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Nephron Clin Pract 2007;106:c82–c88 DOI: 10.1159/000101802

Alport Syndrome and Thin Basement Membrane Nephropathy

Paul Scott Thorner

a, b

a Division of Pathology, Hospital for Sick Children, and b

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont. , Canada

these groups of patients requires a combination of family history and a renal biopsy for electron microscopic examina-tion of the GBM and immunohistochemical staining of the GBM for the � 3, � 4 and � 5 chains of type IV collagen. Muta-tional analysis of the COL4A3 , COL4A4 , and COL4A5 genes, whenever it becomes available, will be a valuable adjunct to the diagnostic workup in these patients. Novel therapeutic approaches may one day provide a treatment or cure for these patients, avoiding the need for transplantation and di-alysis. Copyright © 2007 S. Karger AG, Basel

Alport Syndrome

Alport syndrome (also known as hereditary nephritis) is an inherited progressive nephropathy, often associated with sensorineural deafness and ocular lesions. It ac-counts for 3% of end-stage renal disease (ESRD) in the pediatric population. Alport syndrome arises from muta-tions in the genes encoding type IV collagen and an un-derstanding of that molecule helps to explain the patho-genesis of this disease [1] . Type IV collagen is the prima-ry structural component of all basement membranes, and Alport syndrome can be viewed as a genetic disease of the basement membrane.

Key Words Alport syndrome � Thin basement membrane � Glomerular basement membrane � Type IV collagen

Abstract Both Alport syndrome and thin basement membrane ne-phropathy (TBMN) can be considered as genetic diseases of the GBM involving the � 3/ � 4/ � 5 network of type IV colla-gen. Mutations in any of the COL4A3 , COL4A4 or COL4A5 genes can lead to total or partial loss of this network. Males with mutations in the X-linked COL4A5 gene develop Alport syndrome with progressive renal disease and sometimes ex-tra-renal disease. Females who are heterozygous for a COL4A5 mutation are considered to be carriers for X-linked Alport syndrome. Although their clinical course and GBM ul-trastructural changes can sometimes mimic TBMN, more of-ten it tends to be more progressive than usually seen in TBMN. Males or females who are heterozygous for COL4A3 or COL4A4 mutations usually manifest as TBMN, with non-progressive hematuria, while those who are homozygous or combined heterozygotes develop autosomal-recessive Al-port syndrome. Thus, individuals with TBMN can be consid-ered to be carriers for autosomal-recessive Alport syndrome, but there remain some exceptions in which patients hetero-zygous for COL4A3 or COL4A4 mutations develop autosomal-dominant Alport syndrome. Distinguishing between all

Published online: June 6, 2007

Paul Scott Thorner, MD, PhD Division of Pathology, The Hospital for Sick Children 555 University Avenue Toronto, Ont. M5G 1X8 (Canada) Tel. +1 416 813 5108, Fax +1 416 813 5974, E-Mail paul.thorner@sickkids.ca

© 2007 S. Karger AG, Basel1660–2110/07/1062–0082$23.50/0

Accessible online at:www.karger.com/nec

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Type IV Collagen

Type IV Collagen Genes In humans, six distinct � -chains of type IV collagen

designated � 1(IV)– � 6(IV) have been identified (hereaf-ter referred to as � 1, etc.) each encoded by a different gene designated COL4A1 – COL4A6 , respectively. These genes are large and complex, each comprising � 50 exons. They are paired in a head-to-head fashion with the COL4A1 and COL4A2 genes on chromosome 13q23, COL4A3 and COL4A4 on chromosome 2q36–37 and COL4A5 and COL4A6 on chromosome Xq22.3.

Type IV Collagen Molecules and Networks Each gene encodes an � -chain consisting of a minor

collagenous domain of � 140 residues known as the 7S domain, a major collagenous domain ( � 1,300 residues largely in the form of Gly-X-Y repeats) and a C-terminal noncollagenous domain of � 230 residues known as the NC1 domain [1] . Each collagen molecule is formed from three � -chains. The NC1 domain initiates the assembly and governs the process of � -chain selection. Only three combinations of the 6 different � -chains occur: � 1 2 � 2, � 3 � 4 � 5 and � 5 2 � 6. Collagen molecules (protomers) are then secreted whereupon they self-assemble at the amino terminal forming tetramers and at the C-terminal form-ing dimers. Only three types of type IV collagen net-works are known to exist: � 1 2 � 2 protomers bridge to themselves forming the � 1/ � 2 network; � 3 � 4 � 5 pro-tomers bridge to themselves forming the � 3/ � 4/ � 5 net-work; and � 1 2 � 2 protomers bridge to � 5 2 � 6 protomers forming the � 1/ � 2/ � 5/ � 6 network.

Distribution of Type IV Collagen Networks The � 1/ � 2 network is ubiquitous in basement mem-

branes whereas the other two networks show a restricted distribution that presumably reflects function. The � 3/ � 4/ � 5 network is prominent in sites that serve as filtra-tion barriers, whereas the � 1/ � 2/ � 5/ � 6 network is often found in basement membranes that undergo repeated stretching. The � 3/ � 4/ � 5 network is the predominant one in the glomerular basement membrane (GBM) as well as in several basement membranes in the eye and in-ner ear (see below). The � 1/ � 2/ � 5/ � 6 network is ex-pressed in Bowman’s capsule of the glomerulus and in basement membranes surrounding smooth muscle cells of vessels and viscera. This network is also present in sub-epithelial basement membranes of viscera and the epider-mis.

The type IV collagen networks in certain basement membranes undergo changes in composition during de-velopment, referred to as a ‘developmental switch’ [2] . Developing glomeruli contain only the � 1/ � 2 network until capillary loops form at which time the � 1/ � 2 net-work in GBM is largely replaced by the � 3/ � 4/ � 5 network and the � 1/ � 2/ � 5/ � 6 network appears in Bowman’s cap-sule [2–4] . Similar shifts have been noted in the inner ear [5] and testis. Other basement membranes either do not undergo a shift or this event occurs prenatally and has so far gone undetected.

Genetics of Alport Syndrome

X-linked Alport syndrome accounts for � 85% of all cases [6] and arises from mutations in the COL4A5 gene. Over 350 different mutations have been reported [7] in-cluding large deletions, missense and nonsense muta-tions, small deletions/insertions causing frameshifts, and splice site mutations. No mutational ‘hot spots’ are known. With few exceptions, each family carries a unique muta-tion, but up to 18% of cases are de novo mutations.

Autosomal-recessive Alport syndrome accounts for � 15% of cases and results from homozygous or com-pound heterozygous mutations in the COL4A3 or COL4A4 genes [8] . Over 40 different mutations in these genes have been identified with the same spectrum of mutations as for COL4A5 . Rare examples of autosomal-dominant Alport syndrome have been reported, caused by a mutation in either the COL4A3 or COL4A4 gene [9] .

Fechtner syndrome is a rare disorder formerly consid-ered to be an autosomal-dominant form of Alport syn-drome associated with deafness, macrothrombocytope-nia, congenital cataracts and leukocyte inclusions. More recently, Fechtner syndrome and the related Epstein syn-drome were found to be caused by mutations in the MYH9 gene encoding non-muscle myosin heavy chain IIA [10] .

Clinical and Pathologic Features of Alport Syndrome

Renal Disease The cardinal sign of renal disease in Alport syndrome

is hematuria, microscopic or gross, recurrent or persis-tent, usually first detected in childhood [6] . Only 10% have proteinuria at presentation, but the majority of pa-tients develop this. For X-linked disease, the risk for fe-males progressing to ESRD before 40 years of age is only

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12%, compared to 90% for males [7, 11] . For autosomal-recessive Alport syndrome, both sexes follow a course comparable to males with X-linked disease [12] . Autoso-mal-dominant Alport syndrome shows much greater clinical variability ranging from asymptomatic to ESRD by age 50, but generally milder disease than males with X-linked disease [9] .

The diagnostic biopsy changes for all forms of Alport syndrome are seen by electron microscopy, consisting of variable thinning and thickening of the GBM combined with multilamellation of the lamina densa. Thinning of the GBM is more common in children, in whom multi-lamellation may be very segmental. Measurements need to be compared to standardized values for the patient’s age since the GBM is normally thinner in children than in adults. Later, multilamellation of the GBM becomes widespread; however, some patients continue to show diffuse thinning. In females with X-linked disease, the GBM changes can range from normal to diffuse multi-lamellation.

Hearing Loss Sensorineural hearing loss is the most common extra-

renal manifestation of Alport syndrome, affecting up to 79% of males and 28% of females with X-linked disease [7, 11] . In males, hearing loss can appear in childhood whereas in females, it tends to occur later in life. The hearing loss is bilateral and results from inner ear dys-function. In males, the hearing deficit can progress to involve conversational speech, while females generally have a milder course. In autosomal Alport syndrome, both sexes experience a course similar to that of males with X-linked disease. Pathological studies on the inner ears of Alport syndrome patients have reported degen-erative changes in a variety of locations, including the stria vascularis, organ of Corti, spiral ganglion and spiral ligament [5] .

Ocular Abnormalities Multiple ocular abnormalities occur in Alport syn-

drome [7, 11] . The most common are anterior lenticonus and a ‘dot-and-fleck’ retinopathy. Anterior lenticonus may be exclusive to Alport syndrome. It occurs in � 25% of males and is usually accompanied by nephropathy and deafness. The lens capsule is approximately one third normal thickness and lens distortion results in reduced visual acuity. The retinopathy is more common, occur-ring in up to 85% of males and 15% of females, but with no visual impairment.

Genotype-Phenotype Correlations The clinical heterogeneity in X-linked Alport syn-

drome is related in part to the nature of the type IV col-lagen mutation [7] . The risk of males developing ESRD before 30 years of age is 50% for missense mutations, 70% for splice-site mutations, and 90% for large deletions or nonsense mutations. For females, the situation is less clear [11] . The variability in disease in females may be related to different patterns of X chromosome inactiva-tion of the normal COL4A5 allele. Hearing loss and ocu-lar abnormalities are also more likely to occur in males with large COL4A5 gene rearrangements and mutations that disrupt the reading frame compared to missense mu-tations [7] .

Molecular Pathogenesis of Alport Syndrome

In Alport syndrome, mutations in the COL4A3 , COL4A4 or COL4A5 genes lead to loss or abnormal forms of the � 3, � 4 or � 5 chains, respectively. Since these three chains form a trimeric molecule, a mutation in any one of these chains leads to failure of incorporation of all three. Failure to form the � 3 � 4 � 5 protomer leads to an absence of the � 3/ � 4/ � 5 network and the resultant dis-ease reflects the role of the � 3/ � 4/ � 5 network in special-ized basement membranes in different organs. In the glomerulus, the � 3/ � 4/ � 5 network is produced by the podocyte, and thus the renal disease in Alport syndrome can be thought of as a primary podocyte disorder. For COL4A5 mutations, the � 5 2 � 6 protomer also cannot form and there is loss of the � 1/ � 2/ � 5/ � 6 network, but with COL4A3 and COL4A4 mutations, this network forms normally. These molecular events are reflected at the immunohistochemical level in biopsies. In male pa-tients with X-linked disease, the GBM usually contains the � 1 and � 2 chains only and lacks the � 3, � 4 and � 5 chains, although very occasionally, the � 3, � 4 or � 5 chains are expressed with missense mutations. Female patients with X-linked disease usually show a segmental distribution of the � 3, � 4 and � 5 chains [6] , reflecting Lyonization of the normal or mutant X-chromosome. In autosomal-recessive Alport syndrome, the GBM shows the same findings as with male X-linked patients, al-though up to 20% of patients can have � 3, � 4 and � 5 chains in the GBM [12] . Expression of the � 1 and � 2 chains in the GBM increases with progression of disease [6] . The � 1/ � 2/ � 5/ � 6 network is lost only in X-linked disease. Males show loss of the � 5 and � 6 chains from Bowman’s capsule and the epidermal basement mem-

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brane in skin and vascular smooth muscle, and females show segmental loss. Patients with autosomal disease re-tain expression of the � 5 and � 6 chains in these sites, providing a clue for distinguishing autosomal and X-linked disease by renal or skin biopsy [6] .

Renal Disease The question arises as to why loss of the � 3/ � 4/ � 5 net-

work leads to glomerular disease. Animal model work has shown despite a congenital absence of the � 3/ � 4/ � 5 network, the Alport GBM is initially morphologically and functionally normal [4] . Thus, only the � 1/ � 2 net-work is essential for normal glomerular development, whereas the � 3/ � 4/ � 5 network is more important for long-term maintenance of glomerular structure and function. The � 3 and � 4 chains are more cysteine-rich than � 1 and � 2 chains, allowing loop structures to form between triple helical collagen molecules. Thus, the � 3/ � 4/ � 5 network may provide a structural integrity to the GBM that the � 1/ � 2 network cannot do alone.

The � 3/ � 4/ � 5 network is also more resistant to endo-proteolysis or, alternatively, Alport GBM is more suscep-tible [3] . The matrix metalloproteinase (MMP) family of endopeptidases (including MMP2/gelatinase A, MMP9/gelatinase and MMP12/metalloelastase) plays a promi-nent role in the degradation of extracellular matrix com-ponents. Increased expression of MMP12 has been docu-mented in podocytes in animal and human forms of Al-port syndrome [13] . Expression of TGF � 1 may also play a role in the glomerular disease, as blockade of TGF � 1 reduces GBM changes [14] . Renal failure in Alport syn-drome is closely associated with progressive tubulointer-stitial damage. Animal models have shown that expres-sion of TGF � 1 in the renal interstitium contributes to progressive fibrosis as does increased expression of MMP2 and MMP9 [15] . A more complete understanding of the genes dysregulated in Alport syndrome will come from microarray studies [16] .

Hearing Loss The pathogenesis of deafness in Alport syndrome is

poorly understood. No available animal model shows any hearing deficit analogous to human Alport syndrome. Nevertheless, these models offer the opportunity to ex-amine the inner ear. Murine models have tended to im-plicate changes in the stria vascularis as the source of hearing loss [17] possibly through hypoxia or electrolyte disturbances. Canine model studies have focused more on the spiral ligament based on high expression of the � 3/ � 4/ � 5 network in this site [5] . Cells in this region are

believed to help maintain tension on the basilar mem-brane via their extracellular matrix. Absence of the � 3/ � 4/ � 5 network could lead to reduced tension on the bas-ilar membrane and loss of high frequency sound percep-tion. Studies on human Alport cochlea have suggested loss of function of the organ of Corti, due to separation of cells from the basilar membrane, perhaps secondary to an absent � 3/ � 4/ � 5 network [18] .

Ocular Abnormalities The human lens capsule contains all six chains of type

IV collagen, whereas in Alport syndrome only the � 1 and � 2 chains are expressed [19] . Presumably, the � 3/ � 4/ � 5 network contributes structural integrity that is lost in Al-port syndrome, leading to weakening of the lens capsule and anterior lenticonus. The pathogenesis of the retinal abnormalities in Alport syndrome is unknown but may be related to loss of the � 3/ � 4/ � 5 network, since that is present in the retina.

Therapy in Alport Syndrome

Transplantation Transplantation in Alport syndrome experiences suc-

cess rates comparable to other kidney diseases [6] . A mi-nority ( � 15%) of recipients develop anti-GBM antibodies based on recognition of the � 3/ � 4/ � 5 network in the transplant GBM as a novel antigen, but actual loss of the graft from anti-GBM disease occurs in only � 3% of cas-es. The antibodies can be directed against the � 3, � 4 and/or � 5 chains [20] . Patients with large deletions or non-sense mutations that result in complete absence of the NC1 domain have an increased risk of developing post-transplant anti-GBM nephritis.

Drug Therapy Drug therapy has generally aimed at slowing progres-

sion of the renal disease and most of the work has been done in animal models. Cyclosporine A has been report-ed to be beneficial in Alport syndrome, reducing protein-uria and maintaining renal function up to 10 years [21] . In a canine X-linked model, Cyclosporine A improved renal function, delayed damage to the GBM and reduced glomerulosclerosis [22] . A recent study confirmed cyclo-sporine A decreased proteinuria in patients but resulted in nephrotoxicity [23] .

Use of angiotensin-converting enzyme (ACE) inhibi-tors has been reported to reduce proteinuria in humans [24] . In a canine X-linked model, an ACE inhibitor re-

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duced GBM multilamellation with a � 35% increase in lifespan [25] . In a COL4A3 knockout mouse, treatment reduced proteinuria and interstitial fibrosis and doubled the expected lifespan. The mechanism may be related to lowered expression of TGF � , MMPs and extracellular matrix proteins [26] . An antagonist of chemokine recep-tor 1 that blocks interstitial leukocyte recruitment also reduced interstitial inflammation and fibrosis with in-creased life span [27] .

Administration of a TGF � receptor antagonist or in-hibition of MMP12 has been shown to improve glomeru-lar filtration and GBM morphology in a COL4A3 knock-out mouse [13, 14] . MMP12 was inhibited by an antago-nist of chemokine receptor 2, which is expressed on podocytes and may explain how inhibition of MMP12 reduces GBM damage.

Gene Therapy Alport syndrome is an attractive disease for gene ther-

apy for several reasons: (1) only the kidney disease is life-threatening allowing therapy to be targeted to this organ alone; (2) the isolated circulatory system of the kidney facilitates targeted delivery of a gene transfer vector; (3) turnover of type IV collagen is slow, thus any corrected type IV collagen network may remain effective for some time; (4) GBM of females with X-linked Alport syndrome shows a mixture of normal and abnormal GBM and the prognosis for females is significantly better than for males, implying that even partial correction of the Alport GBM may be beneficial to patients. Animal model studies have shown there is a time window for gene transfer be-fore GBM deterioration begins [4] ; hence, in utero gene transfer should not be necessary. Work using a COL4A3 knockout mouse has confirmed the feasibility of gene therapy, by rescuing the Alport phenotype using a ge-netic construct containing the normal human COL4A3 gene [28] .

To date, gene therapy in Alport syndrome has followed two different approaches: adenoviral vectors and bone marrow stem cells. Intra-arterial or ureteric injections of virus do not efficiently deliver genes to glomeruli. Deliv-ery of adenovirus by slow perfusion (1–2 h) or extracor-poreal renal circuit results in up to 85% of normal glom-eruli expressing a marker gene (LacZ) for up to 4 weeks [29] . The extracorporeal approach was able to transfer a human � 5 transgene to normal porcine kidney with lo-calization of the � 5 chain to GBM [30] . An adenoviral vector containing a canine � 5 transgene injected into bladder smooth muscle was able to restore both � 5 and � 6 chains in a canine X-linked model, implying forma-

tion of the missing � 1/ � 2/ � 5/ � 6 network [31] . It remains to be seen if efficacious results can be obtained in Alport kidney using adenoviral vectors.

Bone marrow-derived stromal stem cells (BMSCs) can exhibit considerable phenotypic plasticity. Following bone marrow transplantation, such cells have taken up residence in glomeruli and differentiated into mesangial cells and possibly even podocytes [32] . BMSCs cultured on type IV collagen showed a degree of podocytic differ-entiation [33] . BMSCs then offer the potential of repair-ing damaged glomeruli in which podocytes are diseased or destroyed.

The few studies published related to Alport syndrome have used COL4A3 knockout models. Unfractionated al-logeneic bone marrow cells injected systemically resulted in partial restoration of GBM ultrastructure and the � 3/ � 4/ � 5 network, with reduced glomerular scarring and interstitial fibrosis, and improved renal function [34, 35] . Bone marrow cells took up residence in glomeruli and appeared either to differentiate into podocytes or fuse with native podocytes. Curiously, use of BMSCs spe-cifically was not beneficial [35] . A similar study found BMSCs did not differentiate into renal parenchymal cells nor populate glomeruli [36] , but there was reduced inter-stitial fibrosis and peritubular capillary loss, possibly re-lated to secretion of TGF � and VEGF by BMSCs, al-though not sufficient to alter the course of the disease. Clearly much work is needed before this type of therapy can be offered to patients but the approach offers great potential for treatment of Alport syndrome.

Thin Basement Membrane Nephropathy

Thin basement membrane nephropathy (TBMN) or thin GBM disease is also sometimes referred to as ‘benign familial hematuria’. These terms are not exactly inter-changeable; not all cases of TBMN are familial, and not all cases of familial hematuria are benign or caused by TBMN. Notwithstanding, cases of TBMN have been shown to have type IV collagen mutations, providing a link between this disease and Alport syndrome.

Genetics of Thin Basement Membrane Nephropathy

In contrast to Alport syndrome, TBMD behaves as an autosomal-dominant disorder and in � 40% of families, segregates with the COL4A3 / COL4A4 loci [37] . Hetero-zygous mutations in both these genes have been identified

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in many cases [8] . Similar to Alport syndrome, mutations are usually different for each family and there is no ‘hot-spot’. Missense, nonsense, splice mutations, deletions and insertions have all been described. Apparently, mutations in the COL4A3 or COL4A4 genes result in a clinical spec-trum of disease, ranging from TBMD to autosomal-dom-inant or -recessive Alport syndrome depending on the nature of the mutation and gene dosage. TBMD can be regarded as the carrier state of autosomal-recessive Al-port syndrome [37] . However, females heterozygous for a COL4A5 mutation are considered to have X-linked Al-port syndrome rather than thin GBM disease.

In some families, there is no linkage to the COL4A3 and COL4A4 genes, but these may be de novo mutations since about one third of cases have no family history. De-tection of mutations is difficult because of the large size of the COL4A3 and COL4A4 genes, the need for direct sequencing of exons to maximize mutation detection, the need to detect splicing mutations, and the large number of polymorphisms that exist in these two genes. The ex-istence of another locus for TBMD has been postulated but not yet identified [37] .

Clinical and Pathologic Features of Thin Basement Membrane Nephropathy

Like Alport syndrome, TBMD is characterized by per-sistent or recurrent hematuria usually presenting in childhood. It is the most common cause of persistent he-maturia in childhood and occurs in at least 1% of the population [37] . In contrast to Alport syndrome, TBMD is generally not associated with significant proteinuria, progression to renal failure, or extrarenal disease [6, 37, 38] . Exceptions do occur but not usually until adulthood when patients can develop proteinuria, hypertension and sometimes renal failure, but for the most part the renal prognosis is excellent.

Light and immunofluorescence microscopy are usu-ally normal or near normal, but by electron microscopy, the GBM shows diffuse thinning compared to normal values for the patient’s age. Marked variability in GBM width and focal multilamellation is more suggestive of Alport syndrome. Carriers of autosomal-recessive Alport syndrome can also show diffuse attenuation of the GBM. Cases that progress to renal failure can show glomerular obsolescence, segmental sclerosis, or other glomerular diseases such as IgA nephropathy [37] .

Molecular Pathogenesis of Thin Basement Membrane Nephropathy

The basis for the thinning is unknown but may differ from Alport syndrome since the distribution of the six � chains of type IV collagen is usually normal in TBMD [6] providing a clue to distinguishing these two diseases. Perhaps there is a quantitative reduction in the amount of the � 3/ � 4/ � 5 network, with similar but less severe consequences as those discussed for Alport syndrome.

It is unclear why some COL4A3 and COL4A4 muta-tions cause autosomal-dominant Alport syndrome rath-er than TBMD [37] or why the same mutation can have different clinical presentations in different families [39] . This may be related to modifier genes [9] , a phenomenon that is believed to influence the course of renal disease in COL4A3 knockout models.

While most patients do not require treatment, not all experience a benign course. The cause of the renal im-pairment in the latter group of patients is unknown. Het-erozygotes in a COL4A3 knockout have been proposed as a model for TBMD [40] . This model shows focal glomer-ulosclerosis, tubulointerstitial fibrosis with upregulation of TGF � and should be beneficial for exploring both pathogenesis and therapeutic options for those patients who develop progressive renal disease in TBMN.

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