Laminin Compensation in Collagen 3(IV) Knockout (Alport) Glomeruli Contributes to Permeability Defects
Dale R. Abrahamson*,
Kathryn Isom*,
Eileen Roach*,
Larysa Stroganova*,
Adrian Zelenchuk*,
Jeffrey H. Miner and
Patricia L. St. John*
* Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas; and Renal Division, Department of Internal Medicine, Washington University, St. Louis, Missouri
Correspondence: Dr. Dale R. Abrahamson, Department of Anatomy and Cell Biology, University of Kansas Medical Center, MS 3038, 3901 Rainbow Boulevard, Kansas City, KS 66160. Phone: 913-588-7000; Fax: 913-588-2710; E-mail: dabrahamson{at}kumc.edu
Received for publication March 17, 2007.
Accepted for publication May 14, 2007.
Alport disease is caused by mutations in genes encoding the3, 4, or 5 chains of type IV collagen, which form the collagenousnetwork of mature glomerular basement membrane (GBM). In theabsence of 3, 4, 5 (IV) collagen, 1, 2 (IV) collagen persists,which ordinarily is found only in GBM of developing kidney.In addition to dysregulation of collagen IV, Alport GBM containsaberrant laminins, which may contribute to the progressive GBMthickening and splitting, proteinuria, and renal failure seenin this disorder. This study sought to characterize furtherthe laminin dysregulation in collagen 3(IV) knockout mice, amodel of Alport disease. With the use of confocal microscopy,laminin 1 and 5 abundance was quantified, and it was found thatthey co-distributed in significantly large amounts in areasof GBM thickening. In addition, labeling of entire glomerulifor laminin 5 was significantly greater in Alport mice thanin wild-type siblings. Reverse transcriptase–PCR fromisolated glomeruli demonstrated significantly more laminin 5mRNA in Alport mice than in wild-type controls, indicating upregulatedtranscription of Lama5. For testing glomerular barrier function,ferritin was injected into 2-wk-old Alport and control mice,and GBM was examined by electron microscopy. Highest ferritinlevels were seen in Alport GBM thickenings beneath effaced podocytefoot processes, but morphologically normal GBM was significantlypermeable as well. We concluded that (1) ultrastructurally normalAlport GBM residing beneath differentiated podocyte foot processesis inherently and abnormally permeable, and (2) upregulationof Lama5 transcription and concentration of laminin 1 and 5within Alport GBM thickenings contribute to abnormal permeabilities.
Alport syndrome is caused by mutations in the COL4A3, COL4A4,or COL4A5 genes, which encodes the type IV collagen 3, 4, and5 chains, respectively, that compose the collagenous networkwithin mature kidney glomerular basement membranes (GBM).1–3When any one of the COL4A3 through A5 genes is mutated, a stable3, 4, 5 (IV) network cannot form, and immunolabeling techniquesshow an absence of these proteins in Alport GBM. In kidney,the disease is marked by hematuria of glomerular origin, thickeningand splitting of the GBM, effacement of podocyte foot processes,progressive proteinuria, and declining renal function that usuallyrequires replacement therapy.1–3 Although several hundredmutations in human COL4A3 through A5 genes have been describedin detail, exactly how these contribute to the glomerular pathogenesisof Alport disease remains unclear. Among other possibilities,a network of collagen 1, 2 (IV), which is present normally indeveloping GBM but removed during maturation,4 has been shownto persist in Alport glomeruli, presumably because of an absenceof collagen 3, 4, 5 (IV).5 The 1, 2 (IV) network has differentstructural properties than 3, 4, 5 (IV) and may confer differentbehavioral signals to the adherent glomerular endothelial cellsand podocytes as well. In addition, the 1, 2 (IV) network seemsto be more susceptible to proteolysis, which over time couldresult in the GBM multilamination commonly seen in Alport kidneys.5Along these lines, recent studies have shown that human andmouse Alport glomeruli express unusually high amounts of a potentprotease, macrophage metalloelastase (MMP-12), and treatmentof Alport mice with a specific inhibitor of this enzyme improvesGBM morphology and glomerular barrier function.6
In addition to the retention of collagen 1, 2 (IV) in AlportGBM, ectopic deposition of laminins has been documented in patientswith Alport and in dog and mouse models of the disease.7 Specifically,abnormal accumulations of laminin 1, 2, and 1 chains have beenreported in Alport GBM,7 whereas normal GBM contains only laminin521 (LM-521) heterotrimers.8 Previously, we showed in 4-wk-oldAlport mice that both glomerular endothelial cells and podocytesare origins for laminin 1 and 1,9 which normally are made bythese cells only during the earliest stages of glomerular development.10Moreover, we localized these proteins by immunoelectron microscopyspecifically to areas of GBM thickening that underlie effacedfoot processes, suggesting that ectopic laminin deposition contributedto or possibly caused the GBM and podocyte deformations.10 Toexamine the laminin dysregulation that takes place during thecourse of Alport disease more thoroughly, here we quantifiedlaminin abundance within normal and Alport GBM using confocalmicroscopy and quantified laminin mRNA by reverse transcriptase–PCR(RT-PCR). In addition, to test the barrier properties of AlportGBM, we injected the ultrastructural tracer ferritin into normaland Alport mice and carried out electron microscopy. Our findingsshow that laminin 5 mRNA and protein were significantly upregulatedin Alport glomeruli. In addition, Alport GBM was significantlymore permeable to ferritin than wild-type GBM, particularlyin areas of thickening and foot process effacement but alsoin areas that were ultrastructurally normal.
Quantification of GBM Laminin Dysregulation
When cryostat sections from 4-wk-old wild-type mouse kidneyswere doubly immunolabeled for laminin 1 and 5 chains and viewedby confocal microscopy, the immunolabels rarely overlapped inglomeruli (Figure 1, a, c, and e). Laminin 1 chain (fluorescein)was concentrated within mesangial areas, whereas laminin 5 (rhodamine)localized specifically to peripheral loop GBM. By contrast,there was extensive label overlap in glomeruli from Alport littermates(Figure 1, b, d, and f), and many peripheral loop GBM containedyellow/orange nodules indicating co-localization of both laminin1 and 5 chains to the same GBM sites (Figure 1f). In addition,the overall immunofluorescence intensities for laminin 5 appearedconsiderably brighter for Alport glomeruli (Figure 1d) whencompared with controls (Figure 1c).
Figure 1. Immunofluorescence micrographs showing distribution of laminin 1 and 5 chains in wild-type (WT; a, c, and e) and Alport glomeruli (b, d, and f). Merged image (f) shows co-localization of laminin 1 and 5 chains to bright yellow nodules of peripheral capillary loop glomerular basement membranes (GBM) of Alport glomeruli (arrows). No co-localization is seen in wild-type glomeruli (e).
For quantification of total glomerular fluorescence intensities,red and green confocal images were each converted to grayscale,and examples for how images were masked and glomerular fluorescencesignals collected are shown in Figure 2. Fluorescence pixeldensities were measured only in areas included within the circularapertures. Despite the appearance of a somewhat brighter signalfor laminin 1 in Alport glomeruli in selected images, no statisticallysignificant differences were observed for total glomerular fluorescenceintensities when compared with normal controls (Figure 3). Conversely,Alport glomeruli displayed an almost six-fold increase in totalimmunofluorescence intensity for laminin 5 chain (Figure 3).
Figure 2. Grayscale images of Figure 1, a and b, showing positioning of measuring aperture (blue circle) and GBM bisecting tool (pink lines) used to quantify confocal immunofluorescence signal intensities.
Figure 3. Histograms showing confocal immunofluorescence intensities for laminin 1 and 5 chains in WT and Alport glomeruli. Highly significant increases were seen for laminin 5 in sections through whole glomeruli and for both laminin 1 and 5 across segments of GBM. n = 17 glomeruli for WT and 18 for Alport; 18 GBM segment intensities for both genotypes.
To compare the relative abundance of laminin chains withinthe GBM nodules characteristic of Alport glomeruli, we alsoquantified immunofluorescence intensities across specific, bisectedGBM segments. This was also accomplished using Scion softwarethat integrated fluorescence intensities across selected widthsof glomerular capillaries (shown as bright pink bars in Figure 2).In these cases, Alport GBM segments contained approximatelyfive-fold more immunolabel for laminin 1 and four-fold morelabel for laminin 5 than seen in bisected GBM from normal littermates(Figure 3).
Conceivably, the increased immunolabeling for laminin 5 seenthroughout Alport GBM could have been due to (1) GBM disorganizationand an accompanying increased epitope exposure to anti-5 or(2) actual increases in laminin 5 protein deposition. To addressthe second possibility, we isolated glomeruli from 4-wk-oldAlport and wild-type mice and analyzed glomerular mRNA by quantitativeRT-PCR. Figure 4a shows a representative preparation obtainedusing the Dynabead isolation technique, which provides a fractiongreatly enriched for glomeruli and with few tubular segments.Our quantitative RT-PCR results showed approximately 50% morelaminin 5 mRNA in Alport glomeruli than in wild-type controls(Figure 4b). Taken together with the confocal quantification,our findings document increased biosynthesis of laminin 5 inAlport glomeruli, and we attribute this to a compensatory responseto the absence of the collagen 3, 4, 5 (IV) network.
Figure 4. (a) Whole-mount view of representative preparation of isolated glomeruli (arrows). Small dots are magnetic beads. (b) Quantitative reverse transcriptase–PCR analysis, showing significant upregulation of laminin 5 mRNA in Alport glomeruli (n = 3 WT and 3 Alport samples).
Abnormal Ferritin Permeability in Alport GBM
For examination of GBM permeability properties, ferritin wasinjected intravenously into 2-wk-old normal and Alport mice,and kidneys were fixed for electron microscopy 40 min afterinjection. As shown in Figure 5a, discrete ferritin particleswere readily observed within GBM of normal mice. However, thesewere found predominantly within the subendothelial layer, andcomparatively few ferritin particles penetrated the full widthof the GBM to the level of the podocytes. For quantificationof ferritin penetrability, GBM was digitally dissected freeof adherent endothelial cells and podocytes, and ferritin particleswere pseudocolored (Figure 5b). GBM were then further dissectedinto their separate subendothelial and subepithelial halves(Figure 5c), and the numbers of ferritin particles within eachlayer were then scored. In all, 1326 ferritin particles wereseen throughout 125 linear µm of GBM of wild-type/heterozygousmice. Our results showed that the outer subepithelial half ofwild-type GBM contained approximately one third the number ofparticles found within the inner, subendothelial half (Figures 5and 6).
Figure 5. Electron micrographs of glomerular capillary wall (a) from 2-wk-old mouse that received an intravenous injection of ferritin. Insert in A shows ferritin (small electron-dense dots) within the GBM. (b) The same length of GBM shown in A digitally microdissected and with ferritin particles pseudocolored pink. (c) GBM further microdissected into subendothelial (subendo) and subepithelial (subep) halves. Insert in (c) shows that relatively few ferritin particles penetrate the subepithelial layer of WT GBM.
Figure 6. Histogram showing numbers of ferritin particles/µm2 GBM in 2-wk-old WT and Alport mice. In Alport mice, significantly more ferritin permeated the total width of GBM, subepithelial halves, and subepithelial segments with normal (nl) GBM and podocyte architecture as well as abnormal (ab) segments.
Undertaking the same approach with Alport mice showed more ferritinwithin the GBM overall (2418 particles were found in 127 linearµm of GBM) and within the subepithelial half in particular(Figures 6 and 7). This was borne out by statistical comparisonsof ferritin counts between Alport and normal GBM, which revealedthat significantly more ferritin was found within Alport GBM,especially subepithelial layers (Figure 6). Average ferritin/µm2within subendothelial layers was also somewhat higher in Alportthan normal GBM, but this apparent difference was not statisticallysignificant (Figure 6).
Figure 7. Glomerular capillary wall from 2-wk-old Alport mice that received ferritin intravenously. Ferritin can be visualized throughout the GBM, including areas of subepithelial projections (*) beneath effaced foot processes. Insert shows higher magnification view of (*). Region to the left of segment marked (a) illustrates abnormal GBM and podocyte architecture, whereas region to the right marked (b) contains normal GBM morphology, podocyte foot process registration (arrowheads), and epithelial slit diaphragms (arrows).
Glomerular capillary walls in 2-wk-old Alport mice containedlarge areas that appear morphologically normal. Specifically,GBM architecture, podocyte foot process registration, and slitdiaphragm formation all were indistinguishable from those ofwild-type mice (Figure 7). Intermingled with these normal areaswere smaller regions containing pronounced GBM deformationsand podocyte abnormalities (Figure 7). We therefore evaluatedferritin penetrability in morphologically normal and abnormalzones of these 2-wk-old Alport glomeruli (Figures 7 and 8).This analysis showed that the outer, subepithelial half withinmorphologically normal areas of Alport glomeruli was significantlymore permeable to ferritin than in wild-type controls (Figure 6).However, maximal permeabilities occurred in the morphologicallyabnormal regions containing subepithelial GBM humps and footprocess effacement (Figures 6 through 8).
Figure 8. Microdissected GBM from regions (a) and (b) from Figure 7, with ferritin pseudocolored pink.
In addition to greatly increased penetration of ferritin into2-wk-old Alport GBM, ferritin was detected in intracellularvesicles within podocytes (Figure 9). At 4 and 6 wk of age,Alport glomeruli progressively became more severely damaged,GBM was greatly thickened and often multilaminated, and podocytesaccumulated large amounts of ferritin within intracellular vesiclesand lysosomes (Figure 10). Ferritin was also readily detectedwithin apical intracellular vesicles of proximal tubule epithelialcells (Figure 11). Ferritin was never detected intracellularlywithin podocytes or tubular epithelial cells in 2- or 4-wk-oldheterozygous or wild-type kidneys.
Figure 9. Glomerular capillary wall from 2-wk-old Alport mouse. Ferritin fully penetrates the GBM and is also seen in intracellular vesicles within podocytes (arrow). Note unusually broad foot processes (fp). En, endothelium.
Figure 10. Glomerulus from 6-wk-old Alport mouse. Podocytes (Po) are severely damaged, and only broadly effaced foot processes (fp) are seen at this age. Numerous ferritin particles are present within intracellular vesicles (*) and lysosomes (arrows).
Figure 11. Apical region of proximal convoluted tubule epithelium from 6-wk-old Alport mouse. Ferritin is seen in apical endocytic vesicles (arrows) at the bases of microvilli (mv), as well as within lysosomes (*).
Earlier studies that were conducted in Alport patients, as wellas in dog and mouse models of the disease, showed laminin dysregulationin GBM of all three species.7 Our previous findings in miceshowed that laminin 1 chain, which is normally restricted tothe mesangium in maturing glomeruli, also immunolocalizes specificallyto the peripheral loop irregularities characteristic of AlportGBM.9 Here, we sought to quantify the magnitude of laminin expressionin Alport mouse glomeruli. Our quantitative immunofluorescenceresults showed no differences in overall glomerular labelingfor laminin 1, but significantly more laminin 1 immunolocalizedto discrete areas of GBM thickening in capillary loops. In addition,considerably more immunolabeling for laminin 5 was found throughoutAlport glomeruli than in wild-type, and especially in the GBMirregularities where laminin 1 was abnormally deposited as well.Quantitative PCR similarly showed an upregulation of laminin5 mRNA, indicating that the increased total glomerular immunofluorescencewas most likely a consequence of activation of Lama5 gene transcription.We also carried out electron microscopic studies using the ultrastructuraltracer ferritin to compare regional permeability propertiesof the developing glomerular capillary wall in Alport mice andnormal littermates. Our ferritin injection results showed thatthe Alport GBM was abnormally permeable and that maximal permeabilitiesoccurred specifically in capillary wall segments containingGBM thickenings and effaced foot processes of podocytes. Nevertheless,significant ferritin permeabilities were also observed in Alportmice in areas where GBM and podocyte foot process ultrastructureappeared entirely normal, signifying that the Alport GBM wasinherently defective in its barrier properties. Taken togetherwith the loss of the collagen 3, 4, 5 (IV) network, our findingsindicate that dysregulation of laminin synthesis and distributionin Alport glomeruli lead to the striking structural and functionaldeficits seen in this disorder.
Mechanisms regulating the synthesis of different laminins andgoverning their microanatomic distribution patterns are poorlyunderstood. Nevertheless, the appearance and disappearance ofvarious laminin isoforms in GBM of developing glomeruli havebeen well described.8 First, basement membranes in the vascularcleft region of comma- and S-shaped nephrons contain LM-111and -411. As glomeruli progress into the capillary loop stage,these isoforms are quickly eliminated and replaced by LM-521,which becomes the only laminin present in the GBM of maturingstage glomeruli and thereafter. Immunoelectron microscopic localizationexperiments have shown that both glomerular endothelial cellsand podocytes synthesize LM-111 and -521 at the appropriatestages of development, but events regulating the isoform switchesat the gene and protein level are unknown.10
Our previous immunofluorescence evidence showed that the disappearanceof LM-111 occurs normally in early glomeruli of Alport kidneys.9However, LM-111 reappears during early capillary loop stages,and immunoelectron microscopy documented that synthesis resumesin both glomerular endothelial cells and podocytes at this stage.Although our findings presented here showed no differences intotal glomerular immunofluorescence intensities for laminin1, there were marked increases in laminin 1 immunolabeling inthe irregular GBM thickenings that typify Alport glomeruli,particularly where immunolabeling also indicated unusually largeamounts of laminin 5. In addition, there were significant increasesin total glomerular immunolabeling for laminin 5, and quantitativeRT-PCR experiments showed marked increases in laminin 5 mRNAin Alport glomeruli as well. This increased tempo in laminin5 synthesis may therefore represent a cellular response to theabsence of the collagen 3, 4, 5 (IV) network that typifies theAlport GBM.
Although exactly how the Alport condition affects glomerularlaminin gene transcription is not known, likely mediators probablyinvolve cell–matrix adhesion receptors, especially theintegrins. The most abundant integrins expressed within theglomerulus include the 11, 21, and 31 heterodimers, and theyeach immunolocalize to restricted compartments: 11 integrinis found chiefly on mesangial cells, 21 occurs largely on endothelialcells, and 31 is highly expressed on basal membranes of podocytes.12Specifically how these proteins direct glomerular cell behaviorsand alter gene transcription patterns in vivo remains incompletelydefined, but studies with knockout mice have been informative.For example, when compared with Alport mice alone, double-knockoutmice null at both the collagen 3 (IV) (Alport mice) and integrin1 alleles have delayed onset and slower progression of disease,improved glomerular morphology, and a reduction of GBM laminin2 and 1, which are also abnormally expressed in Alport GBM.12Because 11 integrin is believed to be expressed chiefly if notexclusively by mesangial cells, how these proteins affect biosyntheticpathways in capillary endothelial cells and podocytes is unclear.Perhaps paracrine signaling from the mesangium somehow mediatesmatrix gene expression in endothelial cells and/or podocytes,as suggested previously.12 However, mesangial cells are embeddedin a matrix rich in collagen 1, 2 (IV) and generally not indirect contact with the GBM per se, so how an absence of 3,4, 5 (IV) in GBM might initiate mesangial cell signaling ismysterious. Another candidate is 31 integrin, which is a provenadhesion receptor for laminins and type IV collagen and locatedin the basal plasma membrane of podocytes.11,13 When integrin3 is deleted in mice, glomerular capillary branching is decreased,and this is accompanied by widespread disorganization and fragmentationof the GBM and a failure of podocyte foot process formation.13Although these changes were originally thought to be due mainlyto a loss of cell–matrix adhesion in mutant podocytes,other studies have now shown that 31 integrin can also associatewith the tetraspanin CD151 cell–cell adhesion complexthat, among other things, can indirectly regulate gene transcriptionas well.14 Clearly, much additional work is required to understandways in which aberrant GBM assembly can affect glomerular cellbehavior, including transcription. Among other things, perhapsfuture studies that carefully inventory the abundance and distributionof the various integrins in Alport glomeruli will be useful.In addition, experiments that specifically examine transcriptionalregulation of the laminin genes may shed light on mechanismsleading to glomerular fibrotic disorders, including Alport disease.
To evaluate permeability properties of the Alport GBM, we injectedferritin into mice, which is an approximately 480-kD proteincomplex and the main intracellular iron storage protein, makingit useful as a quantifiable, electron-dense ultrastructuraltracer. Although ferritin has been used as a glomerular permeabilityprobe in a number of studies carried out in rats,15,16 it hasbeen used infrequently in mice. Recently, however, GBM of 1.5-to 2-wk-old laminin 2 null mice were shown to be abnormallypermeable to ferritin, even in areas where foot process andepithelial slit diaphragm morphology seem intact.17 When weinjected ferritin into 2-wk-old wild-type mice, ferritin wasreadily distinguished within the GBM, although most of it wasrestricted to the inner, subendothelial half. Nevertheless,quantities of ferritin fully penetrated the GBM up to the baseof podocyte foot processes, probably reflecting an incompletelymature filtration barrier in mice at this age. In contrast,when injected into 2-wk-old Alport littermates, significantlymore ferritin permeated the full width of the GBM than in wild-types.Maximal permeabilities were seen in areas of GBM thickeningbeneath abnormally broad foot processes, which are exactly thesame sites where unusually large amounts of laminin 1 and 5were deposited, indicating that the aberrantly distributed laminincontributed to the permeability defect. Importantly, significantlymore ferritin also penetrated the subepithelial half of AlportGBM in areas where GBM architecture and foot process registrationappeared entirely normal. These results therefore indicate thatthe Alport mouse GBM, like that of laminin 2 mutant mice, isinherently and abnormally permeable to circulating macromolecules,and these defects are apparently not due to errors in podocytefoot process formation and/or slit diaphragm integrity.
In addition to the GBM, our ferritin injection studies resultedin abundant localization of tracer intracellularly within vesiclesand lysosomes of Alport podocytes and within intracellular vesiclesin the apical cytoplasm of proximal tubule epithelial cells.Whether ferritin observed in the tubular epithelium breachedthe Alport glomerular filtration barrier by transcytosis throughpodocytes or by extracellular passage through filtration slitpores is unclear, and we are exploring both possibilities.
Animals
Mice that genetically lacked the 3(IV) collagen chain (Alportmice)18 were purchased from Jackson Laboratory (Jax mouse strain129-Col4a3tm1Dec; Bar Harbor, ME). We maintained our colonyby interbreeding heterozygotes, and genotyping was carried outby PCR. Heterozygous and wild-type littermates served as controls.All procedures with animals were approved by the InstitutionalAnimal Care and Use Committee to ensure compliance with theHealth Research Extension Act, Animal Welfare Act, and the PublicHealth Service Policy on Humane Care and Use of Laboratory Animals.
Confocal Microscopy and Image Analysis
Deeply anesthetized Alport and control littermate mice underwentnephrectomies at 4 wk of age, and kidneys were snap-frozen inisopentane chilled in dry ice–acetone bath. Cryostat sections,approximately 10 µm thick, were fixed for 10 min in 100%methanol and immunolabeled with chain-specific rat anti-mouselaminin 1 and rabbit anti-laminin 5 chain antibodies that wereprepared and characterized as described previously.19–21Fluorescein- and rhodamine-conjugated secondary antibodies wereobtained from ICN Biomedicals (Costa Mesa, CA). All slides thatunderwent confocal fluorescence quantification were immunolabeledwith the same mixtures of antibodies on the same day and coverslippedovernight with ProLong anti-fade mounting media (Molecular Probes-Invitrogen,Carlsbad, CA). Slides were then viewed on a Zeiss LSM-510 scanninglaser confocal microscope (Thornwood, NY), and Z-series imageswere captured at 0.2-µm intervals. All images were capturedon the same day using constant laser intensity, confocal apertures,gain, and black-level parameters. Raw confocal images takenfrom the mid-regions of glomeruli on green (immunofluorescencefor laminin 1 chain) and red channels (laminin 5 chain) wereimported into Adobe Photoshop (San Jose, CA) and converted tograyscale, and output levels were set to a range of 0 to 250.Images were masked digitally with an opaque frame containinga fixed circular aperture with a diameter measuring 60 µm(slightly smaller than a mature glomerulus of a 2-wk-old mouse).Glomerular fields that did not fully occupy and extend beyondthe aperture margins were not analyzed. Total pixel densitieswithin only the unmasked (glomerular) areas were then measuredwith Scion software (http://www.scioncorp.com) as a relativemeasure for glomerular immunofluorescence intensities. Meanintensities were statistically compared using a t test. In addition,Scion software was used to bisect selected GBM segments, andfluorescence intensities across these segments were measuredand compared statistically.
Glomerular Isolation and Real-Time PCR
Glomeruli were isolated from three 4-wk-old Alport and threewild-type mice (six separate glomerular preparations) usingthe magnetic bead method.22 Briefly, mice were anesthetizedwith 1 mg/10 g body wt ketamine and 0.15 mg/10 g body wt xylazine.Blood was washed from the mice by cardiac perfusion with HBSSfollowed by perfusion of magnetic beads (Dynabead M-450, Invitrogen;2 x 106 beads/ml in HBSS). Kidneys were removed and minced onice, followed by digestion at 37°C with 1 mg/ml collagenaseand 100 U/ml DNAse I for 30 min. Digested kidneys were filteredtwice with 100 µ Falcon cell strainers, and tissue waspelleted by gentle centrifugation (200 x g, 5 min). Glomerulicontaining Dynabeads were collected using a magnetic particleconcentrator (Dynal, Oslo, Norway), washed, and resuspendedin HBSS three times. The final glomerular enriched fractionwas pelleted, frozen immediately in liquid nitrogen, and storedat –80°C.
Total RNA was isolated from glomeruli using the RNAqueous-Microspin kit (Ambion, Austin, TX). Samples were diluted to 10 ng/mland amplified using QuantiTect SYBR Green RT-PCR kit (Qiagen,Valencia, CA) with the following primers: Laminin 5 forward5'-ACC CAA GGA CCC ACC TGT AG-3' and reverse 5'-TCA TGT GTGCGT AGC CTC TC-3'23 and cyclophilin forward 5'-CAG ACG CCA CTGTCG CTT T-3' and reverse 5'-TGT CTT TGG AAC TTT GTC TGC AA-3'.24Real-time PCR was performed using an iCycler (Bio-Rad, Hercules,CA). The primer sets were validated for efficiency by the comparativeCt method using standard curve analysis.25 PCR products wereanalyzed on agarose gels to confirm size, and melt curve analysiswas performed to reveal a single amplified product. RT-PCR productfor cyclophilin was verified by sequencing using the KUMC BiotechnologySupport Facility.
Ferritin Permeabilities
Horse spleen ferritin (32 mg/ml in 0.15 M NaCl) was injectedintravenously through the saphenous vein into 2-wk-old (100µl/mouse; three Alport, one heterozygous, two wild-type),4-wk-old (150 µl; two Alport, two wild-type), and 6-wk-old(200 µl; two Alport) anesthetized mice. Forty minutesafter injection, kidneys were fixed in situ by clamping thehilus and simultaneously injecting Karnovsky fixative into thecortex. After 5 min, kidneys were removed, and cortices slicedinto 1-mm cubes and fixed for an additional 90 min on ice. Tissuewas then postfixed in ferrocyanide-reduced osmium26 and routinelyembedded in Polybed 812. Ultrathin sections were stained with1:50 bismuth subnitrate for 5 min,27 and multiple glomerularcapillary loops from a minimum of 11 separate glomeruli (fromtwo wild-type mice) and 12 separate glomeruli (from two Alportmice) were examined. Electron micrographic prints of cross-sectionalplanes through capillary walls were scanned into Photoshop;tangential sections were not evaluated further. Ferritin particleson the scans, recognized as discrete electron-dense spots, werepseudocolored, and lengths of GBM were dissected digitally intosubendothelial and subepithelial halves. The numbers of ferritinparticles/µm2 within the separate subendothelial and subepithelialGBM layers were then counted using Scion software, and averageswere compared statistically.
Funds came from National Institutes of Health grants DK052483and DK065123. Confocal images were acquired at the KUMC ConfocalImaging Facility, supported in part by the Kansas IDeA Networkof Biomedical Research Excellence (RR016475).
Portions of this work were presented previously in abstractform (J Am Soc Nephrol 15: 418A, 2004 and Matrix Biol 25: S84–S85,2006).
We thank Dr. Elizabeth Petroske for technical assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
3, 
1 chains have been reported in Alport GBM,7 whereas normal GBM contains only laminin 
1 (LM-521) heterotrimers.8 Previously, we showed in 4-wk-old Alport mice that both glomerular endothelial cells and podocytes are origins for laminin 
