Calcium oxalate stone formation in the inner ear as a result of an Slc26a4 mutation

Abstract

Calcium oxalate stone formation occurs under pathological conditions and accounts for more than 80% of all types of kidney stones. In the current study, we show for the first time that calcium oxalate stones are formed in the mouse inner ear of a genetic model for hearing loss and vestibular dysfunction in humans. The vestibular system within the inner ear is dependent on extracellular tiny calcium carbonate minerals for proper function. Thousands of these biominerals, known as otoconia, are associated with the utricle and saccule sensory maculae and are vital for mechanical stimulation of the sensory hair cells. We show that a missense mutation within the Slc26a4 gene abolishes the transport activity of its encoded protein, pendrin. As a consequence, dramatic changes in mineral composition, size, and shape occur within the utricle and saccule in a differential manner. Although abnormal giant carbonate minerals reside in the utricle at all ages, in the saccule, a gradual change in mineral composition leads to a formation of calcium oxalate in adult mice. By combining imaging and spectroscopy tools, we determined the profile of mineral composition and morphology at different time points. We propose a novel mechanism for the accumulation and aggregation of oxalate crystals in the inner ear.

FIGURE 1.

The S408F mutation resides within a highly conserved region of the pendrin protein and leads to deafness and vestibular dysfunction. A and B, sequencing of Slc26a4 from loop homozygote cDNA revealed a C to T (c.C1439T) mutation causing a serine to phenylalanine amino acid (aa) substitution at position 408 (p.S408F) of the pendrin protein. A hypothetical predicted topology model of pendrin suggests that the pendrin mutation resides in the ninth transmembrane domain. STAS, sulphate transporter and anti-sigma factor antagonist. ConSeq analysis shows that the loop mutation position is within a highly conserved amino acid with the highest value of nine. mPds, mouse Pds. C, auditory brainstem response test on 8-week-old mice reveals that Slc26a4^loop mutants are profoundly deaf according to three frequencies that were tested, 8, 16, and 32 kHz. Output graphs from the 16-kHz examination are shown. n = 21.

FIGURE 2.

The S408F mutation causes a significant reduction of the related anion transport tested for the mouse pendrin (A), as well as for the human pendrin (B). A, change of the fluorescence signal (expressed as maximal fluorescence variations, ΔF_max%) after the Cl⁻ to I⁻ or I⁻ to Cl⁻ substitutions (for details, see “Experimental Procedures”) in HEK 293 Phoenix cells expressing only the EYFP protein (empty) or the EYFP protein and mouse wild-type pendrin (mPds WT) or mutated mouse Pds (mPds S408F). The numbers of the experiments are given (n). Error bars indicate the S.E. Statistical analysis: ***, p < 0.001; *, = p < 0.05; paired Student's t test. Error bars indicate S.E. B, change of the fluorescence signal (expressed as maximal fluorescence variations, ΔF_max%) after the Cl⁻ to I⁻ or I⁻ to Cl⁻ substitutions (for details, see “Experimental Procedures”) in HEK 293 Phoenix cells expressing only the EYFP protein (empty) or the EYFP protein and human wild-type PDS (hPDS WT) or mutated human PDS (hPDS S408F). The numbers of the experiments are given (n). Error bars indicate the S.E. Statistical analysis: ***, = p < 0.001; **, = p < 0.01; n. s. = not significant; paired Student's t test. Error bars indicate S.E. The results of the analysis of variance test with Bonferroni's multiple comparison post-test are reported under “Results.” n represents the number of cells.

FIGURE 3.

Gross malformation of the vestibular gravity receptor. A, wild-type otoconia can be easily detected under a light microscope when looking at isolated inner ears. Two bright reflected areas represent the otoconia of the saccule and utricle of newborn mice. Slc26a4^loop mutant mice lack the bright reflection, and instead, a dark hole appears when looking through the oval window (arrowheads). B, SEM of 2-month-old utricle shows thousands of otoconia (inset) that cover the sensory epithelium in a wild-type mouse, whereas a giant stone is appearing at Slc26a4^loop/loop utricle. C, imaging of the gelatinous matrix (otoconial membrane) reveals its normal structure in wild-type mice, which is characterized by typical pores. In Slc26a4^loop mutants, the otoconial membrane is dissociated and severed. D, high resolution images of the underlying hair cells show that Slc26a4^loop/loop vestibular hair cells appear to be normal. Scale bars equal 500 μm in panel A, 100 μm in B, 5 μm in C and D. n = 4.

FIGURE 4.

Mineral morphology and composition across different progressive stages. A schematic illustration of the inner ear summarizing the morphological differences of the ear stones at three time points, P0, 7 months, and 10 months, is shown (left panel). Left and right panel, SEM images of the minerals from the utricle (left panel) and saccule (right panel). A and B, otoconia from wild-type mice. The image shows the small (∼7 μm) otoconia with typical morphology (inset in A and B). D and E, minerals extracted from the utricle (D) and saccule (E) of newborn Slc26a4^loop mutant mice. The utricle minerals are larger; both types have altered morphology relative to wild-type otoconia and show smooth surfaces. G and H, minerals extracted from 7-month-old Slc26a4^loop mutant mice. Although in the utricle, no changes can be observed as compared with the minerals of newborn mice, the saccule contained an abnormal giant stone with a variety of morphological characteristics. SEM images showed that parts of this stone resemble the morphology of weddellite, calcium oxalate dihydrate (inset in H). J and K, minerals extracted from 10-month-old Slc26a4^loop mutant mice. The minerals in the utricle appear similar to the minerals found at earlier stages (J). In the saccule, a new type of mineral can be found instead of the giant calcitic crystals observed earlier. These minerals have a crystal morphology typical of calcium oxalate dihydrate (weddellite) crystals. C, F, I, and L, normalized FTIR spectra of minerals extracted from the saccule and utricle at different developmental stages. The parts of the spectra in the range below 1000 cm⁻¹ are expanded to reveal the details. C, wild-type crystals. The spectrum of wild-type otoconia shows the typical calcite vibrations at 713, 875, and 1422 cm⁻¹. The spectrum also shows large contribution from organic components at 1638 and around 1047 cm⁻¹. F, spectrum of the mineral extracted from the saccule and utricle in newborn Slc26a4^loop mutant mice. I, spectrum of the mineral extracted from the saccule in Slc26a4^loop mutant mice at the age of 7 months. Note broadening of the peak at 713 cm⁻¹. L, spectrum of the mineral extracted from the saccule in Slc26a4^loop mutant mice at the age of 10 months. Vibrations at 1324 and 776 cm⁻¹ are typical of weddellite crystals. Scale bars equal 50 μm in panels A and B; scale bars equal 100 μm in panels D, E, G, H, J, and K. n ≥ 15.

FIGURE 5.

Ultrastructural morphology of the surface and core of Slc26a4^loop/loop mineralized bodies. A and B, bisected calcite minerals of P0 utricle. A, the interior facet of bisected mineral shows a smooth surface similar to the exterior surface of the mineral. B, high magnification of the interior surface. C–E, surface of different mineralized bodies isolated from Slc26a4^loop/loop saccule presented with high power SEM images. C, calcium carbonate (calcite) minerals of P0 mice have a smooth and clean surface. D, highly disordered calcite minerals of 7-month-old saccules revealed a surface that is pitted and fissured (arrows). This stone contained domains that resemble the morphology of calcium oxalate in the form of weddellite (circled). E, calcium oxalate (weddellite) mineral of 10-month-old saccules show a smooth and clean surface. Scale bars equal 25 μm in panel A, 5 μm in panel B, and 10 μm in panels C–E. n = 6.

FIGURE 6.

Oc90 conglomerates assemble the core of the calcitic giant minerals. A and B, Oc90 immunostaining on P15 paraffin sections of decalcified inner ears. A, wild-type otoconia are labeled with Oc90 (red) and highlight the boundaries of each otoconium. The unlabeled gap between the sensory epithelium (nuclei) and the otoconia represents the gelatinous matrix (otoconial membrane) that supports the otoconia load. B, a Oc90 (red) conglomerate is present in the giant calcitic mineral. Unlike the smooth expression of Oc90 in a single otoconium (inset in A), in the giant mineral, Oc90 expression has different levels of intensity along the mineral body. Furthermore, the giant mineral resides directly on the sensory epithelium, and the gap that corresponds to the otoconial membrane is absent. C, Western blot analysis of Oc90 in the mineral fraction shows a dramatic increase of protein content in the giant minerals. As a control, the non-mineral fraction was loaded on a gel and labeled with HSC70, confirming the equal amount of pooled utricles that were analyzed in each sample. D and E, otogelin immunostaining on P15 paraffin sections of decalcified inner ears. D, in wild-type mice, otogelin (green) is expressed by the supporting cells of the sensory epithelium. The secreted otogelin participates in the otoconial membrane assembly and maintenance. E, in Slc26a4^loop mutant mice, otogelin is expressed by supporting cells; however, the otoconial membrane is severed, disorganized and displaced (arrow in D). Scale bars equal 75 μm in panels A–D. n = 5.

FIGURE 7.

Giant minerals are dislocated in Slc26a4^loop/loop vestibular system. A schematic representation of the inner ear (left panels) illustrates the possible positions of the ectopic mineralized bodies as found in a group of Slc26a4^loop mutant mice. A–F, histological paraffin sections from the vestibular system of P0 mice stained with hematoxylin and eosin. A and B, in wild-type mice, otoconia are restricted to the utricle and saccule, seen as a dense layer of tiny particles over the sensory epithelium. C, other endolymphatic compartments lack any otoconia, as demonstrated with a representative image of a semicircular canal. D and E, in Slc26a4^loop mutant mice, a giant mineral resides on top of the gravity receptors, utricle, and saccule. F, a frequent dislocation of the giant mineralized bodies to the semicircular canals and their cristae is observed. Scale bars equal 75 μm in panels A–F. n = 4.

FIGURE 8.

Proposed working model for calcium oxalate stone formation in the inner ear. The anatomical differences (top panels) between the utricle (green) and saccule (red) are illustrated and summarized in a table. A, under normal conditions, otoconia nucleation begins around E16.5 and reaches its maturation at P7. To support the biomineralization events, otoconia proteins such as Oc90 are secreted to the endolymph prior to and during otoconia nucleation and maturation. B, in Slc26a4^loop mice, the organic fraction of otoconia, including Oc90, is secreted normally to the extracellular space, but the homeostasis of the endolymph is impaired. Pendrin activity is depleted due to the S408F mutation, and the lack of HCO₃⁻ supply leads to acidification of the endolymphatic fluids. This acidification abolishes the reabsorption of calcium by the pH-sensitive calcium channels, TRPV5 and TRPV6 (46). The localization of TRPV5 and TRPV6 in the semicircular canal duct epithelium and in the vestibular dark cells, which share fluid circulation with the utricle, leads to a higher calcium concentration in the utricle as compared with the saccule. The excess calcium ions in the endolymph of the utricle are sequestered by large amounts of Oc90 and deposited into oversized calcite minerals, whereas in the saccule, significantly smaller minerals are formed. C, at progressive ages, wild-type otoconia are maintained with low calcium turnover, whereas in Slc26a4^loop mice, a differential process between saccule and utricle occurs. In the utricle, giant calcitic minerals reside all along the lifespan of the mouse. In the saccule, a gradual change in mineral morphology and composition from calcite into highly disordered calcite at the age of 7 months is observed. The ultrastructural morphology of the highly disordered mineral, a pitted and fissured surface, resembles the morphology of calcite mineral after treatment with an acidic solution. Moreover, this stone contained domains that resembled the morphology of calcium oxalate in the form of weddellite. Between the age of 7 and 10 months, the highly disordered calcite dissolved, and giant calcium oxalate minerals in the form of weddellite were generated. The symmetrical morphology of the weddellite resembles the classical calcium oxalate geological mineral, which is more stable at lower pH as compared with the calcium carbonate. In summary, constant acidification of the saccule leads to dissolution of the calcite mineral that is tied with favorable conditions for calcium oxalate stone formation in the inner ear.