Intracellular calcium increases in vascular smooth muscle cells with progression of chronic kidney disease in a rat model.

Background: Vascular smooth muscle cells (VSMCs) exhibit phenotypic plasticity, promoting vascular calcification and increasing cardiovascular risk. Changes in VSMC intracellular calcium ([Ca 2+ ] i ) are a major determinant of plasticity, but little is known about changes in [Ca 2+ ] i in chronic kidney disease (CKD). We have previously demonstrated such plasticity in aortas from our rat model of CKD and therefore sought to examine changes in [Ca 2+ ] i during CKD progression. Materials and Methods: We examined freshly isolated VSMCs from aortas of normal rats, Cy/+ rats (CKD) with early and advanced CKD, and advanced CKD rats treated without and with 3% calcium gluconate (CKD + Ca 2+ ) to lower parathyroid hormone (PTH) levels. [Ca 2+ ] i was measured with fura-2. Results: Cy/+ rats developed progressive CKD, as assessed by plasma levels of blood urea nitrogen, calcium, phosphorus, parathyroid hormone and fibroblast growth factor 23. VSMCs isolated from rats with CKD demonstrated biphasic alterations in resting [Ca 2+ ] i : VSMCs from rats with early CKD exhibited reduced resting [Ca 2+ ] i , while VSMCs from rats with advanced CKD exhibited elevated resting [Ca 2+ ] i . Caffeine-induced sarcoplasmic reticulum (SR) Ca 2+ store release was modestly increased in early CKD and was more drastically increased in advanced CKD. The advanced CKD elevation in SR Ca 2+ store release was associated with a significant increase in the activity of the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA); however, SERCA2a protein expression was decreased in advanced CKD. Following SR Ca 2+ store release, recovery of [Ca 2+ ] i in the presence of caffeine and extracellular Ca 2+ was attenuated in VSMCs from rats with advanced CKD. This impairment, together with reductions in expression of the Na + /Ca 2+ exchanger, suggest a reduction in Ca 2+ extrusion capability. Finally, store-operated Ca 2+ entry (SOCE) was assessed following SR Ca 2+ store depletion. Ca 2+ entry during recovery from caffeine-induced SR Ca 2+ store release was elevated in advanced CKD, suggesting a role for exacerbated SOCE with progressing CKD. Conclusions: With progressive CKD in the Cy/+ rat there is increased resting [Ca 2+ ] i in VSMCs due, in part, to increased SOCE and impaired calcium extrusion from the cell. Such changes may predispose VSMCs to phenotypic changes that are a prerequisite to calcification.

CT visible internal stone structure, but not Hounsfield unit value, of calcium oxalate monohydrate (COM) calculi predicts lithotripsy fragility in vitro.

Calcium oxalate monohydrate (COM) stones are often resistant to breakage using shock wave (SW) lithotripsy. It would be useful to identify by computed tomography (CT) those COM stones that are susceptible to SW's. For this study, 47 COM stones (4-10 mm in diameter) were scanned with micro CT to verify composition and also for assessment of heterogeneity (presence of pronounced lobulation, voids, or apatite inclusions) by blinded observers. Stones were then placed in water and scanned using 64-channel helical CT. As with micro CT, heterogeneity was assessed by blinded observers, using high-bone viewing windows. Then stones were broken in a lithotripter (Dornier Doli-50) over 2 mm mesh, and SW's counted. Results showed that classification of stones using micro CT was highly repeatable among observers (kappa = 0.81), and also predictive of stone fragility. Stones graded as homogeneous required 1,874 +/- 821 SW/g for comminution, while stones with visible structure required half as many SW/g, 912 +/- 678. Similarly, when stones were graded by appearance on helical CT, classification was repeatable (kappa = 0.40), and homogeneous stones required more SW's for comminution than did heterogeneous stones (1,702 +/- 993 SW/g, compared to 907 +/- 773). Stone fragility normalized to stone size did not correlate with Hounsfield units (P = 0.85). In conclusion, COM stones of homogeneous structure require almost twice as many SW's to comminute than stones of similar mineral composition that exhibit internal structural features that are visible by CT. This suggests that stone fragility in patients could be predicted using pre-treatment CT imaging. The findings also show that Hounsfield unit values of COM stones did not correlate with stone fragility. Thus, it is stone morphology, rather than X-ray attenuation, which correlates with fragility to SW's in this common stone type.

Using Helical CT to Predict Stone Fragility in Shock Wave Lithotripsy (SWL).

Great variability exists in the response of urinary stones to SWL, and this is true even for stones composed of the same mineral. Efforts have been made to predict stone fragility to shock waves using computed tomography (CT) patient images, but most work to date has focused on the use of stone CT number (i.e., Hounsfield units). This is an easy number to measure on a patient stone, but its value depends on a number of factors, including the relationship of the size of the stone to the resolution (i.e., the slicewidth) of the CT scan. Studies that have shown a relationship between stone CT number and failure in SWL are reviewed, and all are shown to suffer from error due to stone size, which was not accounted for in the use of Hounsfield unit values. Preliminary data are then presented for a study of calcium oxalate monohydrate (COM) stones, in which stone structure-rather than simple CT number values-is shown to correlate with fragility to shock waves. COM stones that were observed to have structure by micro CT (e.g., voids, apatite regions, unusual shapes) broke to completion in about half the number of shock waves required for COM stones that were observed to be homogeneous in structure by CT. This result suggests another direction for the use of CT in predicting success of SWL: the use of CT to view stone structure, rather than simply measuring stone CT number. Viewing stone structure by CT requires the use of different viewing windows than those typically used for examining patient scans, but much research to date indicates that stone structure can be observed in the clinical setting. Future clinical studies will need to be done to verify the relationship between stone structure observed by CT and stone fragility in SWL.

Variability of protein content in calcium oxalate monohydrate stones.

BACKGROUND AND PURPOSE: Urinary stones are heterogeneous in their fragility to lithotripter shockwaves. As a first step in gaining a better understanding of the role of matrix in stone fragility, we measured extractible protein in calcium oxalate monohydrate (COM) stones that were extensively characterized by micro-computed tomography (micro CT). MATERIALS AND METHODS: Stones were scanned using micro CT (Scanco mCT20, 34 microm). They were ground, and the protein extracted using four methods: 0.25M EDTA, 2% SDS reducing buffer, 9M urea buffer, and 10% acetic acid. Protein was measured using NanoOrange. The SDS extracts were also examined using polyacrylamide electrophoresis (PAGE). RESULTS: Extracted protein was highest with the SDS or urea methods (0.28% +/- 0.13% and 0.24% +/- 0.11%, respectively) and lower using the EDTA method (0.17% +/- 0.05%; P < 0.02). Acetic acid extracted little protein (0.006 +/- 0.002%; P < 0.001). Individual stones were significantly different in extractability of protein by the different methods, and SDS-PAGE revealed different protein patterns for individual stones. Extracted protein did not correlate with X-ray-lucent void percentage, which ranged from 0.06% to 2.8% of stone volume, or with apatite content. CONCLUSIONS: Extractible stone-matrix protein differs for individual COM stones, and yield is dependent on the extraction method. The presence of X-ray-lucent voids or minor amounts of apatite in stones did not correlate with protein content. The amounts of protein recovered were much lower than reported by Boyce, showing that these methods extracted only a fraction of the protein bound up in the stones. The results suggest that none of the methods tested will be useful for helping to answer the question of whether matrix content differs among stones of differing fragility to lithotripter shockwaves.

Nondestructive analysis of urinary calculi using micro computed tomography.

BACKGROUND: Micro computed tomography (micro CT) has been shown to provide exceptionally high quality imaging of the fine structural detail within urinary calculi. We tested the idea that micro CT might also be used to identify the mineral composition of urinary stones non-destructively. METHODS: Micro CT x-ray attenuation values were measured for mineral that was positively identified by infrared microspectroscopy (FT-IR). To do this, human urinary stones were sectioned with a diamond wire saw. The cut surface was explored by FT-IR and regions of pure mineral were evaluated by micro CT to correlate x-ray attenuation values with mineral content. Additionally, intact stones were imaged with micro CT to visualize internal morphology and map the distribution of specific mineral components in 3-D. RESULTS: Micro CT images taken just beneath the cut surface of urinary stones showed excellent resolution of structural detail that could be correlated with structure visible in the optical image mode of FT-IR. Regions of pure mineral were not difficult to find by FT-IR for most stones and such regions could be localized on micro CT images of the cut surface. This was not true, however, for two brushite stones tested; in these, brushite was closely intermixed with calcium oxalate. Micro CT x-ray attenuation values were collected for six minerals that could be found in regions that appeared to be pure, including uric acid (3515 - 4995 micro CT attenuation units, AU), struvite (7242 - 7969 AU), cystine (8619 - 9921 AU), calcium oxalate dihydrate (13815 - 15797 AU), calcium oxalate monohydrate (16297 - 18449 AU), and hydroxyapatite (21144 - 23121 AU). These AU values did not overlap. Analysis of intact stones showed excellent resolution of structural detail and could discriminate multiple mineral types within heterogeneous stones. CONCLUSIONS: Micro CT gives excellent structural detail of urinary stones, and these results demonstrate the feasibility of identifying and localizing most of the common mineral types found in urinary calculi using laboratory CT.

Helical computed tomography accurately reports urinary stone composition using attenuation values: in vitro verification using high-resolution micro-computed tomography calibrated to fourier transform infrared microspectroscopy.

OBJECTIVES: To assess the ability of helical computed tomography (CT) to differentiate regions of known mineral composition in typical, heterogeneous urinary stones. Interest is substantial in the urologic community in using radiologic imaging to determine accurately the composition of urinary calculi. Recent advances in CT make this a viable prospect, but the heterogeneity of most stones is a complicating factor. METHODS: The ability of micro-CT (a high-resolution laboratory instrument) to identify the mineral composition of stones was confirmed by calibrating micro-CT attenuation values to pure mineral regions of sliced stones using infrared microspectroscopy. Intact human urinary stones were then analyzed by micro-CT, and regions-of-interest of pure mineral were correlated with identical regions-of-interest from quad slice multi-detector row helical CT images. With helical CT, narrow slice widths were used to decrease volume-averaging errors, and bone windows were used so that internal stone structure was visible. RESULTS: When stones were imaged using helical CT at narrow slice widths, mineral-specific regions-of-interest yielded nonoverlapping attenuation values for uric acid (566 to 632 Hounsfield units [HU]), struvite (862 to 944 HU), calcium oxalate (1416 to 1938 HU), and hydroxyapatite (2150 to 2461 HU). CONCLUSIONS: High resolution helical CT yields unique attenuation values for common types of stone mineral, but proper windowing is required to localize regions of homogeneity. The results of this in vitro study suggest that high-resolution helical CT may be able to identify stone composition at patient diagnosis.

Progress in the use of helical CT for imaging urinary calculi.

Helical CT has become the preferred method to diagnose urinary calculi in patients presenting with abdominal or flank pain. Recent in vitro studies have shown that CT also can display the internal structure in stones with remarkable detail. Because some stones respond better to SWL than others, knowing stone structure at diagnosis could be helpful in choosing among treatment options. This paper examines the potential for CT to be used in this way. Older CT technology proved to be problematic, in that all studies using low-resolution CT will suffer from an artifact in which stone size affects apparent CT attenuation values. Thus, the observation that stones with low measured CT attenuation break more easily than stones with high attenuation could be attributable entirely to an artifact of stone size. Most stones are composed of more than one mineral, and heterogeneity of composition may contribute to variability in stone response to SWL. Older technology is not useful in evaluating stone composition, but current and emerging CT machines have sufficient resolution to determine the composition and structure of stones inside the patient, provided proper viewing windows are used. Continuing improvement in image resolution in helical CT promises to provide information about stone composition and structure that will ultimately lead to better care for patients with stone disease.