Distribution of Type IV Collagen in the Cochlea in Alport Syndrome

ORIGINAL ARTICLE Distribution of Type IV Collagen in the Cochlea in Alport Syndrome

Andreas F. Zehnder, MD; Joe C. Adams, PhD; Peter A. Santi, PhD; Arthur G. Kristiansen, MA; Chitsuda Wacharasindhu, MD; Sabine Mann; Raghu Kalluri, PhD; Martin C. Gregory, MD; Clifford E. Kashtan, MD; Saumil N. Merchant, MD

Objective: To determine the distribution of ␣1, ␣3, and brane, in the basement membrane of cochlear blood ves- ␣5 chains of type IV collagen in the cochlea in Alport sels and Schwann cells, and within the spiral limbus. In syndrome. the control ear, we also observed strong staining for ␣3 and ␣5 chains in the basement membrane overlying the Design: Case-control study. basilar membrane and within the spiral ligament. In both cases with Alport syndrome, no immunostaining was ob- Patients: Two patients with sensorineural hearing loss served for ␣3or␣5 chains within the cochlea, whereas due to Alport syndrome. Both patients had known mu- ␣1 staining was present in locations similar to that seen tations in the COL4A5 gene. in the control ear.

Main Outcome Measures: Immunostaining was used Conclusions: The results indicate that isotype switch- to study the distribution of type IV collagen (␣1, ␣3, and ing does not occur within the cochlea in Alport syn- ␣5 chains) within the cochlea. Immunostaining was also drome. The results are also consistent with the hypoth- performed in the cochlear tissues of an unaffected indi- esis that the sensorineural hearing loss in Alport syndrome vidual used as a control. may be due to alterations in cochlear micromechanics and/or dysfunction of the spiral ligament. Results: In the control ear, ␣1 staining was observed in the basement membrane overlying the basilar mem- Arch Otolaryngol Head Neck Surg. 2005;131:1007-1013

LPORT SYNDROME, AN IN- In Alport syndrome, the glomerular BM herited progressive renal within the kidney shows a characteristic disease with sensorineu- ultrastructural abnormality consisting of Author Affiliations: Ear, Nose, ral hearing loss (SNHL) diffuse thickening and splitting of the 2 and Throat Department, and ocular lesions, is lamina densa. During normal develop- University Hospital Basel, Basel, causedA by mutations in the genes (COL4) ment, ␣1 and ␣2 chains predominate in Switzerland (Dr Zehnder); that code for the ␣3, ␣4, or ␣5 chains of the glomerular BM. As glomerular matu- Department of Otolaryngology, type IV collagen.1-3 The disease is geneti- ration progresses, the ␣3, ␣4, and ␣5 Massachusetts Eye and Ear cally heterogeneous with X-linked, auto- chains become the predominant type IV Infirmary, Boston (Drs Zehnder, somal recessive and autosomal dominant collagen chains in glomerular BM, a pro- Adams, Wacharasindhu, and forms. Most cases (approximately 85%) of cess referred to as isotype switching.2,3,5-7 In Merchant, Mr Kristiansen, and Alport syndrome are X-linked and are due Ms Mann); Department of most males with Alport syndrome, this iso- to mutations in the COL4A5 gene located type switch does not occur, as a result of Otology and Laryngology ␣ (Drs Adams and Merchant), on Xq22 that codes for the 5 chain of type which the ␣3, ␣4, and ␣5 chains are usu- Harvard Medical School, and IV collagen. More than 300 mutations of ally absent, as determined by immuno- Center for Matrix Biology, the COL4A5 gene producing X-linked Al- staining. The resulting glomerular BM in Department of Medicine, Beth port syndrome have been reported. Alport syndrome is believed to be defec- Israel Deaconess Medical Type IV collagen is the major structural tive in structure and function, causing Center (Dr Kalluri), Boston; component of basement membrane (BM) hematuria early in life and progressing to Departments of Otolaryngology ␣ and it consists of 3 chains, which make a proteinuria and renal failure. and Neuroscience (Dr Santi) triple-helical molecule.2-4 The trimer com- and Division of Pediatric ␣ ␣ ␣ ␣ In contrast to renal studies, investiga- posed of 1 and 2 chains ( 12- 21)is tions of the human inner ear in Alport syn- Nephrology, Department of present ubiquitously in BMs throughout the Pediatrics (Dr Kashtan), drome have lagged behind because of sev- body. On the other hand, trimers com- University of Minnesota, ␣ ␣ ␣ ␣ ␣ ␣ eral factors: Minneapolis; and Department posed of 3, 4, and 5( 31- 41- 51)or ␣ ␣ ␣ ␣ of Medicine, University of Utah 5 and 6( 52- 61) chains are restricted 1. It is not feasible to take a biopsy Medical Center, Salt Lake City in expression to specific BMs of the kid- sample from the cochlea during life with- (Dr Gregory). ney, inner ear, eye, and other organs. out causing severe SNHL.

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 2. There is a paucity of human postmortem tempo- good speech discrimination. He died at age 57 years from end- ral bone material because temporal bones are not re- stage renal disease, and both temporal bones were removed 4 moved or studied as part of routine autopsies. hours after death. The left temporal bone was examined by light and electron microscopy, and findings were reported previ- 3. When temporal bones are removed and studied af- 8 ter death, tissue alterations are inevitably induced by post- ously. The right temporal bone was fixed in 10% neutral buffered formalin, decalcified using EDTA, trimmed so that only mortem autolysis. the inner ear remained, and was then embedded in paraffin. 4. The human temporal bone contains the delicate Serial sections were cut at a thickness of 8 µm. A representa- membranous labyrinth within an extremely dense shell tive (mid modiolar) section was stained with hematoxylin- of bone, which imposes serious technical challenges in eosin (H&E), while other sections through the cochlea were processing temporal bone tissue in a manner that al- immunostained. lows for preservation of morphology and the ability to immunostain for various proteins. CASE 2 In a recent study,8 our research team examined the Patient 2 was a man aged 31 years with X-linked Alport syn- histopathologic features of the cochlea in 9 patients with drome caused by COL4A5 mutation. This individual had a de- Alport syndrome and described abnormalities of the BM letion of exons 43-47 of the COL4A5 gene. He had bilateral SNHL of cells of the organ of Corti, as well as cellular infilling since the age of 9 years and wore amplification for several years. of the tunnel and extracellular spaces of the organ of Corti His last audiogram at age 25 years showed a downsloping 60- in 8 of the 9 cases. This particular study used a combi- to 110-dB SNHL on the right with speech discrimination of 76% nation of light and electron microscopy to study the mor- and a 60- to 90-dB SNHL on the left with speech discrimina- phologic changes in the cochlea. tion of 90%. He died at the age of 31 years from pancreatitis Knowledge of the distribution of various chains of type and complications of renal failure, and both temporal bones IV collagen in the cochlea may help in the understand- were removed 10 hours after death. The right temporal bone ing of the pathogenesis of SNHL at a cellular level. There was used in the present study. The specimen had been stored in 10% formalin for a number of years. The bone was prepared is a single extant study that examined the distribution by decalcification using EDTA and trimming so that only the of type IV collagen in the normal human inner ear. In inner ear remained, followed by embedding in paraffin and sec- 9 ␣ the study by Kleppel et al, expression of 1 chains was tioning at a thickness of 8 µm. A mid modiolar section was observed in the BM overlying the basilar membrane (ie, stained with H&E, while immunostaining was performed on the BM of cells of the organ of Corti), in the BM of coch- other sections. lear blood vessels, and in the spiral limbus and in the BM of Schwann cells. In contrast, expression of ␣3 and ␣4 CASE 3 chains was restricted to the BM overlying the basilar membrane. The study did not examine expression of ␣5 chains. Case 3 involved a woman aged 57 years with no otologic symp- To our knowledge, no study has described the distribu- toms (control case). This individual died as a result of a hem- tion of type IV collagen within the cochlea in Alport syn- orrhagic cerebral infarct with pulmonary thromboembolism. drome. Both temporal bones were removed 5.5 hours after death, fixed In the present study, we describe the distribution of in 10% formalin, and processed in a manner identical to that type IV collagen (␣1, ␣3, and ␣5 chains) as determined described for cases 1 and 2. Before proceeding with immunostaining on human tempo- by immunostaining within the inner ear of 2 patients with ral bones, we conducted experiments using temporal bones of SNHL due to Alport syndrome. Both patients had known CBA mice to optimize the protocol for immunostaining. The mutations in the COL4A5 gene resulting in Alport syn- temporal bones of CBA mice were fixed in 10% formalin, de- drome. Results indicate that there is selective distribu- calcified in EDTA, embedded in paraffin, and sectioned in a man- tion of ␣3 and ␣5 chains within the cochlea, including ner similar to the processing used for human tissues. Antibod- within the collagen bundles in the spiral ligament and ies against the ␣1, ␣3, and ␣5 chains of type IV collagen were limbus. Results in the cases with Alport syndrome sug- used to immunostain the animal material to optimize the gest that isotype switching for collagen does not occur protocol. The optimized protocol is described in the following in the cochlea, similar to what has been reported in the section. kidney. The implications of our findings with respect to the pathogenesis of SNHL in Alport syndrome are dis- IMMUNOSTAINING cussed herein. Monoclonal antibodies directed against the ␣1, ␣3, and ␣5 chain of collagen IV were used (Wielisa kit; Wieslab AB, Ideon, Lund, METHODS Sweden). We did not have access to antibodies against the ␣2, ␣4, and ␣6 chain of collagen IV, and hence our study was re- CASE 1 stricted to the ␣1, ␣3, and ␣5 chains. Selected slides were de- paraffinized in xylene, rehydrated in decreasing strengths of Patient 1 was a man aged 57 years with X-linked Alport syn- ethyl alcohol, followed by distilled water, and rinsed in a 0.01M drome caused by COL4A5 mutation. This individual had a single- phosphate-buffered saline (PBS; pH 7.3). Each slide was treated base mutation converting a cysteine to serine at position 1564 with 0.0025% protease XXIV solution (Sigma, St Louis, Mo) in the COL4A5 gene. His epidermal BMs lacked expression of and incubated at 37°C in a closed humidity chamber (20 min- the ␣5 chain of type IV collagen. He had bilateral progressive utes for ␣3 and ␣5 and 40 minutes for ␣1; the protease was SNHL that began in his 20s, and he wore hearing aids for the kept at −20°C and thawed just before use). The protease was remainder of his life. His last audiogram at age 53 years showed drained and slides were rinsed 3 times in PBS. Slides for ␣5 were a bilateral, symmetric, relatively flat, 60- to 70-dB SNHL with incubated in cold glycine-urea solution for 5 minutes and then

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 washed 3 times in PBS. The next step for all 3 antibodies was Control Ear incubation in 5% normal horse serum for 30 minutes to block nonspecific immunoglobulin binding. The antibodies were then The cochlea consisted of 2½ turns. An organ of Corti was added in the following dilutions to the sections and left over- ␣ ␣ ␣ present throughout, although it was difficult to differ- night: 1, 1:5; 3, 1:20; and 5, 1:1000. The primary antibod- entiate cells (eg, hair cells and supporting cells) because ies were then drained and the slides rinsed 3 times in PBS, followed by the addition of a biotinylated secondary antibody and of poor preservation associated with processing in par- incubation for 1 hour. After another wash in PBS, avidin-biotin- affin. The stria vascularis showed mild patchy atrophy, horseradish peroxidase (Standard ABC Kit; Vector Laborato- and the cochlear neuronal cells showed a mild loss in the ries, Burlingame, Calif) was applied for 1 hour, followed by a basal turn (compared with normal newborns). The spi- wash in PBS for 10 minutes. The immunoreaction was ampli- ral ligament showed partial atrophy in especially the fied by incubation in 1% biotinylated tyramine and 0.01% hy- middle and apical turns. All the above changes were con- drogen peroxide for 10 minutes.10 After a rinse in PBS, the slides sistent with age-related change. were incubated for another 30 minutes in avidin-biotin- horseradish peroxidase followed by another rinse in PBS. The Case 1 slides were colorized using 0.01% diaminobenzidine and 0.01% hydrogen peroxide for approximately 180 seconds under microscopic control, washed in water, and dehydrated and cov- The cochlea consisted of 2½ turns with diffuse endo- erslips were applied. Negative controls included omission of lymphatic hydrops of a moderate degree in all turns. There the primary antibodies, and positive controls included paraffin- was a zone of separation between the basilar membrane embedded sections of normal renal tissue. and overlying organ of Corti, similar to the changes ob- As described in the “Results” section, we observed strong served in the opposite ear (published previously, case 18). immunostaining with the ␣5 antibody (and the ␣3 antibody While the organ of Corti was present in all turns, its mor- as well) in the spiral ligament where staining had not been pre- phologic features were suboptimum owing to paraffin em- viously described.9 Therefore, we wanted to rule out the pos- bedding, and it was not possible to determine whether sibility of cross-reactivity of the ␣5 antibody. We used the fol- ␣ hair cells were missing and whether there was cellular lowing protocol to obtain purified 5 protein containing the infilling within the tunnel of Corti and extracellular spaces noncollagenous (NC1) domain of the COL4 ␣5 chain. Cells ␣ of Nuel (as was observed in the contralateral ear of this producing the 5NCI protein (EBNA 293 embryonic renal cells) 8 were cultured for 48 hours in serum-free and antibiotic-free individual ). The stria vascularis showed mild patchy at- medium. The serum-free medium contained various secreted rophy, the cochlear neurons showed mild loss in the basal cellular proteins including ␣5NC1. The recombinant FLAG- turn, and the spiral ligament showed partial atrophy in tagged protein was purified at 4°C using the Anti-FLAGM2 af- especially the middle and apical turns. The cochlear vas- finity gel (Sigma). A polypropylene column was filled with 2.5 culature was normal. All these changes were consistent mL of affinity gel, and the serum-free medium was applied to with age-related change. the column following the manufacturer’s specifications. Briefly, approximately 250 mL of serum-free medium was passed over Case 2 the column followed by 2 washes with sterile Tris base (pH 8.0). The FLAG-tagged fusion protein bound to the resin containing anti-FLAGM2 antibody, and the protein then was eluted The cochlea consisted of 2½ turns. There was no endo- from the column into 1.5-mL aliquots with glycine (pH 3.5). lymphatic hydrops. There was mild patchy atrophy of the Glycine was used to denature the antibody and the recombi- stria vascularis, mild to moderate loss of cochlear neu- nant ␣5NC1 protein was eluted into microcentrifuge tubes con- rons in the basal turn, and a separation between the basi- taining 35 µL of Tris base (pH 10) (which neutralizes the pH lar membrane and organ of Corti in many turns. Again, of the recombinant protein-containing glycine solution to mini- suboptimum morphologic features of the processed tis- mize the negative effects of the acidic environment of the pro- sues precluded assessment of cellular architecture within tein). The aliquots were then read on a spectrophotometer at the organ of Corti. Blood vessels supplying the cochlea 280 nm, and those with a significant optical density were pooled appeared normal. and collectively dialyzed 3 times against PBS at 4°C. The purified ␣5NC1 protein was used to preabsorb the ␣5 antibody in a series of sections that were processed for immunostaining as previously described. A BLASTP search of GenBank’s nonre- IMMUNOSTAINING dundant protein database (maintained by the National Center for Biotechnology Information, Bethesda, Md) for the noncol- ␣1 Chain of Type IV Collagen lagenous NC1 domain of COL4␣5 was also performed. Results were similar for the control ear and both cases RESULTS with Alport syndrome. Positive staining was observed in the BM overlying the basilar membrane, the BM of blood vessels within the modiolus, spiral ligament and LIGHT MICROSCOPY stria vascularis, and the BM of Schwann cells within WITH HEMATOXYLIN-EOSIN the modiolus and osseous spiral lamina. Positive stain- Hematoxylin-eosin staining was used to study the inner ing was also observed within the spiral limbus and in ears of 2 patients with SNHL due to Alport syndrome and collagen bundles within the spiral ligament of the of an unaffected individual used as a control. The findings basal turn. The Reissner membrane was also stained, in the representative mid modiolar sections studied from although the staining was within the cytoplasm of the each temporal bone and stained with H&E are described cells of the Reissner membrane rather than just its BM in the following sections. (Table and Figure).

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␣1 ␣3 ␣5

Tissue Control Case 1 Case 2 Control Case 1 Case 2 Control Case 1 Case 2 BM over basilar membrane 1ϩ 1ϩ 2ϩ 2ϩ 00 2ϩ 00 Collagen bundles in spiral ligament 1ϩ (Basal turn) 1ϩ (Basal turn) 1ϩ (Basal turn) 2ϩ 00 2ϩ 00 BM of blood vessels 1ϩ 2ϩ 2ϩ 000000 BM of Schwann cells 2ϩ 2ϩ 1ϩ 1ϩ 00 0 0 0 Stria vascularis 0 0 0 0 0 0 0 0 0 Spiral limbus 2ϩ 2ϩ 1ϩ 2ϩ 00 2ϩ 00 Reissner membrane cells 2ϩ 1ϩ 2ϩ 2ϩ 01ϩ 2ϩ 00

Abbreviations: BM, basement membrane; 0, negative; 1ϩ, positive; 2ϩ, strongly positive.

␣3 Chain of Type IV Collagen trol ear. As might be expected, ␣1 expression was seen in many cochlear tissues, including the BM overlying the basi- There were striking differences between the control ear lar membrane, the BM of Schwann cells, the BM of blood and the 2 specimens with Alport syndrome. In the con- vessels, and extracellular matrix of the spiral ligament and trol ear, strong staining was observed in the BM overly- spiral limbus. These results for the ␣1 chain antibody were ing the basilar membrane and in collagen bundles similar to those published by Kleppel et al.9 Immuno- within both the spiral ligament and spiral limbus, with staining for the ␣3 and ␣5 chains in the control ear was less intense staining in the BM of Schwann cells. No seen only in selected tissues, namely, the BM over the basi- staining was seen in the BM of the cochlear vasculature lar membrane, collagen bundles within the spiral liga- or in the stria vascularis. Staining was also observed ment and spiral limbus, and within Reissner membrane within the cells comprising the Reissner membrane. In cells. No staining was seen within BMs of blood vessels both cases of Alport syndrome, there was a general or within the stria vascularis. While expression of ␣3 (and absence of staining in the cochlea using the ␣3 chain ␣4) type IV collagen within the BM of the basilar mem- antibody. In case 2 (but not in case 1), there was posi- brane and within the spiral limbus was also reported by tive staining of Reissner membrane cells in some turns Kleppel et al,9 the strong staining with the spiral ligament (Table and Figure). seen in the present study was not observed in the study by Kleppel et al.9 The positive immunostaining with the ␣5 Chain of Type IV Collagen spiral ligament and other tissues disappeared when the tissue was preabsorbed with ␣5 type IV collagen, indicating ␣ The results for the 5 chain were similar to those seen that the observed staining was real. Furthermore, a search ␣ for the 3 chain. The control ear showed strong stain- of GenBank’s nonredundant protein database for the non- ing within the BM overlying the basilar membrane, within collagenous NC1 domain of COL4A5 revealed no known the spiral ligament and spiral limbus, and within the homology to any other gene, lending further credence to Reissner membrane cells. No staining was observed in the notion that the ␣5 staining within the spiral ligament the control ear for Schwann cells or blood vessels. When was not a cross-reaction to a similar gene product. We are sections of the control ear were processed after preab- unsure how to interpret the positive staining for ␣3 and ␣ sorption of the antibody using 5 type IV collagen an- ␣5 within the Reissner membrane. Unlike other cochlear tigen, no staining was seen within the cochlea. No stain- tissues, the cytoplasm of the cells appeared to be stained, ␣ ing was noted for the 5 chain antibody in both cases 1 rather than the BM or extracellular matrix. and 2 (Table and Figure). Both cases with Alport syndrome were the result of a COL4A5 mutation. The epidermal BMs of case 1 lacked COMMENT expression of the ␣5 chain of type IV collagen. In case 2, deletion of exons 43 through 47 of the COL4A5 gene would To our knowledge, the distribution of isotypes of type be expected to prevent formation of ␣3-␣4-␣5 trimers that IV collagen within the human inner ear in Alport syn- would be stably expressed in the BM. The cochlear sec- drome has not been previously reported. We had the tions of both cases showed similar results. There was vir- unique opportunity to immunostain cochlear tissues from tual absence of staining for the ␣3 and ␣5 chains, whereas 2 men with Alport syndrome caused by mutations in the positive ␣1 staining was seen in various tissues similar to COL4A5 gene. We hypothesized that the cochlear tis- the control ear. These results are consistent with the hy- sues of both individuals would show absence of isotype pothesis that isotype switching did not occur within the switching, similar to what occurs in renal tissues.2 If our cochlea in both of these individuals with Alport syndrome. hypothesis were correct, we would expect to find posi- The role of various isoforms of type IV collagen within tive immunostaining for the ␣1 chain antibody, but no the inner ear (or other tissues) remains unknown at staining for the ␣3 and ␣5 chain antibody. present. Basement membranes have diverse biological The initial step in our study was to examine the distri- functions such as maintaining tissue architecture, em- bution of the ␣1, ␣3, and ␣5 chains in an unaffected con- bryonic development, and tissue remodelling.2,3,9,11,12 The

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Vessel

RM α-1 SV

SpL µ Basilar Membrane Basilar Membrane 100 m 100 µm 100 µm Vessel

RM α-3

SpL SpL SpL

µ Basilar Membrane Basilar Membrane 100 m 100 µm 100 µm

SV SV

α-5

SpL OoC SpL

Basilar Membrane 100 µm SpL 100 µm 100 µm

After Preabsorption With α5 Antigen

SpL

Basilar Membrane 100 µm

Figure. Results of immunostaining within the cochlea for antibodies against ␣1, ␣3, and ␣5 chains of type IV collagen for the control ear, as well as cases 1 and 2 with Alport syndrome. Expression of ␣1 is similar in the control ear and both cases with Alport syndrome, being present in the basement membrane (BM) overlying the basilar membrane, the BM of blood vessels, and the BM of Schwann cells. Faint staining is also seen in some collagen bundles within the spiral ligament (SpL). Staining in the control ear for ␣3 and ␣5 collagen is similar, with strong staining in the BM over the basilar membrane and in collagen bundles with the spiral ligament. The ␣5 staining in the control ear disappears when the tissue is preabsorbed with ␣5 type IV collagen antigen, indicating that the observed staining is real. The BM of blood vessels does not stain for ␣3or␣5 chains. Both cases of Alport syndrome show lack of ␣3 and ␣5 staining. We are unsure how to interpret the positive staining in the Reissner membrane (RM). When examined with high-powered magnification, the cytoplasm of RM cells appeared to be stained, rather than the BM or extracellular matrix (unlike other cochlear structures). OoC indicates organ of Corti; SV, stria vascularis.

presence of ␣3 and ␣5 type IV collagen within the BM The pathophysiologic mechanisms of SNHL in Al- of the organ of Corti and within the spiral ligament and port syndrome remain unknown. In the past, temporal spiral limbus must confer special properties to these struc- bone studies had failed to identify consistent histopatho- tures that allow them to function properly. What these logic abnormalities within the cochlea.13-16 Our team’s re- special properties might be remains to be elucidated. cent study of the otopathologic changes in a series of 9

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 cases of Alport syndrome showed 2 characteristic histo- the hearing loss. Although it is not possible to mea- pathological abnormalities: a separation between the basi- sure endolymphatic potential in humans, it is feasible lar membrane and the overlying BM of the organ of Corti to make these measurements in animals. and cellular in-filling of the tunnel and extracellular spaces These findings underscore the need for a valid ani- of the organ of Corti.8 We had hypothesized that these mal model for hearing loss in Alport syndrome. While abnormalities result in SNHL by altering cochlear mi- several canine and murine models of Alport syndrome cromechanics. The present study shows that the BM of have been described,23 the reported phenotypes have the organ of Corti contains ␣3 and ␣5 chains of type IV not demonstrated hearing loss or the characteristic collagen in the normal control case. In Alport syn- cochlear histopathologic abnormalities seen in drome, both ␣3 and ␣5 chains are absent and the BM con- humans. No hearing loss was found in a mouse model tains only the ␣1 chain. We believe that this defective of autosomal Alport syndrome,12 and these mice BM permits normal cochlear function early in life but re- showed significant thickening of BMs surrounding sults in architectural and functional abnormalities over blood vessels in the stria vascularis, a finding that was time, leading to SNHL. For example, the BM may fail to not seen in the human cases.8 In the canine model, provide adequate adhesion between cells of the organ of Harvey et al22 reported no hearing loss between Corti and the basilar membrane so that basilar mem- affected and unaffected animals up to the age of 5 brane motion is not properly tuned by the outer hair cells. months. One affected dog tested at age 7 months Such a hypothesis is consistent with clinical observa- showed a threshold shift of 25 to 40 dB, which was tions that speech discrimination usually remains excel- thought to be perhaps due to hypocalcemia or uremia. lent in Alport syndrome and the SNHL generally does not The authors did not comment on the morphologic fea- exceed 60 to 70 dB.17,18 tures of the organ of Corti in the affected dogs, espe- The finding that ␣3 and ␣5 are present within the cially the separation between the BM and basilar mem- normal spiral ligament but absent in Alport syndrome brane and cellular in-filling within the tunnel of Corti also enables one to propose an alternative hypothesis that have been observed in humans. for the SNHL. It has become increasingly clear in In conclusion, in the normal human cochlea, ␣1 recent years that the spiral ligament plays an impor- type IV collagen was present ubiquitously in BMs, tant role in regulating the metabolic and ionic milieu while expression of ␣3 and ␣5 chain was restricted to of the inner ear.19-21 The fibrocytes of the ligament are the BM overlying the basilar membrane and to the col- richly endowed with a variety of proteins and enzymes lagen bundles within the spiral ligament and spiral and coupled together by gap junctional systems that limbus. In the 2 cases with X-linked Alport syndrome permit recycling of potassium as well as other (un- due to mutations in the COL4A5 gene, the distribution known) functions. Thus, it is conceivable that lack of of ␣1 type IV collagen was similar to that seen in a the normal ␣3, ␣4, and ␣5 isotypes of type IV collagen control ear, whereas there was absence of staining for leads to dysfunction of the ligament, with loss of endo- ␣3 and ␣5 chains. The results indicate that isotype lymphatic potential or other abnormalities in the ionic switching does not occur within the cochlea in Alport content of endolymph, in turn resulting in dysfunction syndrome. The results are also consistent with the of hair cells within the organ of Corti. A mechanical hypotheses that the SNHL in Alport syndrome may be dysfunction of the spiral ligament as the cause of SNHL due to alterations in cochlear micromechanics or dys- in Alport syndrome has been hypothesized by Harvey et function of the spiral ligament. al,22 using a canine model of X-linked Alport syndrome caused by a nonsense mutation of the COL4A5 gene. The authors suggested that loss of the ␣3, ␣4, and ␣5 network within the spiral ligament would reduce ten- Submitted for Publication: June 14, 2005; accepted July sion on the basilar membrane leading to hearing loss. 7, 2005. We note that at the light microscopic level, neither the Correspondence: Saumil N. Merchant, MD, Massachu- 2 cases in the present study nor any of the cases in our setts Eye and Ear Infirmary, 243 Charles St, Boston, MA group’s previous study8 showed morphologic evidence 02114-3096 ([email protected]). of abnormalities or degeneration within the spiral liga- Author Contributions: Dr Merchant had full access to all ment that was greater than what was expected for age. the data in the study and takes responsibility for the integ- Additional studies will be needed to ascertain the rity of the data and the accuracy of the data analysis. possible mechanisms of hearing loss in Alport syn- Financial Disclosure: None. drome. Both hypotheses (abnormalities of cochlear Funding/Support: We are grateful to Axel Eliasen for his micromechanics or spiral ligament dysfunction) generous support of this work. This work was also sup- would predict that evoked otoacoustic emissions ported by the National Institute of Diabetes and Diges- would be abnormal or absent in ears with hearing loss tive and Kidney Diseases, Bethesda, Md (Drs Kashtan and due to Alport syndrome because functional outer hair Kalluri); the Curtis L. Atkin Memorial Fund (Dr Gregory) cells are necessary for otoacoustic emissions.16 A and the National Institute of Deafness and Other Com- potential method of differentiating between the 2 munications Disorders National Temporal Bone, Hear- hypotheses would be to measure endolymphatic ing and Balance Pathology Resource Registry, Boston, potential. A normal endolymphatic potential in the Mass. presence of SNHL would argue against metabolic dys- Acknowledgment: We thank Derin Wester, PhD, for pro- function of the spiral ligament as being responsible for viding the audiometric data for case 2.

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 IV collagen chains during postnatal development in the murine cochlea. Hear Res. REFERENCES 1996;100:21-32. 12. Cosgrove D, Samuleson G, Meehan DT, et al. Ultrastructural, physiological, and 1. Alport AC. Hereditary familial congenital haemorrhagic nephritis. BMJ. 1927;1: molecular defects in the inner ear of a gene-knockout mouse model for autoso- 504-506. mal Alport syndrome. Hear Res. 1998;121:84-98. 2. Kashtan CE. Alport syndrome: an inherited disorder of renal, ocular and coch- 13. Fujita S, Hayden RC. Alport’s syndrome: temporal bone report. Arch Otolaryngol. lear basement membranes. Medicine (Baltimore). 1999;78:338-360. 1969;90:453-466. 3. Hudson BG, Tryggvason K, Sundaramoorthy M, Neilson EG. Alport syndrome, 14. Myers GJ, Tyler HR. The etiology of deafness in Alport’s syndrome. Arch Goodpasture’s syndrome, and type IV collagen. N Engl J Med. 2003;348:2543- Otolaryngol. 1972;96:333-340. 2556. 15. Smith RJH, Steel KP, Barkway C, Soucek S, Michaels L. A histologic study of 4. Kalluri R, Cosgrove D. Assembly of type IV collagen: insights from ␣3(IV) collagen- nonmorphogenetic forms of hereditary hearing impairment. Arch Otolaryngol Head deficient mice. J Biol Chem. 2000;275:12719-12724. Neck Surg. 1992;118:1085-1094. 5. Miner JH, Sanes JR. Collagen IV a3, a4 and a5 chains in rodent basal laminae: 16. Schuknecht HF. Pathology of the Ear. 2nd ed. Philadelphia, Pa: Lea and Febiger; 1993. sequence, distribution, association with laminins, and developmental switches. 17. Rintelmann W. Auditory manifestations of Alport’s disease syndrome. Trans Sect J Cell Biol. 1994;127:879-891. Otolaryngol Am Acad Ophthalmol Otolaryngol. 1976;82:375-387. 6. Kalluri R, Shield CF, Todd P, Hudson BG, Neilson EG. Isoform switching of type 18. Gleeson MJ. Alport’s syndrome: audiological manifestations and implications. IV collagen is developmentally arrested in X-linked Alport syndrome leading to J Laryngol Otol. 1984;98:449-465. increased susceptibility of renal basement membranes to endoproteolysis. J Clin 19. Weber PC, Cunningham CD III, Schulte BA. Potassium recycling pathways in the Invest. 1997;99:2470-2478. human cochlea. Laryngoscope. 2001;111:1156-1165. 7. Harvey SJ, Zheng K, Sado Y, et al. Role of distinct type IV collagen networks in 20. Adams JC. Clinical implications of inflammatory cytokines in the cochlea: a tech- glomerular development and function. Kidney Int. 1998;54:1857-1866. nical note. Otol Neurotol. 2002;23:316-322. 8. Merchant SN, Burgess BJ, Adams JC, et al. Temporal bone histopathology in 21. Shen Z, Liang F, Hazen-Martin DJ, Schulte BA. BK channels mediate the voltage- Alport syndrome. Laryngoscope. 2004;114:1609-1618. dependent outward current in type I spiral ligament fibrocytes. Hear Res. 2004; 9. Kleppel MM, Santi PA, Cameron JD, Wieslander J, Michael AF. Human tissue 187:35-43. distribution of novel basement membrane collagen. Am J Pathol. 1989;134: 22. Harvey SJ, Mount R, Sado Y, et al. The inner ear of dogs with X-linked nephritis 813-825. provides clues to the pathogenesis of hearing loss in X-linked Alport syndrome. 10. Adams JC. Biotin amplification of biotin and horseradish peroxidase signals in Am J Pathol. 2001;159:1097-1104. histochemical stains. J Histochem Cytochem. 1992;40:1457-1463. 23. Kashtan CE. Animal models of Alport syndrome (editorial comments). Nephrol 11. Cosgrove D, Kornak JM, Samuelson G. Expression of basement membrane type Dial Transplant. 2002;17:1359-1361.

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