteinuria andhas an impact on secondary changes related to nephrotic-rangeproteinuria, we compared UAE and renal function parameters betweenproteinuric MWF and consomic MWF-6SHR at 32 wk of age (n = 9to 10). Finally, because previous studies in the MWF straindemonstrated a lower number of nephrons compared with Wistarrats (17,18), the total number of glomeruli was compared inall three strains. However, in this analysis, to exclude aninfluence of secondary changes as a result of a loss of damagedglomeruli during the determination of glomeruli number in adultrats, we used an additional set of young rats at 4 wk of age(n = 7, respectively).
Development of the Consomic Strain
The consomic strain was derived from our MWF/Rkb and SHR/Rkbcolonies. To develop the consomic strain, we crossed the MWFstrain with the SHR strain in accordance with our linkage results(15) and introgressed the whole RNO6 from SHR into the MWF background(19). In a first step, an F1 population was generated betweenone male MWF rat and female SHR. Male heterozygous offspringswere backcrossed with female MWF to conserve the Y chromosomefrom MWF. In each of seven backcross generations in total, themale breeders with the highest number of homozygous markersof the MWF background and concomitant heterozygous for RNO6were crossed with four MWF female rats, respectively, by sequentialmarker-assisted backcrossing (19). To fix RNO6 from SHR intothe MWF background, we then performed several intercrosses betweenbackcross male and female rats that were homozygous for allMWF chromosomes except RNO6 and heterozygous for RNO6. Finally,the purity of the consomic MWF-6SHR strain was confirmed bytotal genome screen analysis with 240 microsatellite markers.In the MWF-6SHR colony, no genetic contaminations of SHR inthe background genome could be detected. Genotyping was performedby standard methods as previously reported (5).
Phenotyping
For urine analysis, rats were placed in metabolic cages for2 d. The first day was used for adaptation, and urine was collectedfor the last 24 h for biochemistry analysis. UAE was measuredwith a rat-specific ELISA technique as described previously(20). In addition to UAE, the urinary excretion of low molecularweight (LMW) proteins was analyzed as previously reported (21).In brief, 4 µg of total urinary protein together with3 µl of Precision Plus Protein Standard (Bio-Rad, Hercules,CA) and 2 µg of rat serum albumin (Sigma, St. Louis, MO)as a control were resolved on a 10% SDS-PAGE. The gels thenwere stained with Coomassie blue and photographed using differentexposure times to ensure unsaturated band intensities. The negativeswith the optimal exposure subsequently were scanned under standardconditions. The digital analysis of the resulting images wasdone with ImageJ (http://rsb.info.nih.gov/ij/) using the gelanalysis features.
SBP was measured at 24 wk of age by a noninvasive tail-cuffmethod in awake rats using a computer-assisted oscillatory detectiondevice (TSE, Bad Homburg, Germany) as described previously (20).These measurements involved two training sessions on 2 d followedby up to 18 (minimum 12) recordings in awake rats on 3 consecutivedays performed as previously reported (20). Subsequently, ratswere killed at 24 wk, and blood was drawn from the aorta forthe determination of serum creatinine, urea, cholesterol, andtriglycerides with standard methods. Both kidneys and the heartwere excised. The body, total kidney, and heart weights weredetermined. For light microscopy evaluation, a midcoronal sectionof the left kidney was fixed and embedded in paraffin for histologystudies (5,22). The 3-µm sections of the kidneys werestained with the periodic acid-Schiff technique for the determinationof glomerulosclerosis and tubulointerstitial damage indicesby semiquantitative scoring methods as reported (5,22). RIFwas determined after staining of sections with Sirius red followingprevious recommendations (23). In addition, we determined thenumbers of both surface glomeruli with direct contact to thesurface of the kidney and the total number of all glomerulipresent in the renal cortex corticis zone but without directsurface contact (i.e., superficial glomeruli) in three midcoronalsections for each rat as reported (5).
Determination of Glomerular Number
After perfusion fixation with 4% phosphate-buffered formaldehyde,one kidney was used for estimating glomerular number in 4-wk-oldrats. The kidney was dehydrated in graded ethanol and embeddedin glycolmethacrylate (Technovite 7100; Hereus Kulzer, Wehrheim,Germany). Using a Microm HM 355 microtome, each kidney was cutexhaustively in 20-µm-thick sections. Every 30th sectionand its adjacent section (nine to 11 section pairs) were selectedusing systematic, uniformly random sampling (24). The sampledsection pairs were mounted on one slide and stained with periodicacid-Schiff and Mayer’s hematoxylin. Counting was performedusing an Olympus BX-50 microscope at a magnification of x113with an automated Märzhäuser Multi Control 2000 specimenstage (Märzhäuser, Wetzlar-Steindorf, Germany) anda fast digital camera (Pixelink PL-A686C) connected to a computer(Dell Optiplex GX110) with newCAST software (Visiopharm, Hørsholm,Denmark) to superimpose the counting frame. Glomeruli were countedin six consecutive section pairs starting with the third becauseof the problem of artificial edges in the first two sectionsand the last sections. Therefore, a sampling fraction Ps/Pfwas introduced: Ps is the number of points that hit all kidneytissue, and Pf is the number of points that hit only kidneytissue that was used for glomerular counting.
The number of glomeruli was estimated by the physical fractionator(25). The glomeruli were counted if they were present insidethe two-dimensional unbiased counting frame in one section (thesampling frame) but not in the adjacent section plane (the look-upsection) and vice versa. On average, 187 glomeruli (Q–)were counted per kidney. The total number of glomeruli per kidney[N(glom)] was calculated using the following formula:
The factor was introduced becauseglomeruli were counted both ways in the disector.
Area sampling fraction (ASF) was calculated as the countingframe area [A(frame)] divided by the step lengths in the x andy direction (dx · dy) of the counting frame. The errorvariance of this technique that is used for counting glomeruliwas estimated to be 8% (24).
RNA Extraction and cDNA Synthesis
RNA was isolated from kidneys by the TRIzol reagent (Invitrogen,Karlsruhe, Germany) according to the manufacturer’s instructionsand was resuspended in DEPC-treated H2O. First-strand cDNA synthesiswas carried out on 2 µg of total RNA in a 20-µlreaction using the First Strand cDNA Synthesis Kit (FermentasLife Sciences, St. Leon-Rot, Germany) following the manufacturer’srecommendations.
Collagen III mRNA Expression Analysis
To quantify mRNA expression of collagen III in kidney, we usedthe real-time quantitative reverse transcriptase (TaqMan) PCRmethod. Appropriate primers and fluorogenic probes were designedwith the Primer Express software. The ABI PRISM 7000 SDS instrumentin conjunction with the ABI TaqMan Universal Master Mix (AppliedBiosystems, Darmstadt, Germany) was used to perform the assays.The reaction volume was 25 µl with a final concentrationof 900 nM for the primers and 200 nM for the cDNA probes. PCRconditions were used as recommended by the manufacturer. Thefluorogenic probes were synthesized by TIB Molbiol (Berlin,Germany), and the primers were obtained from Proligo (Paris,France; Table 1).
Table 1. Overview of primers and fluorogenic probes for quantitative collagen III mRNA analysis by TaqMan-PCRa
Relative quantification was done using the standard curve method.For each gene, a PCR fragment that contained the sequence ofthe TaqMan system was generated. Seven serial 1:10 dilutionsof this fragment served as a standard curve that was assayedtogether with the corresponding unknown samples on each plate.Every sample was measured in triplicate. To normalize our expressiondata, we used porphobilinogen deaminase as a housekeeping gene(GenBank accession no. X06827) (26).
Statistical Analyses
Statistical analysis was performed using one-way ANOVA followedby Bonferroni adjustment and by nonparametric Mann-Whitney Utest. Data are means ± SEM, and P < 0.05 was consideredsignificant.
The evaluation of early-onset albuminuria in MWF at 8 wk ofage demonstrated a marked 55-fold increase in MWF compared withSHR (18.1 ± 2.0 versus 0.3 ± 0.1 mg/24 h; P <0.0001; Figure 1). This increase was completely abolished inMWF-6SHR, which showed similar UAE levels of 0.7 ± 0.1mg/24 h compared with SHR. To analyze the gene dosage effectof the SHR allele on the early onset of UAE at 8 wk, we studiedone group of F1 hybrid rats (n = 8) that were derived from MWFand consomic MWF-6SHR. In this analysis, a significant increasein UAE to 5.7 ± 1.7 mg/24 h was found in F1 rats in comparisonwith SHR and MWF-6SHR (P < 0.05; Figure 1). The F1 rats,however, demonstrated UAE levels that were significantly belowthe values that were observed in parental MWF (P < 0.05;Figure 1).
Figure 1. Urinary albumin excretion (UAE) of spontaneously hypertensive rats (SHR), Munich Wistar Frömter rats (MWF), consomic MWF-6SHR, and F1 rats that were derived from MWF and MWF- 6SHR at 8 wk of age. *P < 0.0001, #P < 0.05 versus other groups.
Further time-course analysis of UAE between weeks 8 and 24 revealeda further marked increase of UAE in MWF, particularly betweenweeks 14 and 18. In this time period, MWF reached UAE levelsat the nephrotic range of approximately 160 mg/24 h. In agingSHR, UAE remained low at 24 wk (2.3 ± 1.1 mg/24 h), whereasonly a moderate increase in UAE levels was observed in MWF-6SHRbetween weeks 14 (2.9 ± 3.5 mg/24 h) and 24 (16.6 ±15.2 mg/24 h; Figure 2). Overall, in comparison with parentalMWF, UAE was markedly suppressed in consomic MWF-6SHR at alltime points investigated (P < 0.0001). The analysis of urinaryexcretion of LMW proteins in SHR revealed that the amount ofexcreted LMW proteins is larger than UAE (albumin/LMW proteinratio 0.41 ± 0.04; Figure 2). In contrast, the high proteinexcretion in MWF is attributable largely to albumin, whereasthe contribution of LMW proteins to total protein excretionis only minor (albumin/LMW ratio 9.50 ± 0.55; P <0.001 versus SHR). Consomic MWF-6SHR demonstrated an almostequal distribution of both protein species; the increased albuminto LMW protein ratio observed in MWF clearly is suppressed inthis strain (albumin/LMW protein ratio 1.59 ± 0.42; P< 0.01 versus MWF and SHR, respectively; Figure 2).
Figure 2. (A) Time-course analysis of UAE of SHR (), consomic MWF-6SHR (
), and MWF () at 14, 18, and 24 wk of age. *P < 0.0001 versus SHR and MWF-6SHR; #P < 0.05 versus age-matched SHR. (B) Comparison of the urinary albumin/low molecular weight (LMW) protein ratio between SHR (), MWF-6SHR (), and MWF () at 24 wk of age. *P < 0.001 versus SHR; #P < 0.0004 versus MWF-6SHR. Below the bar graph, representative lanes from SDS-PAGE experiments are displayed for each strain. A, pure rat serum albumin control; M, molecular weight marker.
At 24 wk of age, no significant difference in body weight wasobserved among strains (Table 2). Both absolute and relativekidney weight in relation to body weight were significantlylower in MWF compared with SHR (Table 2). SBP differed significantlyamong strains (P < 0.0001) at 24 wk of age. Both MWF andconsomic MWF-6SHR exhibited lower SBP compared with SHR, whereasSBP was lower in MWF compared with consomic rats (P = 0.034;Table 2). The significant differences observed in absolute andrelative heart weight among the three strains mirrored the differencesthat were seen in SBP (Table 2). No significant difference inserum creatinine and creatinine clearance was found among strains,whereas serum urea differed significantly among strains (Table 2).MWF showed the highest and MWF-6SHR exhibited intermediate serumurea concentrations (Table 2). Both serum triglycerides andcholesterol concentrations were significantly higher in MWFand MWF-6SHR compared with SHR (P < 0.001; Table 2). A significantreduction in cholesterol levels but not in triglycerides wasseen in MWF-6SHR compared with MWF (Table 2) at the age of 24wk.
Table 2. Overall characteristics of parental MWF, SHR, and consomic MWF-6SHR rats at 24 wk of agea
The determination of total glomerular number per kidney in youngrats at 4 wk of age revealed overall a significant differencebetween strains (P < 0.005) with a significantly lower number(approximately –30%) in MWF compared with SHR (27,000± 3500 versus 37,000 ± 5900; P = 0.006; Figure 3).Glomerular number in consomic MWF-6SHR (34,600 ± 5700)was significantly higher compared with MWF (P = 0.04) and notstatistically different from SHR (Figure 3).
Figure 3. Total number of glomeruli per kidney in SHR (), consomic MWF-6SHR (
), and MWF (). *P = 0.006 versus SHR; #P = 0.04 versus MWF-6SHR.
The results that were obtained for renal histology analysisin adult animals at 24 wk of age are summarized in Table 3.The number of surface glomeruli (i.e., with direct contact tothe surface) but not the total number of superficial glomeruliwas significantly reduced in the MWF-6SHR strain compared withMWF. Glomerulosclerosis index and tubulointerstitial damageindex were elevated significantly in MWF compared with SHR andsignificantly reduced in MWF-6SHR compared with MWF (Table 3).Quantitative analysis of RIF revealed a similar pattern witha significant reduction of fibrosis in the MWF-6SHR consomicstrain (Table 3; Figure 4A). In addition, the increased collagenIII mRNA expression that was observed in MWF compared with SHRwas normalized in MWF-6SHR (Figure 4B).
Figure 4. (A) Sirius red staining for renal interstitial fibrosis (RIF) of a representative MWF (1), consomic MWF-6SHR (2), and SHR (3). (B) Relative collagen III mRNA expression in kidney of MWF, SHR, and consomic MWF-6SHR. *P < 0.01 versus MWF-6SHR and SHR.
Further follow-up of a subgroup of MWF and consomic MWF-6SHRto week 32 demonstrated the manifestation of overt proteinuriain MWF (354.6 ± 112.9 mg/24 h) and also a significantincrease in UAE in consomic rats (48.8 ± 41.7 mg/24 h).However, the maximum levels that were observed in consomic MWF-6SHRwere well below the nephrotic range that was observed in MWF(Figure 5). In addition, serum urea, cholesterol, and triglycerideconcentrations were significantly lower and creatinine clearancewas significantly higher in MWF-6SHR at 32 wk of age (P <0.05; Figure 5).
In our previous cross-breeding study between MWF and SHR, wemapped a QTL on RNO6 that was linked to albuminuria in younganimals at 8 wk of age and to both elevated UAE and increasedRIF in adult backcross animals at 24 wk of age (15). We thereforeproposed that this QTL could be important not only for the manifestationof early onset albuminuria but also for the development of overtproteinuria and decline in renal function. An important subsequentstep to test this hypothesis is the generation of consomic strainsin which an entire chromosome is introgressed into the isogenicbackground of another inbred strain using marker-assisted selection(27). Therefore, the generation of MWF-6SHR was of particularimportance for testing the functional relevance of the QTL onRNO6, because we had shown that UAE in MWF probably is influencedby up to 11 different QTL (5,15). Here, we can demonstrate thatthe genetic exchange of one QTL has a striking effect on thedevelopment of overt proteinuria in MWF. It has been well establishedthat MWF develop overt proteinuria with age with progressiveglomerular injury and proteinuric renal disease. Moreover, thisstrain represents a valuable model for functional analysis ofglomerular damage and repair mechanisms and intervention studiesin progressive renal failure (28–33). In agreement withthese studies, we confirmed in this study that MWF from ourcolony reached UAE levels in the nephrotic range of approximately150 mg/24 h (34) between 14 and 24 wk followed by a furthermore than two-fold progression of UAE in week 32. In contrastto our previous results in younger animals (5,15) and in agreementwith other reports (17,21), we determined in this set of experimentslower SBP values in the MWF strain and significant higher SBPin SHR. It is interesting that SBP was significantly increased,albeit moderately, in MWF-6SHR compared with MWF, suggestingthat RNO6 from SHR contains a BP-increasing QTL that was notdetected in the original linkage study (15). The latter notionshould be viewed with caution until this finding is evaluatedin further studies that involve the characterization of congeniclines. Currently, it should not be dismissed, however, thatan important conclusion can be derived from this finding, namelythat the protective effect on the development of proteinuriathat was observed in the consomic strain cannot be attributedto a decrease in SBP in this strain.
Nevertheless, in consomic MWF-6SHR, the development of nephrotic-rangeproteinuria clearly was suppressed and analysis of structuralchanges at 24 wk showed that glomerular and tubulointerstitialdamage as well as RIF and collagen III expression were reducedsignificantly in consomic rats. Subsequently, the decline ofrenal function and progression of dyslipidemia that were observedin aging 32-wk-old MWF also were improved significantly in consomicrats.
The suppressive effect on UAE that was conferred by introgressionof RNO6 from SHR occurred very early in MWF-6SHR at 8 wk ofage, when consomic and SHR were indistinguishable with regardto UAE, demonstrating levels below the 1-mg/24 h range. Thesubsequent comparison between MWF-6SHR and the contrasting lowUAE strain SHR showed a continuous but modest increase of UAEin MWF-6SHR that became significant at week 18 (Figure 2). Therefore,the development of elevated UAE is delayed significantly andin magnitude clearly suppressed in the consomic strain. In addition,the comparison between UAE and LMW protein excretion at week24 by gel electrophoresis in the three strains revealed thatthe increased urinary albumin to LMW protein ratio of MWF clearlywas suppressed in the consomic strain, thus confirming the reversalof albuminuria in these rats.
It is interesting that the evaluation of early-onset albuminuriain young F1 hybrid rats that were derived from MWF and consomicMWF-6SHR revealed a gene dosage effect on UAE. These F1 hybridscarry a heterozygous genotype on RNO6 (i.e., they carry oneallele from SHR and one from MWF). Young F1 rats exhibited asignificant increase of UAE compared with SHR and MWF-6SHR;they demonstrated approximately one third of UAE levels thatwere observed in MWF. This finding indicates that one SHR alleleimpairs the increase of UAE, whereas two alleles are requiredto suppress fully the development of early-onset albuminuriain MWF-6SHR.
In backcrossed rats in our QTL mapping study, we observed thatthe MWF allele of the QTL on RNO6 also was linked to the presenceof increased numbers of superficial and surface glomeruli (15),which represent additional traits that are inherited in theMWF rat (35) together with a significant reduction in glomerularnumber (17,18). Although we could confirm a reduced number ofglomeruli with direct contact to the surface in MWF-6SHR, thetotal number of superficial glomeruli in the outer cortex zonewas not affected significantly. It is interesting, however,that transfer from RNO6 into the MWF background resulted ina significant increase in total glomerular number per kidneyin consomic MWF-6SHR compared with MWF. These findings pointto the possibility that a causative gene on RNO6 is linked toearly-onset albuminuria, and reduced glomerular number in theMWF strain. Alternatively, the selective breeding of the MWFstrain for enrichment in surface glomeruli (35) may have selectedand captured by chance this important UAE QTL on RNO6. Our comparisonof renal structural damage between the consomic strain and SHRindicated that glomerulosclerosis and RIF were reduced onlypartially in MWF-6SHR, whereas tubulointerstitial damage wassimilar in MWF-6SHR and SHR. However, despite the low and normalUAE levels that were observed in SHR, secondary renal changesas a result of hypertension in the SHR strain may hamper thiscomparison. Therefore, it also is of further interest to comparethe protective effect on renal structural damage between consomicor subsequently congenic strains with a normotensive referencestrain.
Comparative mapping analysis (36) between the 99 and 95% confidenceintervals for placement of this QTL (Figure 3 of reference [15])with the human genome maps this region to human chromosomes14q23.1 to 14q31.3. This region contains 146 annotated genes,46 of which are predicted or based on expressed sequence tagdata and require further confirmation (Supplementary Table 1).Currently, we are not able to give any indication which gene(s)within the RNO6 region is (are) responsible for early-onsetalbuminuria. However, the development of MWF-6SHR validatesthis region and provides the rationale to develop further congenicrats that carry smaller intervals, thereby reducing the numberof genes within the QTL for further investigation.
The homologous region in the human genome, 14q23.1 to q31.3,is different from UAE QTL that were identified recently on humanchromosomes 5q, 7q, 8q, 12q, 19p, 21p, and 22q by genome-widelinkage analysis (6,7,10). This does not exclude, however, itspotential clinical relevance considering that UAE in humansrepresents a highly complex phenotype that is influenced bymultiple interactions between genetic susceptibilities and environmentalfactors. Strain-specific genetic differences in inbred animalmodels may reflect phenotypic subtypes of a more complex UAEphenotype that is observed in human populations, which exhibitgreater genetic and phenotypic heterogeneity than the inbredlines that are used for QTL identification. The systematic analysisof consomic strains using the SS rat model as a recipient andthe normotensive Brown-Norwayrat as a donor strain within thePrograms for Genomic Applications funded by the National Heart,Lung, and Blood Institute (data are available at http://pga.mcw.edu)demonstrated also a protective effect of RNO6 on the developmentof UAE in SS, whereas BP was not affected (http://pga.mcw.edu).In contrast, transfer of RNO6 from Brown-Norway rats into theFHH rat model of renal damage, which also is analyzed in thisprogram, had no effect on either UAE or BP (http://pga.mcw.edu).Therefore, the identification of the RNO6 UAE QTL in the MWFand SS rat models may be indicative of the existence of a subtype-specificgenetic predisposition, which has not yet been determined throughlinkage analysis in human populations because of genetic heterogeneityand the complexity of the UAE phenotype. The confirmation ofthe pivotal role of the RNO6 locus for the development of UAEin the MWF model may guide targeted analysis in humans by moreadvanced and powerful genetic techniques that involve single-nucleotidepolymorphism and haplotype analyses in the corresponding regionon human chromosome 14 in clinical studies. At the same time,the establishment of MWF-6SHR and the demonstration of its phenotypiccharacteristics provide the next essential step for the identificationof the causative gene(s) by breeding congenic lines and furtherfunctional analysis. The power of using rat models to dissectthe genetics of UAE and renal disease and to identify new targetswas highlighted recently by the identification of Rab38 forelevated UAE in the FHH rat model (37) and Fcgr3 as a determinantof susceptibility to immunologically mediated glomerulonephritisin WKY rats (38).
From this study, we conclude that the removal of one importantsusceptibility locus for UAE from the complex interplay amongseveral UAE QTL leads to a marked suppression of proteinuricrenal disease in the MWF strain. We propose that the availabilityof consomic MWF-6SHR will complement the MWF strain as a valuablemodel for the isolation and characterization of putative candidategenes and the genetic dissection for disease pathways in proteinuricrenal disease.
This study was supported by grants KGCV1, 01GS0416, and NGFN2from the Bundesministerium für Bildung und Forschung (BMBF).
We acknowledge the contributions of Sabine Wunderlich, ClaudiaPlum, and Norbert Hint for excellent laboratory assistance andof Bettina Lack for excellent support in animal breeding.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
Q–) were counted per kidney. The total number of glomeruli per kidney [N(glom)] was calculated using the following formula:
was introduced because glomeruli were counted both ways in the disector. 
), consomic MWF-6SHR (
), and MWF (
) at 14, 18, and 24 wk of age. *P < 0.0001 versus SHR and MWF-6SHR; #P < 0.05 versus age-matched SHR. (B) Comparison of the urinary albumin/low molecular weight (LMW) protein ratio between SHR (
