Type I collagen glomerulopathy: postnatal collagen deposition follows glomerular maturation

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1 & 27 International Soiety of Nephrology original artile Type I ollagen glomerulopathy: postnatal ollagen deposition follows glomerular maturation AC Brodeur 1,2, DA Wirth 1, CL Franklin 3, LW Reneker 4, JH Miner 5 and CL Phillips 1,2 1 Department of Biohemistry, University of Missouri, Columbia, Missouri, USA; 2 Department of Child Health, University of Missouri, Columbia, Missouri, USA; 3 Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA; 4 Department of Ophthalmology, University of Missouri, Columbia, Missouri, USA and 5 Renal Division, Washington University Shool of Mediine, St Louis, Missouri, USA In hroni renal disease, the progressive aumulation of ollagen and other extraellular matrix proteins in the mesangium results in fibrosis, glomeruloslerosis, and eventual renal failure. Mie defiient in proa2(i) ollagen are not only a model of osteogenesis imperfeta but also aumulate fibrillar homotrimeri type I ollagen in the mesangium. This aumulation spreads to the subendothelial spae in the peripheral apillary loops. Piosirius red staining of kidney setions demonstrates that in omparison to wild-type mie, Col1a2-defiient homozygous and heterozygous mie exhibit abnormal glomerular ollagen deposition in a gene dosage-dependent manner. The glomerulopathy initiates during the first postnatal week, appears progressive following the pattern of glomerular maturation and results in albuminuria in severely affeted animals. In situ hybridization revealed no gross differenes in steady-state proa1(i) and proa2(i) ollagen mrna levels among the three genotypes. Quantitative reverse transriptase-polymerase hain reation, however, using whole kidney setions showed a twofold inrease in steady-state proa1(i) ollagen mrna in 1-month homozygous Col1a2-defiient animals ompared with wild-type and heterozygous animals. We suggest that glomerular ollagen deposition seen in the osteogenesis imperfeta model mie is, in part, owing to pretranslational mehanisms and may represent an over ompensation of wound healing. Kidney International (27) 71, doi:1.138/sj.ki.52173; published online 14 Marh 27 KEYWORDS: glomeruloslerosis; podoyte; glomerular development; albuminuria; type I ollagen Correspondene: CL Phillips, Department of Biohemistry, University of Missouri-Columbia, M743 Medial Siene Building, Columbia, Missouri 65212, USA. phillipsl@missouri.edu Reeived 2 September 26; revised 18 Deember 26; aepted 17 January 27; published online 14 Marh 27 A novel type I ollagen glomerulopathy was identified in the Col1a2-defiient mouse model, haraterized by the deposition of homotrimeri type I ollagen in the renal mesangium. 1 Under normal physiologi onditions, very little type I ollagen is in the glomerulus. 2,3 In hroni renal disease, progressive aumulation of ollagen and other extraellular matrix (ECM) omponents in the mesangium results in fibrosis, glomeruloslerosis, and renal failure. 4 6 The primary event leading to development of the glomerulopathy in Col1a2-defiient mie appears to be deposition of homotrimeri type I ollagen in the mesangial matrix, similar to the progressive glomerular deline following injury and may represent overompensation of the glomerular wound healing response. 7 9 To haraterize further the type I ollagen glomerulopathy, we defined the pattern of disease initiation and progression, and began investigating the mehanisms responsible for glomerular ollagen deposition in Col1a2-defiient mie. The predominant isotype of type I ollagen is a heterotrimer omposed of two proa1(i) ollagen hains and one similar, but genetially distint, proa2(i) ollagen hain [a1(i) 2 a2(i)]. 1,11 The homotrimeri type I ollagen isotype ontaining three proa1(i) ollagen hains, [a1(i) 3 ] is a minor isotype, whose role is not well understood. 1,11 Homotrimer is found embryonially 2,12 in small amounts in skin, 13 in ertain tumors and ultured aner ell lines, 12,14,15 and also during wound healing. 7 Cultured mesangial ells produe homotrimeri type I ollagen, further suggesting that homotrimer plays a role in wound healing. 7,8 The Col1a2-defiient mouse model, otherwise known as the oim mouse (osteogenesis imperfeta model) is homozygous for a spontaneous nuleotide deletion in the Col1a2 gene, resulting in a frameshift altering the arboxy propeptide of the proa2(i) ollagen hain. Although the arboxy propeptide is not present in mature type I ollagen, it is responsible for assoiation of the a2(i) hain with the a1(i) hains during assembly of the triple helix The glomerulus, a network of apillaries, serves as the filtration unit of the kidney, with mesangial ells omposing 3 4% of the ell population, funtioning to reate and integrate a strutural saffold for the apillary network. 2,8 Kidney International (27) 71,

2 o r i g i n a l a r t i l e AC Brodeur et al.: Type I ollagen glomerulopathy Mesangial ells serete ECM proteins to provide a framework for the saffold. Glomeruli form in a entrifugal pattern around the renal medulla. At birth, mouse kidneys have numerous glomeruli, yet only a fration are funtional. Between birth and 21 days of age, glomeruli gain funtion regionally, 19 with initial funtioning glomeruli in the juxtamedullary region of the deep ortex. 2,21 As the mouse develops, there is stepwise indution of new nephrons and glomeruli gain funtional ativity, progressing until full maturation when glomeruli in the outer ortex assume the majority of glomerular funtion A similar progression of glomerular maturation ours in other mammalian speies, inluding humans. 21,23,24 Several studies using animal models demonstrate that glomerular ollagen deposition an result from either inreased pretranslational expression of type I ollagen (transriptional upregulation) or dereased matrix metalloproteinase (MMPs) expression and/or ativity, or both In this study, we sought to determine whether the ollagen glomerulopathy in the Col1a2-defiient model was progressive with pre- or postnatal initiation and whether pretranslational upregulation of type I ollagen expression was involved in glomerular ollagen deposition. Through histologi examination, urine analyses, and quantitative reverse transriptase-polymerase hain reation (RT-PCR), we determined that the type I ollagen glomerulopathy in Col1a2-defiient mie initiates postnatally, progresses with age and glomerular maturation, is assoiated with a gene dose effet, impairs renal funtion, and is, in part, owing to inreased steady-state proa1(i) ollagen mrna levels. RESULTS Heterozygous mie demonstrate ollagen glomerulopathy Pirosirius red-stained setions of Col1a2-defiient, heterozygous, and wild-type mie were examined by light mirosopy to determine lesion sore. Heterozygous mie exhibited ollagen deposition in their glomeruli harateristi of the type I ollagen glomerulopathy identified in the Col1a2-defiient mie, though their lesions were less severe (Figure 1). Glomerulopathy initiation We determined the lesion sore of heterozygous and Col1a2- defiient mouse kidneys at 1 day, 1 week, 2 weeks, and 1 month of age to determine whether initiation of glomerular fibrillar ollagen deposition ourred postnatally. Figure 2 highlights the postnatal initiation of ollagen deposition within the renal glomeruli. Glomeruli of 1-day-old mie lak fibrillar ollagen deposition aross all genotypes. However, by 1 week of age, fibrillar ollagen deposition was present in Col1a2-defiient and heterozygous mie. By 1 month of age, Col1a2-defiient mie exhibited lesions that ranged from mild to severe, whereas heterozygous mie had mild to moderate lesions. Interestingly, the younger Col1a2-defiient mie had glomerular ollagen deposition in the juxtamedullary region in ontrast to older mie, where lesions were throughout the ortex. Morphometry To investigate further disease progression, we developed a morphometry-mapping system to examine severity and loalization of individual lesions. Morphometri analyses (Figure 3, Supplementary Material) onfirmed that the type I ollagen glomerulopathy is progressive and exhibits a gene dose effet. At 2 weeks of age, 55% of Col1a2-defiient glomeruli were affeted, whih inreased to 95% by 1 month of age. In omparison, only 8% of heterozygous glomeruli were affeted at 2 weeks of age inreasing to 53% by 1 month. Polynomial orthogonal ontrast analysis of the morphometri data onfirmed a linear gene dose response. Differenes also existed in lesion severity and perent of glomeruli affeted between the ortial and juxtamedullary regions within individual kidneys. The morphometry sore in the juxtamedullary region was signifiantly greater than the ortial region in Col1a2-defiient mie at 2 weeks and 1 month of age (Figure 4a). The mean juxtamedullary morphometry sore in 2-week Col1a2-defiient animals was.94, whereas the 1-month juxtamedullary morphometry sore was 2.1, demonstrating disease progression. Similar findings were true of perent glomeruli affeted (Figure 4b). At 2 weeks of age, 73% of juxtamedullary glomeruli were affeted as ompared with 36% of ortial glomeruli in Col1a2-defiient mie. In ontrast, by 1 month of age 97% of juxtamedullary and 93% of ortial glomeruli were affeted. A similar pattern exists in heterozygous mie, though the shift in affeted glomeruli from predominantly juxtamedullary to both juxtamedullary and ortial involvement was not as dramati. These data suggest a entrifugal pattern of a b Figure 1 Deposition of type I ollagen in heterozygous and Col1a2-defiient glomeruli. Pirosirius red-stained setions of (a) wild-type (lesion sore ), (b) heterozygous (lesion sore 1), and () Col1a2-defiient (lesion sore 4) kidneys from 1-month-old mie. Arrows indiate glomeruli. Asterisks denote affeted (Pirosirius red þ ) glomeruli. 986 Kidney International (27) 71,

3 AC Brodeur et al.: Type I ollagen glomerulopathy o r i g i n a l a r t i l e 1 day 2 weeks 1 month a b +/+ d e f +/ g h i / Figure 2 Initiation of type I ollagen deposition in glomeruli ours postnatally. Pirosirius red stain of (a, d, and g) 1-day-old, (b, e, and h) 2-week-old, and (, f, and i) 1-month-old mie. (a ) Wild-type ( þ / þ ) mie do not demonstrate ollagen deposition and have lesion sores of. (d f) Heterozygous ( þ / ) mie show evidene of disease at 2 weeks (inset: enlargement of indiated glomeruli (e)), demonstrating lesion sores of at 1 day, 1 at 2 weeks, and 1 at 1 month. (g i) Col1a2-defiient ( / ) mie also show evidene of deposition at 2 weeks with a lesion sore of at 1 day, 1 at 2 weeks, and 4 at 1 month. Arrows indiate glomeruli. Asterisks denote affeted (Pirosirius red þ ) glomeruli. % glomeruli affeted per field / +/ / +/ 2 weeks 1 month Figure 3 The type I ollagen glomerulopathy in heterozygous mie demonstrate a gene dose effet (Po.1) that is progressive with age (Po.1). The perent of affeted glomeruli per field is signifiantly greater in Col1a2-defiient ( / ) than heterozygous ( þ / ) kidneys at both 2 weeks and 1 month of age. Signifiant differenes in the perent of glomeruli affeted between kidneys examined at 2 weeks and 1 month of age is also shown irregardless of genotype. glomerular ollagen deposition as the animal ages, with disease initiation ourring primarily in the juxtamedullary region and progressing to involve both the juxtamedullary and ortial regions of the kidney. Lesion development oinides with glomerular maturation, whih progresses from the juxtamedullary region outward toward the ortex These data taken together onfirm that initiation of the type I ollagen glomerulopathy ours postnatally before 1 week of age and that initiation of disease oinides with the maturation of glomeruli into funtional filtration units. 22 Subendothelial type I ollagen aumulation Ultrastrutural examination of glomerular ollagen deposition in wild-type and severely affeted Col1a2-defiient (lesion sore G4) kidney setions revealed that the fibrillar ollagen was extraellular, aumulating in the mesangial matrix as well as between the fenestrated endothelial ells and the glomerular basement membrane within the glomeruli (Figure 5). In addition, there was podoyte foot proess effaement in areas demonstrating severe fibrillar ollagen deposition and marked separation of endothelial ells from the basement membrane. However, in all setions, the glomerular basement membrane remained intat. Albuminuria Analysis of albumin exretion in urine of wild-type and Col1a2-defiient mie demonstrated signifiant inreases in albumin exretion in Col1a2-defiient mie (Table 1). Col1a2-defiient mie had a mean albumin/reatinine ratio of mg albumin/mg reatinine, whereas wild-type had a mean ratio of.499 mg albumin/mg reatinine. Mie with higher lesion sores had greater albuminuria than those with less severe lesion sores. These data suggest that inreasing deposition of homotrimeri ollagen into the glomeruli results in impaired renal funtion. Kidney International (27) 71,

4 o r i g i n a l a r t i l e AC Brodeur et al.: Type I ollagen glomerulopathy Morphometry Sore b a % affeted per field weeks 1 month jm jm jm Heterozygous COL1A2 defiient Heterozygous 1 2 weeks 1 month % affeted per field Morphometry Sore jm COL1A2 defiient jm Heterozygous jm COL1A2 defiient jm jm Heterozygous COL1A2 defiient Figure 4 The type I ollagen glomerulopathy initiates postnatally and glomeruli sequentially beome affeted in a entrifugal pattern from the juxtamedullary (jm) to the ortial () region in a distribution onsistent with glomerular maturation and initiation of funtion. (a) Demonstrates that there is a signifiant differene between the morphometry sore in jm and glomeruli at 2 weeks and 1 month of age in Col1a2-defiient mie with more severely affeted glomeruli loated in the jm region where glomerular funtion initiates. (b) Demonstrates a signifiant differene between perent of glomeruli affeted per field between jm and glomeruli in Col1a2-defiient mie at 2 weeks of age whereas greater than 9% of jm and glomeruli per field are affeted by 1 month of age. Conversely, the heterozygous animals lak a signifiant differene at the earlier time point but demonstrate a signifiant differene in both lesion severity and perent glomeruli affeted per field between jm and glomeruli at 1 month of age. Further, the lesion severity and perent glomeruli affeted is signifiantly different between the 2 week and 1 month ages (y), onfirming the progressive nature of the glomerulopathy (Po.5, Po.5, Po.1). a p GBM b p m GBM p e m GBM Figure 5 (a) Eletron mirosopy of glomeruli from wild-type and (b and ) Col1a2-defiient mie (lesion sore 4) demonstrate that the Col1a2-defiient glomeruli exhibit deposition of extraellular fibrillar type I ollagen () into the subendothelial spae displaing the fenestrated endothelium (e) from the underlying glomerular basement membrane (b). Fibrils demonstrating ross striation of organized ollagen (b; inset) were also present. In areas of severe ollagen deposition and expansion of the subendothelial spae, podoyte (p) foot proess effaement is seen (). It is hypothesized that the type I ollagen is being produed by the mesangial (m) ell followed by deposition in the subendothelial spae. In all mie examined, the glomerular basement membrane (GBM) was intat. Wild-type provided for omparison (a). Steady-state proa1(i) and proa2(i) ollagen mrna In situ hybridization with Col1a1 and Col1a2 antisense probes to wild-type and Col1a2-defiient mouse kidneys demonstrated no differenes in temporal or spatial distribution of Col1a1 and Col1a2 mrnas (Supplementary Material). To assess quantitatively whether type I ollagen deposition in the glomerulus of Col1a2-defiient mie is assoiated with inreased steady-state proa1(i) and proa2(i) ollagen mrna levels, we evaluated Col1a2-defiient, heterozygous and wildtype mie at 1 week, 2 weeks, and 1 month of age by quantitative RT-PCR (Figure 6). A signifiant inrease in steady-state proa1(i) ollagen mrna levels was seen in 1-month old Col1a2-defiient mie as ompared with agemathed heterozygous and wild-type animals. In ontrast, a signifiant differene in steady-state proa2(i) ollagen mrna was not seen. Figure 6 also demonstrates a signifiant 988 Kidney International (27) 71,

5 AC Brodeur et al.: Type I ollagen glomerulopathy o r i g i n a l a r t i l e Table 1 Col1a2-defiient mie demonstrate albuminuria as ompared with wild-type animals Genotype n Albumin (lg)/reatinine (mg) (range) Wild-type ( ) Col1a2-defiient ( ) Lesion sore Po.1. Po.5. a RNA opy number b RNA opy number COL1A1 / +/ +/+ / +/ +/+ / +/ +/+ 1 week 2 weeks 1 month COL1A2 / +/ +/+ / +/ +/+ / +/ +/+ 1 week 2 weeks 1 month Figure 6 Quantitative RT-PCR demonstrates a signifiant inrease in (a) proa1(i) ollagen mrna expression in 1 month Col1a2-defiient ( / ) animals when ompared with age-mathed heterozygous ( þ / ) and wild-type ( þ / þ ) animals (Po.5). It also demonstrates a derease in proa(i) ollagen mrna expression with age independent of genotype (Po.1) and a signifiant differene in (b) proa2(i) ollagen mrna expression when omparing 1 month and 1 week animals for all three genotypes. derease in proa1(i) and proa2(i) ollagen mrna with inreasing age aross all three genotypes. DISCUSSION The original haraterization of the type I ollagen glomerulopathy in Col1a2-defiient mie demonstrated, by immunohistohemistry, that the fibrillar material deposited in the glomerulus was type I ollagen and suggested that heterozygous mie did not exhibit glomerular ollagen deposition. 1 However, revision of the lesion soring system to inlude the examination of kidney setions stained with Pirosirius red revealed that heterozygous mie indeed aumulate glomerular fibrillar ollagen, though to a lesser degree. Heterozygous mie, like Col1a2-defiient mie, exhibit a range of lesion sores, though the age-dependent inrease was less severe, suggesting that the loss of proa2(i) ollagen or the presene of homotrimeri type I ollagen has a gene dosage effet. It is not an unommon phenomenon for heterozygous animals to be less severely affeted than their homozygous ounterparts and has been shown to our in several disorders inluding ahondroplasia. 32,33 We also showed that the deposition of type I ollagen initiates postnatally, before 1 week of age, and that the progression follows a pattern similar to glomerular maturation. Initially, within 1 week of life, affeted glomeruli were onfined to the juxtamedullary region. However, by 1 month of age, affeted glomeruli were evident throughout the juxtamedullary and ortial regions in Col1a2-defiient mie, following the pattern for funtional maturation of glomeruli, whih is omplete by 21 days of age. 19 The glomerulopathy progressed in a entrifugal pattern, oiniding with initiation of glomerular funtion and maturation. 2,21 Further, as Col1a2-defiient mie age, inreasing amounts of fibrillar ollagen were deposited resulting in pathologial onsequenes; Col1a2-defiient mie exhibit miroalbuminuria and mie with greater lesion sores have a higher degree of miroalbuminuria. With age, Col1a2-defiient mie demonstrate inreasing severity in glomerular lesions and funtional impairment of their kidneys, onsistent with glomerular injury. Ultrastrutural analyses of Col1a2-defiient kidneys demonstrated fibrillar ollagen deposition in the subendothelial spae between the fenestrated endothelium and the basement membrane, though basement membrane appeared normal. Regions of severe deposition exhibited podoyte foot proess effaement. Podoytes funtion in ultrafiltration and establishment of the glomerular barrier to protein. Foot proess effaement and overall derease in podoyte number has been shown to result in inreased proteinuria, a hallmark of glomerular injury. 34,35 Similar findings were seen in a mouse model of glomeruloslerosis in whih the transforming growth fator (TGF)-b1 transgene under the ontrol of a murine albumin promoter expressed high plasma TGF-b1 levels as well as a subendothelial type I ollagen aumulation in the glomeruli and proteinuria in the severely affeted mie. 36,37 We hypothesize that the deposition of type I ollagen in the mesangial matrix and the subendothelial spae results in impaired podoyte/endothelial ell interations, podoyte foot proess effaement, inreased proteinuria, and is responsible for the progressive glomerular funtional deline. 38 In situ hybridization analyses of Col1a2-defiient, heterozygous, and wild-type kidneys demonstrated no temporal or Kidney International (27) 71,

6 o r i g i n a l a r t i l e AC Brodeur et al.: Type I ollagen glomerulopathy spatial differenes in proa1(i) and proa2(i) ollagen steadystate mrna levels. However, subsequent quantitative RT- PCR was performed due to insensitivity of in situ hybridization to quantitative hanges and demonstrated a signifiant inrease in proa1(i) ollagen mrna in 1-month Col1a2- defiient animals, as ompared with age-mathed heterozygous and wild-type animals. The presene of inreased steady-state proa1(i) and proa2(i) ollagen mrna levels normally at early ages may be due to developmental hanges and thus mask any differenes that might be ourring due to initiation of the glomerulopathy. Furthermore, the quantitative RT-PCR analysis was performed on whole kidney setions, and to larify whether proa1(i) and proa2(i) ollagen steady-state mrna inreases our with initiation of disease, quantitative RT-PCR analyses needs to be performed on isolated glomeruli. Under normal physiologi onditions, a balane exists between synthesis and degradation of type I ollagen and other ECM omponents. To maintain homeostasis, synthesis and degradation must remain balaned. However, in disease states, the balane is often disrupted resulting in inreased synthesis and/or dereased degradation, and thus, aumulation of ECM omponents. 39 Steady-state levels of proa1(i) ollagen mrna were inreased by twofold in 1-month-old Col1a2-defiient animals relative to age-mathed heterozygous and wild-type animals, suggesting that the aberrant aumulation is due, at least in part, to a pretranslational inrease in type I ollagen gene expression. Type I ollagen degradation is regulated by MMPs and their assoiated tissue inhibitor of metalloproteinases (TIMPs). In a rat model of proliferative glomerulonephritis glomerular type I and IV ollagen deposition is assoiated with dereased MMP-9 steady-state mrna expression without differenes in expression of TIMP-1, MMP-2, MMP-13, MT1-MMP, type I ollagen, or type IV ollagen steady-state mrna. 3 This ontrasts the ICR-derived glomerulonephritis mie (ICGN) mouse model of idiopathi nephriti syndrome that demonstrates proteinuria, hypoproteinemia, and hyperlipidemia as well as type I ollagen deposition in their glomeruli. 28,29,31 ICGN mie have dereased MMP-1, 2, and 9 ativity 28 with a orresponding derease in MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 protein. 29 Subsequent studies of ICGN mie also demonstrate inreased steady-state proa1(i) ollagen and proa1(iii) ollagen mrna levels relative to ontrol animals. 31 Therefore, deposition of ECM omponents in the ICGN mouse result from inreased pretranslational synthesis as well as dereased degradative apaity of renal MMPs. The TGF-b transgeni mie also demonstrate glomerular type I ollagen deposition assoiated with inreased steady-state a1(i) mrna levels in young mie and inreased ativity of MMP-2 and TIMP-1 in severely affeted animals indiating that in the presene of elevated TGF-b, glomerular ollagen deposition is the result of an imbalane in pretranslational synthesis and degradative apaities mediated by MMP/TIMP interations. 25 Despite inreased proa1(i) ollagen mrna levels, we annot rule out that dereased MMP enzyme expression and/or ativity may ontribute to the glomerular type I ollagen aumulation in Col1a2-defiient animals. Partiularly, as 1-month heterozygous animals also demonstrate glomerular deposition without a signifiant inrease in ollagen gene expression as measured by quantitative RT-PCR. Our findings are also onsistent with the onept that the glomerular ollagen deposition seen in the Col1a2-defiient mie may represent overompensation of the glomerular wound healing response. Wild-type rat mesangial ells when subjeted to tissue ulture produe type I ollagen of whih more than 5% is homotrimeri. We postulate that the type I ollagen aumulation in Col1a2-defiient mie reflets an injury response to the initiation of inreased glomerular apillary pressures that ours with glomerular maturation (funtional ativation). Inreased glomerular apillary pressure is known to be tightly assoiated with inreased ECM prodution, mesangial expansion, and progression of glomeruloslerosis. 4,41 Moreover, we previously demonstrated that Col1a2-defiient mie have redued biomehanial integrity of the aorta. 42,43 If the glomerular apillaries have similar dereased vasular integrity then funtional ativation of the glomeruli may indue the wound response owing to altered mehanial strain on the apillaries. Though TGF-b, onnetive tissue growth fator, and the phosphatidylinositol 3 kinase/akt-signaling pathway are impliated in ellular sensing of mehanial strain in mesangial ells, whether these fators or even mehanial strain plays a role in homotrimeri type I ollagen deposition remains to be eluidated. 41,44 In summary, we demonstrated that the type I ollagen glomerulopathy affets heterozygous animals in a dose response manner, initiates postnatally following a pattern of glomerular maturation, and is progressive resulting in impaired renal funtion. We also suggest mehanistially that the glomerulopathy is due, in part, to pretranslational upregulation of proa1(i) ollagen mrna expression and suggests that further investigation is needed to determine in vivo expression and ativity of MMPs in Col1a2-defiient mie. MATERIALS AND METHODS Animals Homozygous B6C3Fe a/a- Col1a2 oim/j (Col1a2-defiient, / ); heterozygous ( / þ ) and wild-type ( þ / þ ) mie were purhased from Jakson Laboratory (Bar Harbor, ME). Animals were housed in an AAALAC aredited animal faility, provided with water and food (Purina 58 Formulab Diet; Purina Mills In., Rihmond, IN) ad libitum, and ared for in aordane with an approved University of Missouri Animal Care and Use protool. þ / þ, þ /, and / genotypes were determined by PCR-restrition fragment-length polymorphism analysis. 45 Animals were divided into four age groups, 1 month (n ¼ 52 mie), 2 weeks (n ¼ 56), 1 week (n ¼ 55), and 1 day (n ¼ 26) of age. Animals were killed and kidneys were harvested. The right kidney was snap-frozen in liquid nitrogen and stored at 81C. The left kidney was fixed in 1% neutral-buffered formalin for 48 h. 99 Kidney International (27) 71,

7 AC Brodeur et al.: Type I ollagen glomerulopathy o r i g i n a l a r t i l e Mirosopy Formalin-fixed kidneys embedded in paraffin were setioned longitudinally (5 mm) and stained with Pirosirius red stain. Glomeruli within individual setions were examined in a blinded fashion and a glomerular lesion sore for eah individual kidney was determined. Glomerular lesion sores were determined using the following sale: G1 mild lesions; p5% of glomeruli affeted; G2 moderate lesions; p5% of glomeruli affeted; G3 moderate lesions; X5% glomeruli affeted; G4 severe lesions; X5% of glomeruli affeted. A positive orrelation was demonstrated between the amount of ollagen deposited in the glomeruli and the lesion sore assigned to the mouse. 1 Morphometry mapping Pirosirius red-stained kidney setions from 1-month- (n ¼ 1 / (lesion sore G2 4), n ¼ 1 þ / þ (lesion sore G), and n ¼ 1 þ / (lesion sore G1 4)) and 2-week-(n ¼ 1 / (lesion sore G1 3), n ¼ 1 þ / þ (lesion sore G), and n ¼ 1 þ / (lesion sore G1)) old mie were examined in a blinded fashion to assess individual glomerulus morphometry lesion distribution and severity within longitudinal setions (see Supplementary Material). Longitudinal setions were assessed using four fields of original magnifiation 2 that braketed the renal ortex, from the juxtamedullary juntion to the renal apsule. Eah field was divided into juxtamedullary and ortial halves (zones). Within eah zone, we determined number of glomeruli, number of glomeruli without ollagen deposition (assigned a morphometry sore of M ¼ ), with o25% of glomerular area oupied by ollagen (sore M ¼ 1), with 25 5% of glomerular area oupied by ollagen (sore M ¼ 2), with 5 75% of glomerular area oupied by ollagen (sore M ¼ 3), and with 75 1% of glomerular area oupied by ollagen (sore M ¼ 4). A total sore for eah animal was then determined by multiplying the number of glomeruli in eah ategory by their respetive morphometry sore, adding the sores, and dividing by the total number of glomeruli. For example, if 4 glomeruli were found in the four ortial fields examined and 2 had no ollagen deposition, 1 had morphometry sores of 2, eight had morphometry sores of 3, and two had morphometry sores of 4, the total sore was ((2 ) þ (1 2) þ (8 3) þ (2 4))/4 ¼ 1.3. Mean juxtamedullary and ortial glomerular morphometry sores, mean juxtamedullary and ortial glomerular numbers, mean number of affeted glomeruli, and the perentage of affeted glomeruli were determined for eah genotype at 2 weeks and 1 month of age. Eletron mirosopy Kidneys were harvested and fixed in 4% paraformaldehyde, 4% glutaraldehyde, and.1 M aodylate buffer and tissue proessing arried out as desribed previously. 46 Thin setions were stained with lead itrate plus uranyl aetate for transmission eletron mirosopy. 47 Reagents were purhased from Polysienes In. (Warrington, PA). Albumin to reatinine ratio To determine whether Col1a2-defiient mie exhibit miroalbuminuria, urine was olleted before killing, stored at 21C and urine albumin onentration measured using the Albuwell M kit (Exoell, Philadelphia, PA) following a 1:13 dilution of the urine sample (Animal Models of Diabeti Compliations Consortium, September, 26). Creatinine onentration was analyzed using an automated Jaffe alkaline pirate assay on a urine sample diluted 1:2. Quantitative RT-PCR Snap-frozen kidneys from 1-month- (n ¼ 6 / (lesion sore G3 4), n ¼ 6 þ / þ (lesion sore G), and n ¼ 6 þ / (lesion sore G1 4)), 2-week- (n ¼ 7 / (lesion sore G1 4), n ¼ 6 þ / þ (lesion soreg),andn ¼ 6 þ / (lesion sore G1)), and 1-week- (n ¼ 6 / (lesion sore G1 2), n ¼ 6 þ / þ (lesion sore G), and n ¼ 7 þ / (lesion sore G1)) old mie were homogenized in TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) using a TissueLyser homogenizer (QIAGEN, Valenia, CA), and total RNA isolated aording to manufaturer s protool (Invitrogen Corporation). Total RNA (5 mg) was reverse transribed with oligo(dt) primers aording to manufaturer s protool (Supersript, Invitrogen Corporation). PCR primer sequenes for Col1a1 and Col1a2 are found in Table 2. Primer sequenes for hypoxanthine guanine phosphoribosyltransferase have been previously reported. 48 Col1a1 and Col1a2 gene expression was quantified using real-time RT-PCR (LightCyler s ; Rohe Diagnosti Corporation, Basel, Switzerland) in a 2 ml PCR volume that ontained DNA (2 ng/ml),.5 mm of forward and reverse primers, 3 mm MgCl 2, SYBR & green, and QuantiTet TM SYBR & Green PCR Master Mix, whih ontains dntp mix, HotStart Taq DNA Polymerase, reation buffer I, and SYBR & green (QIAGEN). 49,5 Copy number of the desired genes was determined through generation of a standard urve using known onentrations, opies of the PCR-Blunt II-TOPO (Invitrogen Corporation) plasmid, whih ontains the amplion of interest that was previously loned. The PCR reations for Col1a1 and Col1a2 were inubated at 951C for 15 min to ativate the polymerase followed by 4 yles (15 s denaturation, 941C; 3 s annealing, 61C; 3 s extension, 721C). Fluoresene was monitored at the end of eah extension phase at 81C (5 s) and values were normalized to hypoxanthine guanine phosphoribosyltransferase levels. Table 2 Primer sequenes used for in situ hybridization and quantitative reverse transriptase-pcr analysis Primer GI number Sequene Nuleotide segment Amplion size ISH and RT-PCR COL1A1 forward AGC CTG AGT CAG CAG ATT GAG A bp COL1A1 reverse CTT GCA GTG ATA GGT GAT GTT CT ISH COL1A2 forward CTT CTT GGT GCT CCC GGT ATT CT bp COL1A2 reverse TTT TGG AGC AGC CAT CGA CTA RT-PCR COL1A2 forward TGA AGT GGG TCT TCC AGG TCT TTC bp COL1A2 reverse CAC CCT TGT TAC CGG ATT CTC CTT ISH, in situ hybridization; RT-PCR, reverse transriptase-polymerase hain reation. Kidney International (27) 71,

8 o r i g i n a l a r t i l e AC Brodeur et al.: Type I ollagen glomerulopathy Statistis Statistial analyses were performed using SAS (SAS Institute In., Cary, NC). Morphometry data were analyzed as a split plot in spae. The main plot ontains effets of gene, age, and the interations between genotype and age. The subplot ontains effets of side (ortial versus juxtamedullary) and all possible interations between side and the main plot effets. Polynomial orthogonal ontrasts were performed to test for linear and/or quadrati gene dose effet. Urine data was analyzed as a one-way ompletely randomized design analysis of variane. The RT-PCR data was analyzed as a ompletely randomized design in whih genotype and age were arranged as a 3/3 fatorial (three genotypes, three ages). Owing to heterogeneous varianes among albumin to reatinine ratios and among opy numbers, log transformations were used to stabilize the variation. Data presented are the atual mean and standard error but the differenes within genotype and lesion sore were analyzed using the transformed data sets. Mean differenes were asertained using Fisher s least signifiant differene. All results are presented as mean7s.e. Differenes were onsidered to be statistially signifiant at Po.5. ACKNOWLEDGMENTS We are grateful to Stephanie Carleton, Brent Pfeiffer, and Anna Roberts-Pilgrim for assistane in speimen olletion, Allison Howard and Dr Matt Myles for assistane in the RT-PCR analyses, Jeanette Cunningham for assistane with eletron mirosopy, Howard Wilson and Elaine Brodeur for assistane in generating the digital and fine artwork depited, Dr Charles Wiedmeyer for urine analysis, and Dr Mark Ellersiek for the statistial analysis. This work was supported by Leda J Sears Trust Foundation Grant and NIH/NIDDK, R21DK SUPPLEMENTARY MATERIAL Supplementary information is available on Kidney International website. REFERENCES 1. 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