A crystallin mutant cataract with mineral deposits.

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.

Connexin Mutants Cause Cataracts Through Deposition of Apatite.

Cataracts are lens opacities that are among the most common causes of blindness. It is commonly believed that cataracts develop through the accumulation of damage to lens proteins. However, recent evidence suggests that cataracts can result from calcium ion accumulation and the precipitation of calcium-containing salts. To test for the presence of precipitates and to identify their components, we studied the lenses of mice that develop cataracts due to mutations of connexin46 and connexin50. Micro-computed tomography showed the presence of radio-dense mineral in the mutant lenses, but not in wild-type lenses. Three-dimensional reconstructions of the scans showed that the distribution of the radio-dense mineral closely paralleled the location and morphology of the cataracts. The mutant lens homogenates also contained insoluble particles that stained with Alizarin red (a dye that stains Ca2+ deposits). Using attenuated total internal reflection micro-Fourier transform infrared spectroscopy, we identified the mineral as calcium phosphate in the form of apatite. Taken together, these data support the novel paradigm that cataracts are formed through pathological mineralization within the lens.

Multimodal imaging reveals a unique autofluorescence signature of Randall's plaque.

Kidney stones frequently develop as an overgrowth on Randall's plaque (RP) which is formed in the papillary interstitium. The organic composition of RP is distinct from stone matrix in that RP contains fibrillar collagen; RP in tissue has also been shown to have two proteins that are also found in stones, but otherwise the molecular constituents of RP are unstudied. We hypothesized that RP contains unique organic molecules that can be differentiated from the stone overgrowth by fluorescence. To test this, we used micro-CT-guided polishing to expose the interior of kidney stones for multimodal imaging with multiphoton, confocal and infrared microscopy. We detected a blue autofluorescence signature unique to RP, the specificity of which was also confirmed in papillary tissue from patients with stone disease. High-resolution mineral mapping of the stone also showed a transition from the apatite within RP to the calcium oxalate in the overgrowth, demonstrating the molecular and spatial transition from the tissue to the urine. This work provides a systematic and practical approach to uncover specific fluorescence signatures which correlate with mineral type, verifies previous observations regarding mineral overgrowth onto RP and identifies a novel autofluorescence signature of RP demonstrating RP's unique molecular composition.

Claudin-2 deficiency associates with hypercalciuria in mice and human kidney stone disease.

The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2-null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2-null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.

Structure-Property Effects in the Generation of Transient Aqueous Benzoic Acid Anhydrides by Carbodiimide Fuels.

The design of dissipative systems, which operate out-of-equilibrium by consuming chemical fuels, is challenging. As yet, there are a few examples of privileged fuel chemistries that can be broadly applied in abiotic systems in the same way that ATP hydrolysis is exploited throughout biochemistry. The key issue is that designing nonequilibrium systems is inherently about balancing the relative rates of coupled reactions. The use of carbodiimides as fuels to generate transient aqueous carboxylic anhydrides has recently been used in examples of new nonequilibrium materials and supramolecular assemblies. Here, we explore the kinetics of formation and decomposition of a series of benzoic anhydrides generated from the corresponding acids and EDC under typical conditions (EDC = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride). The reactions can be described by a simple mechanism that merges known behavior for the two processes independently. Structure-property effects in these systems are dominated by differences in the anhydride decomposition rate. The kinetic parameters allow trends in concentration-dependent properties to be simulated, such as reaction lifetimes, peak anhydride concentrations, and yields. For key properties, there are diminishing returns with the addition of increasing amounts of fuel. These results should provide useful guidelines for the design of functional systems making use of this chemistry.

Analysis of Counterfeit Coated Tablets and Multi-Layer Packaging Materials Using Infrared Microspectroscopic Imaging.

An effective method for detecting and characterizing counterfeit finished dosage forms and packaging materials is described in this study. Using attenuated total internal reflection Fourier transform infrared spectroscopic imaging, suspect tablet coating and core formulations as well as multi-layered foil safety seals, bottle labels, and cigarette tear tapes were analyzed and compared directly with those of a stored authentic product. The approach was effective for obtaining molecular information from structures as small as 6 µm.

The Advantages of an Attenuated Total Internal Reflection Infrared Microspectroscopic Imaging Technique for the Analysis of Polymer Laminates.

Until recently, the analysis of polymer laminates using infrared microspectroscopy involved the painstaking separation of individual layers by dissection or by obtaining micrometer thin cross-sections. The latter usually requires the expertise of an individual trained in microtomy and even then, the very structure of the laminate could affect the outcome of the spectral results. The recent development of attenuated total internal reflection (ATR) infrared microspectroscopy imaging has provided a new avenue for the analysis of these multilayer structures. This report compares ATR infrared microspectroscopy imaging with conventional transmission infrared microspectroscopy imaging. The results demonstrate that the ATR method offers improved spatial resolution, eliminates a variety of competing optical processes, and requires minimal sample preparation relative to transmission measurements. These advantages were illustrated using a polymer laminate consisting of 11 different layers whose thickness ranged in size from 4-20 µm. The spatial resolution achieved by using an ATR-FTIR (Fourier transform infrared spectroscopy) imaging technique was diffraction limited. Contrast in the ATR images was enhanced by principal component analysis.

Analysis of thin-film polymers using attenuated total internal reflection-Raman microspectroscopy.

Two methods commonly employed for molecular surface analysis and thin-film analysis of microscopic areas are attenuated total reflection infrared (ATR-IR) microspectroscopy and confocal Raman microspectroscopy. In the former method, the depth of the evanescent probe beam can be controlled by the wavelength of light, the angle of incidence, or the refractive index of the internal reflection element. Because the penetration depth is proportional to the wavelength of light, one could interrogate a smaller film thickness by moving from the mid-infrared region to the visible region employing Raman spectroscopy. The investigation of ATR Raman microspectroscopy, a largely unexplored technique available to Raman microspectroscopy, was carried out. A Renishaw inVia Raman microscope was externally modified and used in conjunction with a solid immersion lens (SIL) to perform ATR Raman experiments. Thin-film polymer samples were analyzed to explore the theoretical sampling depth for experiments conducted without the SIL, with the SIL, and with the SIL using evanescent excitation. The feasibility of micro-ATR Raman was examined by collecting ATR spectra from films whose thickness measured from 200 to 60 nm. Films of these thicknesses were present on a much thicker substrate, and features from the underlying substrate did not become visible until the thin film reached a thickness of 68 nm.

Biopsy proven medullary sponge kidney: clinical findings, histopathology, and role of osteogenesis in stone and plaque formation.

Medullary sponge kidney (MSK) is associated with recurrent stone formation, but the clinical phenotype is unclear because patients with other disorders may be incorrectly labeled MSK. We studied 12 patients with histologic findings pathognomonic of MSK. All patients had an endoscopically recognizable pattern of papillary malformation, which may be segmental or diffuse. Affected papillae are enlarged and billowy, due to markedly enlarged inner medullary collecting ducts (IMCD), which contain small, mobile ductal stones. Patients had frequent dilation of Bellini ducts, with occasional mineral plugs. Stones may form over white (Randall's) plaque, but most renal pelvic stones are not attached, and have a similar morphology as ductal stones, which are a mixture of calcium oxalate and apatite. Patients had no abnormalities of urinary acidification or acid excretion; the most frequent metabolic abnormality was idiopathic hypercalciuria. Although both Runx2 and Osterix are expressed in papillae of MSK patients, no mineral deposition was seen at the sites of gene expression, arguing against a role of these genes in this process. Similar studies in idiopathic calcium stone formers showed no expression of these genes at sites of Randall's plaque. The most likely mechanism for stone formation in MSK appears to be crystallization due to urinary stasis in dilated IMCD with subsequent passage of ductal stones into the renal pelvis where they may serve as nuclei for stone formation.

Contrasting histopathology and crystal deposits in kidneys of idiopathic stone formers who produce hydroxy apatite, brushite, or calcium oxalate stones.

Our previous work has shown that stone formers who form calcium phosphate (CaP) stones that contain any brushite (BRSF) have a distinctive renal histopathology and surgical anatomy when compared with idiopathic calcium oxalate stone formers (ICSF). Here we report on another group of idiopathic CaP stone formers, those forming stone containing primarily hydroxyapatite, in order to clarify in what ways their pathology differs from BRSF and ICSF. Eleven hydroxyapatite stone formers (HASF) (2 males, 9 females) were studied using intra-operative digital photography and biopsy of papillary and cortical regions to measure tissue changes associated with stone formation. Our main finding is that HASF and BRSF differ significantly from each other and that both differ greatly from ICSF. Both BRSF and ICSF patients have significant levels of Randall's plaque compared with HASF. Intra-tubular deposit number is greater in HASF than BRSF and nonexistent in ICSF while deposit size is smaller in HASF than BRSF. Cortical pathology is distinctly greater in BRSF than HASF. Four attached stones were observed in HASF, three in 25 BRSF and 5-10 per ICSF patient. HASF and BRSF differ clinically in that both have higher average urine pH, supersaturation of CaP, and calcium excretion than ICSF. Our work suggests that HASF and BRSF are two distinct and separate diseases and both differ greatly from ICSF.

Characterization of silver halide fiber optics and hollow silica waveguides for use in the construction of a mid-infrared attenuated total reflection fourier transform infrared (ATR FT-IR) spectroscopy probe.

Advances in fiber optic materials have allowed for the construction of fibers and waveguides capable of transmitting infrared radiation. An investigation of the transmission characteristics associated with two commonly used types of infrared-transmitting fibers/waveguides for prospective use in a fiber/waveguide-coupled attenuated total internal reflection (ATR) probe was performed. Characterization of silver halide polycrystalline fiber optics and hollow silica waveguides was done on the basis of the transmission of infrared light using a conventional fiber optic coupling accessory and an infrared microscope. Using the fiber optic coupling accessory, the average percent transmission for three silver halide fibers was 18.1 ± 6.1% relative to a benchtop reflection accessory. The average transmission for two hollow waveguides (HWGs) using the coupling accessory was 8.0 ± 0.3%. (Uncertainties in the relative percent transmission represent the standard deviations.) Reduced transmission observed for the HWGs was attributed to the high numerical aperture of the coupling accessory. Characterization of the fibers/waveguides using a zinc selenide lens objective on an infrared microscope indicated 24.1 ± 7.2% of the initial light input into the silver halide fibers was transmitted. Percent transmission obtained for the HWGs was 98.7 ± 0.1%. Increased transmission using the HWGs resulted from the absence or minimization of insertion and scattering losses due to the hollow air core and a better-matched numerical aperture. The effect of bending on the transmission characteristics of the fibers/waveguides was also investigated. Significant deviations in the transmission of infrared light by the solid-core silver halide fibers were observed for various bending angles. Percent transmission greater than 98% was consistently observed for the HWGs at the bending angles. The combined benefits of high percent transmission, reproducible instrument responses, and increased bending tolerance indicated HWGs should be preferred in the construction of a fiber/waveguide-coupled ATR probe.

Infrared microspectroscopy using prism-based spectrographs and focal plane array detection.

Several prism-based spectrographs employing a mercury cadmium telluride (MCT) focal plane array detector have been interfaced to an infrared microscope. In the combined system, the area-defining aperture of the microscope also served as the entrance slit to the spectrograph. This investigation considered the fundamental limits of diffraction for both the spectrograph and microscope in order to determine both the spatial and spectral resolution of the system as a whole. Experimental results for spectral resolution, spectral range, and peak-to-peak noise have been presented. Finally, the dynamic capabilities of one spectrograph/microscope combination were investigated.

Analysis of counterfeit pharmaceutical tablet cores utilizing macroscopic infrared spectroscopy and infrared spectroscopic imaging.

Advantages and limitations of analyzing authentic and counterfeit pharmaceutical tablets with both macro (nonimaging) attenuated total internal reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and micro ATR-FT-IR spectroscopic imaging have been evaluated. The results of this study demonstrated that micro ATR imaging was more effective for extracting formulation information (sourcing), whereas a macro ATR approach was better suited for counterfeit detection (screening). More importantly, this study demonstrated that a thorough analysis of the counterfeit core can be achieved by combining the results of both techniques.

Renal histopathology and crystal deposits in patients with small bowel resection and calcium oxalate stone disease.

We present here the anatomy and histopathology of kidneys from 11 patients with renal stones following small bowel resection, including 10 with Crohn's disease and 1 resection in infancy for unknown cause. They presented predominantly with calcium oxalate stones. Risks of formation included hyperoxaluria (urine oxalate excretion greater than 45 mg per day) in half of the cases, and acidic urine of reduced volume. As was found with ileostomy and obesity bypass, inner medullary collecting ducts (IMCDs) contained crystal deposits associated with cell injury, interstitial inflammation, and papillary deformity. Cortical changes included modest glomerular sclerosis, tubular atrophy, and interstitial fibrosis. Randall's plaque (interstitial papillary apatite) was abundant, with calcium oxalate stone overgrowth similar to that seen in ileostomy, idiopathic calcium oxalate stone formers, and primary hyperparathyroidism. Abundant plaque was compatible with the low urine volume and pH. The IMCD deposits all contained apatite, with calcium oxalate present in three cases, similar to findings in patients with obesity bypass but not an ileostomy. The mechanisms for calcium oxalate stone formation in IMCDs include elevated urine and presumably tubule fluid calcium oxalate supersaturation, but a low calcium to oxalate ratio. However, the mechanisms for the presence of IMCD apatite remain unknown.

The advantages of an attenuated total internal reflection infrared microspectroscopic imaging approach for kidney biopsy analysis.

The benefits of an attenuated total reflection Fourier transform infrared (ATR-FTIR) imaging approach for kidney biopsy analysis are described. Biopsy sections collected from kidney-stone formers are analyzed at the initial stages of stone development to provide insights into stone growth and formation. The majority of tissue analysis currently conducted with IR microspectroscopy is performed with a transflection method. The research presented in this manuscript demonstrates that ATR overcomes many of the disadvantages of transflection or transmission measurements for tissue analysis including an elimination of spectral artifacts. When kidney biopsies with small mineral inclusions are analyzed with a transflection approach, specular reflection and the Christiansen effect (anomalous dispersion) can occur, leading to spectral artifacts. Another effect specific to the analysis of mineral inclusions present in kidney biopsies is known as the reststrahlen effect whereby the inclusions become strong reflectors near an absorption band. ATR eliminates these effects by immersing the sample in a high index medium. Additionally, the focused beam size for ATR is decreased by a factor of four when a germanium internal reflection element is used, allowing the acquisition of spectra from small mineral inclusions several micrometers in diameter. If quantitative analysis of small mineral inclusions is ultimately desired, ATR provides the photometrically accurate spectra necessary for quantification.

Rapid molecular imaging using attenuated total internal reflection planar array infrared spectroscopy for the analysis of counterfeit pharmaceutical tablets.

A planar array infrared (PA-IR) spectrograph containing an attenuated total internal reflection (ATR) accessory has been constructed in order to permit rapid analysis of poorly transmitting materials. The technique has been optimized to allow molecular spectroscopic information to be collected in roughly 2 seconds with a corresponding peak-to-peak noise value as low as 2.14 x 10(-4) absorbance units. Additionally, up to 150 spectra could be extracted from sample sizes as large as 6 mm where each spatial element measured 40 x 200 microm at the sample position. An application study for this technique entailed developing an embedding method that allows cross-sectioned pharmaceutical tablets to be brought into intimate contact with the internal reflection element (IRE) of the accessory. A supplemental investigation involved calculating the yield strength of multiple IRE materials in order to determine the maximum amount of pressure that can be applied to a sample without damaging the IRE. Finally, feasibility was demonstrated for using the instrument/accessory as a means to rapidly authenticate suspected counterfeit pharmaceutical tablets.

Intra-tubular deposits, urine and stone composition are divergent in patients with ileostomy.

Patients with ileostomy typically have recurrent renal stones and produce scanty, acidic, sodium-poor urine because of abnormally large enteric losses of water and sodium bicarbonate. Here we used a combination of intra-operative digital photography and biopsy of the renal papilla and cortex to measure changes associated with stone formation in seven patients with ileostomy. Papillary deformity was present in four patients and was associated with decreased estimated glomerular filtration rates. All patients had interstitial apatite plaque, as predicted from their generally acid, low-volume urine. Two patients had stones attached to plaque; however, all patients had crystal deposits that plugged the ducts of Bellini and inner medullary collecting ducts (IMCDs). Despite acid urine, all crystal deposits contained apatite, and five patients had deposits of sodium and ammonium acid urates. Stones were either uric acid or calcium oxalate as predicted by supersaturation, however, there was a general lack of supersaturation for calcium phosphate as brushite, sodium, or ammonium acid urate because of the overall low urine pH. This suggests that local tubular pH exceeds that of bulk urine. Despite low urine pH, patients with an ileostomy resemble those with obesity bypass, in whom IMCD apatite crystal plugs are found. They are, however, unlike these bypass patients in having interstitial apatite plaque. IMCD plugging with sodium and ammonium acid urate has not been found previously and appears to correlate with formation of uric acid stones.

Attenuated total internal reflection Fourier transform infrared spectroscopy: a quantitative approach for kidney stone analysis.

The impact of kidney stone disease is significant worldwide, yet methods for quantifying stone components remain limited. A new approach requiring minimal sample preparation for the quantitative analysis of kidney stone components has been investigated utilizing attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FT-IR). Calcium oxalate monohydrate (COM) and hydroxylapatite (HAP), two of the most common constituents of urinary stones, were used for quantitative analysis. Calibration curves were constructed using integrated band intensities of four infrared absorptions versus concentration (weight %). The correlation coefficients of the calibration curves range from 0.997 to 0.93. The limits of detection range from 0.07 +/- 0.02% COM/HAP where COM is the analyte and HAP is the matrix, to 0.26 +/- 0.07% HAP/COM where HAP is the analyte and COM is the matrix. This study shows that linear calibration curves can be generated for the quantitative analysis of stone mixtures provided the system is well understood especially with respect to particle size.

Histopathology and surgical anatomy of patients with primary hyperparathyroidism and calcium phosphate stones.

Using a combination of intra-operative digital photography and micro-biopsy we measured renal cortical and papillary changes in five patients with primary hyperparathyroidism and abundant calcium phosphate kidney stones. Major tissue changes were variable papillary flattening and retraction, dilation of the ducts of Bellini, and plugging with apatite deposits of the inner medullary collecting ducts and ducts of Bellini. Some of the papillae in two of the patients contained plentiful large interstitial deposits of Randall's plaque and where the deposits were most plentiful we found overgrowth of the attached stones. Hence, this disease combines features previously described in brushite stone formers--dilation, plugging of ducts and papillary deformity--with the interstitial plaque and stone overgrowth characteristic of routine idiopathic calcium oxalate stone formers, suggesting that these two patterns can coexist in a single patient.

Prism-based infrared spectrographs using modern-day detectors.

A comparison of prism-based spectrographs to grating-based spectrographs is made when each of the systems is coupled to a modern-day liquid-nitrogen-cooled photovoltaic array detector. A comparison of the systems is also made using a room-temperature microbolometer array detector. Finally, infrared microspectroscopy of samples whose size is approximately 10 micrometers will be demonstrated using a prism spectrograph outfitted with both types of detectors. The results of the study show that prism-based spectrographs offer an economical alternative to grating-based systems when spectral coverage is more critical than spectral resolution. The results also demonstrate that spectra with good signal-to-noise ratios can be collected on any of the systems with a total integration time of 10 seconds or less.