We previously demonstrated that endothelin A receptor (ETAR) signaling plays a major role in regulating strial pathology in the COL4A3 knockout mouse model for Alport syndrome. Blocking the receptor prevented accumulation of extracellular matrix (ECM) in the strial capillary basement membranes (SCBMs) including laminin α2. Herein, we explored whether α-dystroglycan receptors for laminin α2 are present on cultured strial pericytes, marginal cells, and intermediate cells, as well as in intact stria vascularis. We performed comparative analysis of gene expression in isolated glomeruli and stria vascularis from wild type and Alport mice using RNA-seq. We examined the effects of endothelin treatment of pericytes on cytoskeletal dynamics and CDC42 activation using immunofluorescence, and on cell signaling using phosphoantibody microarrays. mRNA encoding α-dystroglycan receptors are present in strial cell lines as well as RNA from micro dissected stria vascularis. These same receptors were found adjacent to the capillary basement membranes on strial endothelial cells, marginal cells, intermediate cells, and pericytes in vivo by immunofluorescence. RNA-seq analysis of RNA from wild type and Alport glomeruli and stria vascularis revealed similar regulation of genes implicated in the pathogenesis of both organs. Treatment of pericytes with endothelin-1 (ET-1) resulted in changes in cell signaling consistent with observed reduction of filamentous to globular actin ratios and cell adhesion. Therefore, the induction of laminin α2 likely contributes to strial pathology in Alport syndrome via signaling through α-dystroglycan receptors. ET-1 activation of ETARs directly contributes to altered cell signaling in strial pericytes, resulting in changes in actin cytoskeletal dynamics.

Alterations in mitochondrial function are the linchpin in numerous disease states including in the development of chemotherapy-induced neuropathic pain (CIPN), a major dose-limiting toxicity of widely used chemotherapeutic cytotoxins. In CIPN, mitochondrial dysfunction is characterized by deficits in mitochondrial bioenergetics (e.g., decreased ATP production) that are thought to drive the degeneration of the peripheral nerve sensory axon terminal sensory arbors in the skin (the intraepidermal nerve fibers; IENFs) and induce abnormal spontaneous discharge in peripheral nerve sensory axons. Preserving mitochondrial function is anticipated to prevent CIPN. We have now discovered that the G-protein-coupled receptor, A3adenosine receptor subtype (A3AR), is expressed on the mitochondrial outer membrane. Ex vivo application of a highly selective A3AR agonist, MRS5980, to saphenous nerve microfilaments harvested from male oxaliplatin-treated rats reversed the loss in ATP production underscoring mitoprotective effects resulting from A3AR activation on mitochondria. Moreover, in vivo administration of A3AR agonists to rats during oxaliplatin treatment was associated with reduced IENF loss and a lower incidence of spontaneous discharge in peripheral afferent axons. These effects are accompanied by improved mitochondrial ATP production in primary afferent sensory axons and overall inhibition of the development of neuropathic pain. These data identify for the first time mitochondrial A3AR and indicate that activation of A3AR protects mitochondrial function in primary afferent sensory axons against chemotherapy-induced neurotoxicity. Repurposing A3AR agonists that are already in clinical trials as anticancer agents as adjunct to chemotherapeutics will address a major unmet medical need for which there are no FDA-approved drugs.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for alpha 1 beta 1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. (c) 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hearing loss is the most common form of sensory deficit. It occurs predominantly due to hair cell (HC) loss. Mammalian HCs are terminally differentiated by birth, making HC loss incurable. Here, we show the pharmacogenetic downregulation of Cldn9, a tight junction protein, generates robust supernumerary inner HCs (IHCs) in mice. The putative ectopic IHCs have functional and synaptic features akin to typical IHCs and were surprisingly and remarkably preserved for at least fifteen months >50% of the mouse’s life cycle. In vivo, Cldn9 knockdown using shRNA on postnatal days (P) P1-7 yielded analogous functional putative ectopic IHCs that were equally durably conserved. The findings suggest that Cldn9 levels coordinate embryonic and postnatal HC differentiation, making it a viable target for altering IHC development pre- and post-terminal differentiation.

Endolymphatic hydrops, increased endolymphatic fluid within the cochlea, is the key pathologic finding in patients with Meniere's disease, a disease of episodic vertigo, fluctuating hearing loss, tinnitus, and aural fullness. Endolymphatic hydrops also can occur after noise trauma and its presence correlates with cochlear synaptopathy, a form of hearing loss caused by reduced numbers of synapses between hair cells and auditory nerve fibers. Here we tested whether there is a mechanistic link between these two phenomena by using multimodal imaging techniques to analyze the cochleae of transgenic mice exposed to blast and osmotic challenge. In vivo cochlear imaging after blast exposure revealed dynamic increases in endolymph that involved hair cell mechanoelectrical transduction channel block but not the synaptic release of glutamate at the hair cell-auditory nerve synapse. In contrast, ex vivo and in vivo auditory nerve imaging revealed that synaptopathy requires glutamate release from hair cells but not endolymphatic hydrops. Thus, although endolymphatic hydrops and cochlear synaptopathy are both observed after noise exposure, one does not cause the other. They are simply co-existent sequelae that derive from the traumatic stimulation of hair cell stereociliary bundles. Importantly, these data argue that Meniere's disease derives from hair cell transduction channel blockade.

Altered expression of peripheral myelin protein 22 (PMP22) results in demyelinating peripheral neuropathy. PMP22 exhibits a highly restricted tissue distribution with marked expression in the myelinating Schwann cells of peripheral nerves. Auditory and vestibular Schwann cells and the afferent neurons also express PMP22, suggesting a unique role in hearing and balancing. Indeed, neuropathic patients diagnosed with PMP22-linked hereditary neuropathies often present with auditory and balance deficits, an understudied clinical complication. To investigate the mechanism by which abnormal expression of PMP22 may cause auditory and vestibular deficits, we studied gene-targeted PMP22 -null mice. PMP22 -null mice exhibit an unsteady gait, have difficulty maintaining balance, and live for only ∼3–5 weeks relative to unaffected littermates. Histological analysis of the inner ear revealed reduced auditory and vestibular afferent nerve myelination and profound Na + channel redistribution without PMP22. Yet, Na + current density was unaltered, in stark contrast to increased K + current density. Atypical postsynaptic densities and a range of neuronal abnormalities in the organ of Corti were also identified. Analyses of auditory brainstem responses (ABRs) and vestibular sensory-evoked potential (VsEP) revealed that PMP22 -null mice had auditory and vestibular hypofunction. These results demonstrate that PMP22 is required for hearing and balance, and the protein is indispensable for the formation and maintenance of myelin in the peripheral arm of the eighth nerve. Our findings indicate that myelin abnormalities and altered signal propagation in the peripheral arm of the auditory nerve are likely causes of auditory deficits in patients with PMP22-linked neuropathies.

Alport syndrome (AS), a type IV collagen disorder, leads to glomerular disease and, in some patients, hearing loss. AS is treated with inhibitors of the renin–angiotensin system; however, a need exists for novel therapies, especially those addressing both major pathologies. Sparsentan is a single‐molecule dual endothelin type‐A and angiotensin II type 1 receptor antagonist (DEARA) under clinical development for focal segmental glomerulosclerosis and IgA nephropathy. We report the ability of sparsentan to ameliorate both renal and inner ear pathologies in an autosomal‐recessive Alport mouse model. Sparsentan significantly delayed onset of glomerulosclerosis, interstitial fibrosis, proteinuria, and glomerular filtration rate decline. Sparsentan attenuated glomerular basement membrane defects, blunted mesangial filopodial invasion into the glomerular capillaries, increased lifespan more than losartan, and lessened changes in profibrotic/pro‐inflammatory gene pathways in both the glomerular and the renal cortical compartments. Notably, treatment with sparsentan, but not losartan, prevented accumulation of extracellular matrix in the strial capillary basement membranes in the inner ear and reduced susceptibility to hearing loss. Improvements in lifespan and in renal and strial pathology were observed even when sparsentan was initiated after development of renal pathologies. These findings suggest that sparsentan may address both renal and hearing pathologies in Alport syndrome patients. © 2023 Travere Therapeutics, Inc and The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A point-of-care (POC) device to measure mouse glucose and lipid profiles is an important unmet need for cost-effective, immediate decision making in research. We compared metabolic analyte profiles obtained using a human clinical POC device with those from a veterinary laboratory chemical analyzer (LCA). Unfasted terminal blood samples were obtained by cardiac puncture from C57Bl/6J mice used in a diet-induced obesity model of type 2 diabetes mellitus; age-matched C57Bl/6J controls; a transgenic mouse model of Alzheimer's disease on a C57BL/6J background (16 wk old); and aged C57BL/6J mice (24 to 60 wk old). Aliquots of the blood were immediately assayed onsite using the POC device. Corresponding serum aliquots were sent analyzed by LCA. Measures from the POC and LCA devices were compared by using the Bland-Altman and Passing-Bablok methods. Of a total of 40 aliquots, LCA results were within reported reference ranges for each model. POC results that fell beyond the device range were excluded from the analyses. The coefficient of determination and Passing-Bablok analysis demonstrated that POC glucose and HDL had the best agreement with LCA. The Bland-Altman analysis found no value-dependent bias in glucose and no significant bias in HDL. The remaining lipid analytes (cholesterol and triglyceride) showed significant bias. Until an improved, validated mouse POC device with lipid profile capability is available, the POC device that we tested appears adequate for screening glucose and HDL in mouse blood. Disadvantages of this clinical POC device are the narrow human ranges relative to ranges found in mice and its limited precision as compared with the LCA. This study demonstrates that when the samples are within the device range limits, this human POC device can accurately track metabolic syndrome and be used to compare patterns in glucose and HDL.

Organelle crosstalk is vital for cellular functions. The propinquity of mitochondria, ER, and plasma membrane promote regulation of multiple functions, which include intracellular Ca2+flux, and cellular biogenesis. Although the purposes of apposing mitochondria and ER have been described, an understanding of altered organelle connectomics related to disease states is emerging. Since inner ear outer hair cell (OHC) degeneration is a common trait of age-related hearing loss, the objective of this study was to investigate whether the structural and functional coupling of mitochondria with subsurface cisternae (SSC) was affected by aging. We applied functional and structural probes to equal numbers of male and female mice with a hearing phenotype akin to human aging. We discovered the polarization of cristae and crista junctions in mitochondria tethered to the SSC in OHCs. Aging was associated with SSC stress and decoupling of mitochondria with the SSC, mitochondrial fission/fusion imbalance, a remarkable reduction in mitochondrial and cytoplasmic Ca2+levels, reduced K+-induced Ca2+uptake, and marked plasticity of cristae membranes. A model of structure-based ATP production predicts profound energy stress in older OHCs. This report provides data suggesting that altered membrane organelle connectomics may result in progressive hearing loss.

Previous work demonstrates that the hearing loss in Alport mice is caused by defects in the stria vascularis. As the animals age, progressive thickening of strial capillary basement membranes (SCBMs) occurs associated with elevated levels of extracellular matrix expression and hypoxia-related gene and protein expression. These conditions render the animals susceptible to noise-induced hearing loss. In an effort to develop a more comprehensive understanding of how the underlying mutation in the COL4A3 gene influences homeostasis in the stria vascularis, we performed vascular permeability studies combined with RNA-seq analysis using isolated stria vascularis from 7-week old wild-type and Alport mice on the 129 Sv background. Alport SCBMs were found to be less permeable than wild-type littermates. RNA-seq and bioinformatics analysis revealed 68 genes were induced and 61 genes suppressed in the stria from Alport mice relative to wild-type using a cut-off of 2-fold. These included pathways involving transcription factors associated with the regulation of pro-inflammatory responses as well as cytokines, chemokines, and chemokine receptors that are up- or down-regulated. Canonical pathways included modulation of genes associated with glucose and glucose-1-PO4 degradation, NAD biosynthesis, histidine degradation, calcium signaling, and glutamate receptor signaling (among others). In all, the data point to the Alport stria being in an inflammatory state with disruption in numerous metabolic pathways indicative of metabolic stress, a likely cause for the susceptibility of Alport mice to noise-induced hearing loss under conditions that do not cause permanent hearing loss in age/strain-matched wild-type mice. The work lays the foundation for studies aimed at understanding the nature of strial pathology in Alport mice. The modulation of these genes under conditions of therapeutic intervention may provide important pre-clinical data to justify trials in humans afflicted with the disease.

Abstract Background and Aims In Alport syndrome (AS), endothelin type A receptor (ETAR) activation plays a role in both renal and inner ear pathologies. Currently, angiotensin converting enzyme inhibitors or angiotensin II type 1 receptor (AT1R) blockers are the standard of care for patients with AS; however, these drugs have not been shown to improve hearing. Previously, we showed that sparsentan (SP), a dual ETAR/AT1R inhibitor, prevented increases in proteinuria, fibrosis, glomerulosclerosis, and glomerular basement membrane dysmorphology. The AS mouse model lifespan was extended when SP treatment was started at 3 weeks (W) of age (pre-proteinuric). Here we compare the effect of SP and the AT1R blocker losartan (LS) on lifespan and proteinuria in AS mice treated from 4 W, and on hearing loss and associated inner ear pathology in AS mice treated from 3 W to 8.75 W. Method Wild type (WT) and AS mice were treated daily with vehicle (V), 60, 120, or 200 mg/kg SP (SP60, SP120, or SP200) by oral gavage, or 20 mg/kg LS by daily oral gavage from 3-4 W of age and in drinking water at 10 mg/kg from 4-8.75 W. Three studies were conducted: early intervention for hearing from 3-8.75 W (V, SP120, and LS, n=5) or for assessment of laminin α2, laminin α5, and collagen IVα1 expression in stria vascularis from 3-7 W (V, SP200, and LS, n=7), and for lifespan with treatment from 3 W (V, n=10) or from 4 W (LS, SP60, or SP120, n=5). Urinary protein/creatinine (UP/C) was assessed weekly during the lifespan study. In the hearing study, the auditory brainstem response (ABR) assessed hearing ability and sensitivity to noise at 8-9 W (n=5). The cochleae were preserved and transmission electron microscopy was used to assess severity of strial pathology and to measure strial capillary basement membrane (SCBM) thickness (n=5-7). Accumulation of extracellular matrix in SCBM was determined in mice treated with V, SP200, or LS by immunofluorescence microscopy using antibodies to laminin α2, laminin α5, or collagen IVα1. Results In AS mice treated with SP from 4 W, SP120 significantly (P<0.05) increased median lifespan by 34 days compared to LS-treated mice, and by 50.5 days compared to V-treated mice (Figure 1). AS mice treated with SP60 or LS initiated at 4 W did not have a significant difference in lifespan compared to V. At 8 W, SP120 but not SP60 or LS significantly (P<0.05) attenuated the increase in UP/C compared to V (UP/C mg/mg mean±SD 8 W: 47±16 V; 31±6 LS; 61±44 SP60; 20±3 SP120). Notably, development of UP/C at 11 W in AS-SP120 mice was significantly attenuated (P<0.05) compared to that in AS-LS mice (UP/C mg/mg mean±SD 11 W: 58±8 LS; 77±51 SP60; 18±10 SP120). In hearing studies, although both SP120 and LS significantly (P<0.05) prevented SCBM thickening compared to AS-V mice (mean SCBM width±SD nm: 57.8±2.1 WT-V; 67.6±5.5 AS-V; 54.7±2.4 AS-SP120; 55.0±5.9 AS-LS), post-noise ABR thresholds at 16 kHz were significantly improved only in AS-SP120 mice compared to AS-V mice (*P<0.05; Figure 2). AS-LS post-noise hearing did not differ from that of AS-V mice. Moreover, SP200 tended to ameliorate increases in expression levels of laminin α2, laminin α5, and collagen IVα1 to a greater extent than LS in the SCBM of AS mice. Finally, dysmorphology of the stria vascularis was noted in LS but not SP120-treated AS mice. Conclusion SP120 provided significant nephroprotection in AS mice when treatment was started at 4 W, significantly extending lifespan beyond that of mice treated with LS while delaying the development of proteinuria. SP120 treatment in AS mice reduced the SCBM width. Moreover, SP120 treatment significantly attenuated noise-induced hearing loss in AS mice whereas LS was without effect. These pre-clinical studies, if successfully translated to the clinic, may suggest that sparsentan offers a novel, dual-therapeutic approach in AS by reducing both renal injury and hearing loss.

In 129 Sv autosomal Alport mice, the strial capillary basement membranes (SCBMs) progressively thicken between 5 and 9 weeks of age resulting in a hypoxic microenvironment with metabolic stress and induction of pro-inflammatory cytokines and chemokines. These events occur concomitant with a drop in endocochlear potential and a susceptibility to noise-induced hearing loss under conditions that do not permanently affect age/strain-matched littermates. Here we aimed to gain an understanding of events that occur before the onset of SCBM thickening. Alport stria has normal thickness and shows levels of extracellular matrix (ECM) molecules in the SCBMs commensurate with wild-type mice. Hearing thresholds in the 3-week Alport mice do not differ from those of wild-type mice. We performed RNAseq analysis using RNA from stria vascularis isolated from 3-week Alport mice and wild type littermates. Data was processed using Ingenuity Pathway Analysis software and further distilled using manual procedures. RNAseq analysis revealed significant dysregulation of genes involved in cell adhesion, cell migration, formation of protrusions, and both actin and tubulin cytoskeletal dynamics. Overall, the data suggested changes in the cellular architecture of the stria might be apparent. To test this notion, we performed dual immunofluorescence analysis on whole mounts of the stria vascularis from these same animals stained with anti-isolectin gs-ib4 (endothelial cell marker) and anti-desmin (pericyte marker) antibodies. The results showed evidence of pericyte detachment and migration as well as the formation of membrane ruffling on pericytes in z-stacked confocal images from Alport mice compared to wild type littermates. This was confirmed by TEM analysis. Earlier work from our lab showed that endothelin A receptor blockade prevents SCBM thickening and ECM accumulation in the SCBMs. Treating cultured pericytes with endothelin-1 induced actin cytoskeletal rearrangement, increasing the ratio of filamentous to globular actin. Collectively, these findings suggest that the change in type IV collagen composition in the Alport SCBMs results in cellular insult to the pericyte compartment, activating detachment and altered cytoskeletal dynamics. These events precede SCBM thickening and hearing loss in Alport mice, and thus constitute the earliest event so far recognized in Alport strial pathology.

Potassium (K+) channels shape the response properties of neurons. Although enormous progress has been made to characterize K+ channels in the primary auditory neurons, the molecular identities of many of these channels and their contributions to hearing in vivo remain unknown. Using a combination of RNA sequencing and single molecule fluorescent in situ hybridization, we localized expression of transcripts encoding the sodium-activated potassium channels KNa1.1 (SLO2.2/Slack) and KNa1.2 (SLO2.1/Slick) to the primary auditory neurons (spiral ganglion neurons, SGNs). To examine the contribution of these channels to function of the SGNs in vivo, we measured auditory brainstem responses in KNa1.1/1.2 double knockout (DKO) mice. Although auditory brainstem response (wave I) thresholds were not altered, the amplitudes of suprathreshold responses were reduced in DKO mice. This reduction in amplitude occurred despite normal numbers and molecular architecture of the SGNs and their synapses with the inner hair cells. Patch clamp electrophysiology of SGNs isolated from DKO mice displayed altered membrane properties, including reduced action potential thresholds and amplitudes. These findings show that KNa1 channel activity is essential for normal cochlear function and suggest that early forms of hearing loss may result from physiological changes in the activity of the primary auditory neurons.

Local and privileged expression of dendritic proteins allows segregation of distinct functions in a single neuron but may represent one of the underlying mechanisms for early and insidious presentation of sensory neuropathy. Tangible characteristics of early hearing loss (HL) are defined in correlation with nascent hidden hearing loss (HHL) in humans and animal models. Despite the plethora of causes of HL, only two prevailing mechanisms for HHL have been identified, and in both cases, common structural deficits are implicated in inner hair cell synapses, and demyelination of the auditory nerve (AN). We uncovered that Na+-activated K+ (KNa) mRNA and channel proteins are distinctly and locally expressed in dendritic projections of primary ANs and genetic deletion of KNa channels (Kcnt1 and Kcnt2) results in the loss of proper AN synaptic function, characterized as HHL, without structural synaptic alterations. We further demonstrate that the local functional synaptic alterations transition from HHL to increased hearing-threshold, which entails changes in global Ca2+ homeostasis, activation of caspases 3/9, impaired regulation of inositol triphosphate receptor 1 (IP3R1), and apoptosis-mediated neurodegeneration. Thus, the present study demonstrates how local synaptic dysfunction results in an apparent latent pathological phenotype (HHL) and, if undetected, can lead to overt HL. It also highlights, for the first time, that HHL can precede structural synaptic dysfunction and AN demyelination. The stepwise cellular mechanisms from HHL to canonical HL are revealed, providing a platform for intervention to prevent lasting and irreversible age-related hearing loss (ARHL).

Presbycusis or age-related hearing loss (ARHL) is the most common sensory deficit in the human population. A substantial component of the etiology stems from pathological changes in sensory and non-sensory cells in the cochlea. Using a non-obese diabetic (NOD) mouse model, we have characterized changes in both hair cells and spiral ganglion neurons that may be relevant for early signs of age-related hearing loss (ARHL). We demonstrate that hair cell loss is preceded by, or in parallel with altered primary auditory neuron functions, and latent neurite retraction at the hair cell-auditory neuron synapse. The results were observed first in afferent inner hair cell synapse of type I neurites, followed by type II neuronal cell-body degeneration. Reduced membrane excitability and loss of postsynaptic densities were some of the inaugural events before any outward manifestation of hair bundle disarray and hair cell loss. We have identified profound alterations in type I neuronal membrane properties, including a reduction in membrane input resistance, prolonged action potential latency, and a decrease in membrane excitability. The resting membrane potential of aging type I neurons in the NOD, ARHL model, was significantly hyperpolarized, and analyses of the underlying membrane conductance showed a significant increase in K+ currents. We propose that attempts to alleviate some forms of ARHL should include early targeted primary latent neural degeneration for effective positive outcomes.

Lysyl oxidase like-2 (LOXL2) is an amine oxidase with both intracellular and extracellular functions. Extracellularly, LOXL2 promotes collagen and elastin crosslinking, whereas intracellularly, LOXL2 has been reported to modify histone H3, stabilize SNAIL, and reduce cell polarity. Although LOXL2 promotes liver and lung fibrosis, little is known regarding its role in renal fibrosis. Here we determine whether LOXL2 influences kidney disease in COL4A3 (-/-) Alport mice. These mice were treated with a small molecule inhibitor selective for LOXL2 or with vehicle and assessed for glomerular sclerosis and fibrosis, albuminuria, blood urea nitrogen, lifespan, pro-fibrotic gene expression and ultrastructure of the glomerular basement membrane. Laminin alpha 2 deposition in the glomerular basement membrane and mesangial filopodial invasion of the glomerular capillaries were also assessed. LOXL2 inhibition significantly reduced interstitial fibrosis and mRNA expression of MMP-2, MMP-9, TGF-b1, and TNF-alpha. LOXL2 inhibitor treatment also reduced glomerulosclerosis, expression of MMP-10, MMP-12, and MCP-1 mRNA in glomeruli, and decreased albuminuria and blood urea nitrogen. Mesangial filopodial invasion of the capillary tufts was blunted, as was laminin a2 deposition in the glomerular basement membrane, and glomerular basement membrane ultrastructure was normalized. There was no effect on lifespan. Thus, LOXL2 plays an important role in promoting both glomerular and interstitial pathogenesis associated with Alport syndrome in mice. Other etiologies of chronic kidney disease are implicated with our observations.

It is hypothesized that ferrocene (FC)-loaded nanocarriers (FC-NCs) are safe label-free contrast agents for cochlear biodistribution study by transmission electron microscopy (TEM). To test this hypothesis, after engineering, the poly(epsilon-caprolactone)/polyglycolide NCs are tested for stability with various types and ratios of sugar cryoprotectants during freeze-drying. Their physicochemical properties are characterized by UV-visible spectroscopy, dynamic light scattering, Fourier transform infrared spectroscopy, and scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDS). The biodistribution of the FC-NCs in the cochlear tissue after intratympanic injection in guinea pigs is visualized by TEM. Auditory brainstem responses are measured before and after 4-day treatments. These FC-NCs have 153.4 ± 8.7 nm, 85.5 ± 11.2%, and -22.1 ± 1.1 mV as mean diameters, percent drug association efficiency, and zeta potential, respectively (n = 3). The incorporation of FC into the NCs is confirmed by Fourier transform infrared spectroscopy and SEM/EDS spectra. Lactose (3:1 ratio, v/v) is the most effective stabilizer after a 12-day study. The administered NCs are visible by TEM in the scala media cells of the cochlea. Based on auditory brainstem response data, FC-NCs do not adversely affect hearing. Considering the electrondense, radioactive, and magnetic properties of iron inside FC, FC-NCs are promising nanotemplate for future inner ear theranostics.

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ETARs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ETAR antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ETAR blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ETARs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology.