Accurate 24-h urine cystine quantification for patients on cystine-binding thiol drugs.

Cystinuria is a rare disorder resulting in development of recurrent kidney stones, adversely affecting patient quality of life. The goal of cystinuria management is to reduce stone formation by increasing cystine solubility in urine, which includes lowering the urinary cystine level below its solubility limit. Treatment usually involves alkalinization of the urine and often requires initiating pharmacotherapy with a cystine-binding thiol drug (CBTD) such as tiopronin; however, proper dose adjustment requires accurate measurement of urinary cystine. The goal of this study was to validate a novel high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for quantification of cystine in the urine of patients with cystinuria receiving a CBTD. Urine samples were collected over 24 h from 24 patients and separated into 2 aliquots. Chromatographic separation of samples was conducted and separation of cystine from the cysteine-tiopronin drug complex was complete in < 3 min. The method was validated for accuracy, precision, linearity, limit of detection (LOD), and limit of quantification (LOQ). Mean accuracy range was 97.7-102.3%; intermediate precision was high with relative percent difference values calculated at 1.2-9.3%; the calibration curve resulted in a linear response throughout the concentration range (R2 = 0.998); and the LOD and LOQ were 0.002 and 0.005 mg/mL, respectively. Mean (range) cystine concentrations measured were 111.10 (51.31-179.46) and 242.21 (61.14-741.80) g/L in Aliquots A and B, respectively. The HPLC-MS/MS method presented here indicates that urine cystine can be reliably quantified in patients receiving a CBTD.

Spontaneous hair cell regeneration in the neonatal mouse cochlea in vivo.

Loss of cochlear hair cells in mammals is currently believed to be permanent, resulting in hearing impairment that affects more than 10% of the population. Here, we developed two genetic strategies to ablate neonatal mouse cochlear hair cells in vivo. Both Pou4f3(DTR/+) and Atoh1-CreER™; ROSA26(DTA/+) alleles allowed selective and inducible hair cell ablation. After hair cell loss was induced at birth, we observed spontaneous regeneration of hair cells. Fate-mapping experiments demonstrated that neighboring supporting cells acquired a hair cell fate, which increased in a basal to apical gradient, averaging over 120 regenerated hair cells per cochlea. The normally mitotically quiescent supporting cells proliferated after hair cell ablation. Concurrent fate mapping and labeling with mitotic tracers showed that regenerated hair cells were derived by both mitotic regeneration and direct transdifferentiation. Over time, regenerated hair cells followed a similar pattern of maturation to normal hair cell development, including the expression of prestin, a terminal differentiation marker of outer hair cells, although many new hair cells eventually died. Hair cell regeneration did not occur when ablation was induced at one week of age. Our findings demonstrate that the neonatal mouse cochlea is capable of spontaneous hair cell regeneration after damage in vivo. Thus, future studies on the neonatal cochlea might shed light on the competence of supporting cells to regenerate hair cells and on the factors that promote the survival of newly regenerated hair cells.

Regulation of p27Kip1 by Sox2 maintains quiescence of inner pillar cells in the murine auditory sensory epithelium.

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27(Kip1), a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27(Kip1), p27(Kip1)-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27(Kip1) gradually declined with age. In addition, deletion of Sox2 or p27(Kip1) did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27(Kip1) promoter support that Sox2 directly activates p27(Kip1) transcription in postmitotic IPCs. Hence, in contrast to the well known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27(Kip1) to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27(Kip1) expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.

Polycystin-1 is required for stereocilia structure but not for mechanotransduction in inner ear hair cells.

The polycystic kidney disease-1 (Pkd1) gene encodes a large transmembrane protein (polycystin-1, or PC-1) that is reported to function as a fluid flow sensor in the kidney. As a member of the transient receptor potential family, PC-1 has also been hypothesized to play a role in the elusive mechanoelectrical transduction (MET) channel in inner ear hair cells. Here, we analyze two independent mouse models of PC-1, a knock-in (KI) mutant line and a hair cell-specific inducible Cre-mediated knock-out line. Both models exhibit normal MET channel function at neonatal ages despite hearing loss and ultrastructural abnormalities of sterecilia that remain properly polarized at adult ages. These findings demonstrate that PC-1 plays an essential role in stereocilia structure and maintenance but not directly in MET channel function or planar cell polarity. We also demonstrate that PC-1 is colocalized with F-actin in hair cell stereocilia in vivo, using a hemagglutinin-tagged PC-1 KI mouse model, and in renal epithelial cell microvilli in vitro. These results not only demonstrate a novel role for PC-1 in the cochlea, but also suggest insight into the development of polycystic kidney disease.

Vacuolization and alterations of lysosomal membrane proteins in cochlear marginal cells contribute to hearing loss in neuraminidase 1-deficient mice.

The neuraminidase-1 (Neu1) knockout mouse model is a phenocopy of the lysosomal storage disease (LSD) sialidosis, characterized by multisystemic and neuropathic symptoms, including hearing loss. We have characterized the auditory defects in Neu1(-/-) mice and found that hearing loss involves both conductive and sensorineural components. Auditory brainstem response (ABR) thresholds were significantly elevated in Neu1(-/-) mice at P21 (48-55 dB), and hearing loss appeared progressive (53-66 dB at P60). At these ages Neu1(-/-) mice accumulated cerumen in the external ear canal and had a thickened mucosa and inflammation in the middle ear. In cochleae of adult wild-type mice, Neu1 was expressed in several cell types in the stria vascularis, the organ of Corti, and spiral ganglion. Progressive morphological abnormalities such as extensive vacuolization were detected in the Neu1(-/-) cochleae as early as P9. These early morphologic changes in Neu1(-/-) cochleae were associated with oversialylation of several lysosomal associated membrane proteins (Lamps) in the stria vascularis. A marked increase in the expression and apical localization of Lamp-1 in marginal cells of the stria vascularis predicts exacerbation of lysosomal exocytosis into the endolymph. Consequently, the endolymphatic potential in Neu1(-/-) mice was reduced by approximately 20 mV at ages P31-P44, which would cause dysfunction of transduction in sensory hair cells. This study suggests a molecular mechanism that contributes to hearing loss in sialidosis and identifies potential therapeutic targets.