Search

The presence of infectious microorganisms in urinary stones is commonly inferred from stone composition, especially by the presence of struvite in a stone. The presence of highly carbonated apatite has also been proposed as a marker of the presence of bacteria within a stone. We retrospectively studied 368 patients who had undergone percutaneous nephrolithotomy, and who also had culture results for both stone and urine. Urine culture showed no association with stone mineral content, but stone culture was more often positive in struvite containing stones (73% positive) and majority apatite stones (65%) than in other stone types (54%, lower than the others, P

Shock wave lithotripsy (SWL) is the only noninvasive method for stone removal. Once considered as a primary option for the treatment of virtually all stones, SWL is now recognized to have important limitations that restrict its use. In particular, the effectiveness of SWL is severely limited by stone burden, and treatment with shock waves carries the risk of acute injury with the potential for long-term adverse effects. Research aiming to characterize the renal response to shock waves and to determine the mechanisms of shock wave action in stone breakage and renal injury has begun to suggest new treatment strategies to improve success rates and safety. Urologists can achieve better outcomes by treating at slower shock wave rate using a step-wise protocol. The aim is to achieve stone comminution using as few shock waves and at as low a power level as possible. Important challenges remain, including the need to improve acoustic coupling, enhance stone targeting, better determine when stone breakage is complete, and minimize the occurrence of residual stone fragments. New technologies have begun to address many of these issues, and hold considerable promise for the future.

Previous in vitro studies of acoustic coupling in shock wave lithotripsy (SWL) have shown that air pockets trapped at the surface of the treatment head significantly reduce transmission of shock wave (SW) energy to the focal zone of the lithotripter, reducing the effectiveness of stone breakage. Since there are no reliable means to monitor the quality of coupling during SWL, we looked for a practical protocol to improve how coupling is achieved. In vitro studies were performed using a Dornier DoLi-50 lithotripter. LithoClear gel was used to couple the treatment head to the acoustic window of a clear acrylic test tank. Numerous methods of applying gel were tested including common sense variations of routine protocols typically used with patients. For each method the coverage of air pockets (% defects) was determined using digital imaging. Different coupling regimes were tested for effect on the breakage of gypsum model stones. The quality of acoustic coupling was affected by how the gel was handled--how it was dispensed and applied, and whether the gel was applied only to the treatment head or to both the lithotripter water cushion and the test tank (surrogate patient). Dispensing gel from a squeeze bottle for application by hand created significantly more defects than when a large volume (approximately 250 ml) of gel from the stock jug was applied as a mound to just the treatment head (26.5+/-2.7 vs. 1.2+/-0.5% defects, P0.001). The efficiency of stone breakage was better when gel was applied from the stock jug compared to application by hand (P0.006). Poor coupling was substantially improved by using the inflation feature of the water cushion to collapse air pockets, but this strategy was not a substitute for establishing good coupling at the outset. The quality of coupling in shock wave lithotripsy can be improved by minimizing the handling of the coupling medium. Hand application of coupling gel is clearly not the best way to prepare for lithotripsy. Better results can be obtained by delivering lithotripsy gel as a bolus to the treatment head alone, and allowing it to spread upon contact between the treatment head and the skin. These in vitro tests also suggest that the inflation feature of the lithotripter may be useful in reducing defects in coupling.

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.

Increased ambient pressure (excess hydrostatic pressure) was used to regulate cavitation in the aqueous media surrounding isolated red blood cells and aluminum foils used as targets in studies of the mechanisms of shock wave (SW) damage in shock wave lithotripsy (SWL). Foils or cells were placed in a cylindrical chamber (bronze-aluminum alloy) connected to a regulator and nitrogen source. The ends of the cylinder were capped with planar, plastic plates 12.7 mm thick. Tests performed with a PVDF membrane hydrophone showed that SW's passed through the plastic without significant loss of amplitude or change in waveform. Pitting of foils, a form of damage that can only be due to cavitation, was eliminated by very high (∼70 atm) added pressure. It took substantially greater added pressure to reduce damage to foils than has been reported to reduce damage to cells, suggesting differences in how cavitation is regulated in bulk fluid versus a solid surface. This report describes the design of the pressure chamber used in these studies and explains how SW exposures of isolated cells and foil targets were performed.

We recently demonstrated that endothelial cells are more susceptible than renal tubular epithelial cells to oxidant injury and that renal tubular epithelial cells with proximal tubular characteristics including porcine proximal tubular epithelial cells, opossum kidney proximal tubular epithelial cells, and normal human kidney cortical epithelial cells are more susceptible to oxidant injury than the distal nephron-derived Madin Darby canine kidney cell line. To determine the basis of this differential response, we evaluated several antioxidant defenses in the five cell lines. Glutathione levels were not significantly different among the five cell lines, but catalase and glutathione reductase levels were significantly (p less than 0.01) lower in endothelial cells compared to all renal tubular epithelial cells. Among renal tubular epithelial cells, Madin Darby canine kidney cells had significantly (p less than 0.05) higher glutathione peroxidase activity. To further evaluate the role of antioxidant defenses in limiting oxidant injury, we determined two responses to oxidant injury (ATP depletion and 51Cr release) when glutathione was depleted with buthionine sulfoxamine and when catalase was inhibited with aminotriazole. Oxidant-induced ATP depletion was accentuated when catalase was inhibited as well as when glutathione was depleted with buthionine sulfoxamine. In contrast, inhibition of catalase had little or no effect on 51Cr release, whereas glutathione depletion resulted in accentuated 51Cr release. We conclude that the increased susceptibility of endothelial cells to oxidant injury as compared with epithelial cells is associated with lower antioxidant defenses. Disruption of the glutathione redox cycle results in accentuated ATP depletion and lytic injury, whereas inhibition of catalase results in accentuated ATP depletion with little effect on lytic injury.(ABSTRACT TRUNCATED AT 250 WORDS)

This protocol describes the use of the pH-sensitive, intracellularly trapped dye 2′,7′-bis (2-carboxyethyl), 5 (and -6) carboxyfluorescein (BCECF), to characterize the pH regulating mechanisms in cultured human kidney proximal tubule cells. This is a reliable method for intracellular pH measurements and is applicable to single cells, cell suspensions, and confluent cultures.

A method for measuring cell volume and fluid transport rate in Madin-Darby canine kidney (MDCK) cysts is described. The method uses Nomarski optics and video microscopy to record changes in cyst dimensions in an in vitro superfusion system.

A procedure is described for the establishment and propagation of epithelial cell rich cultures derived from normal human kidney cortex (NHK-C cells). Cells are harvested from tissue fragments of donor human kidney by progressive enzymatic dissociation. NHK-C cultures are morphologically heterogeneous but exhibit, predominantly, the functional characteristics of cells of the kidney proximal tubule.

Background The global prevalence and incidence of renal calculi is reported to be increasing. Of the patients that undergo surgical intervention, nearly half experience symptomatic complications associated with stone fragments that are not passed and require follow-up surgical intervention. In a clinical simulation using a clinical prototype, ultrasonic propulsion was proven effective at repositioning kidney stones in pigs. The use of ultrasound to reposition smaller stones or stone fragments to a location that facilitates spontaneous clearance could therefore improve stone-free rates. The goal of this study was to determine an injury threshold under which stones could be safely repositioned. Methods Kidneys of 28 domestic swine were treated with exposures that ranged in duty cycle from 0%–100% and spatial peak pulse average intensities up to 30 kW/cm2 for a total duration of 10 min. The kidneys were processed for morphological analysis and evaluated for injury by experts blinded to the exposure conditions. Results At a duty cycle of 3.3%, a spatial peak intensity threshold of 16,620 W/cm2 was needed before a statistically significant portion of the samples showed injury. This is nearly seven times the 2,400-W/cm2 maximum output of the clinical prototype used to move the stones effectively in pigs. Conclusions The data obtained from this study show that exposure of kidneys to ultrasonic propulsion for displacing renal calculi is well below the threshold for tissue injury.

The 3rd International Urolithiasis Research Symposium, organized by the International Kidney Stone Institute, was held in Indianapolis, IN, USA, on 3–4 December 2009. The Symposium included special sessions on Nephrocalcinosis, Pediatric Stone Disease, Hypercalciuria and Plaque, and Stone Analysis. There were additionally two sessions on Shock Wave Lithotripsy, looking at renal injury and the mechanisms underlying tissue damage by shock waves. The Symposium also included two live stone surgeries that allowed participants to see two distinctly different pathologies on live video, with added commentary by experts in the auditorium showing biopsy results from similar patients. A main theme running throughout the Symposium was that we have much to learn about the pathologies underlying the different forms of stone disease. For too long these pathologies have been lumped together, but it is quite clear from recent studies in patients that trying to study ‘stone disease’ in a ‘calcium stone’ patient group that includes, for example, both idiopathic calcium oxalate stone formers and patients who form calcium oxalate stones as a consequence of bowel disease is very much a study of ‘‘apples and oranges.’’ Such confusion has clouded work in the past. In contrast, new clarity in this field promises to allow an acceleration of study of the diseases that result in urinary calculi. Included in this second installment of papers from the Symposium is a series comparing renal tissue calcifications between human disease and animal models. Miller et al. show how endoscopic viewing of the calyceal system can allow one to know with certainty which radiologic calcifications are nephrocalcinosis and which are urinary stones, while Khan reviews various animal models of renal calcification. Then Evan et al. relate the pathologic findings of two new mouse models to those found in humans with idiopathic calcium oxalate stone disease. Matlaga et al. give us a review of pediatric stone diseases, and Daudon et al. lead off a series of papers on stone analysis with a comprehensive review of phosphate stones and what is known of their etiology. We end with four papers on the general topic of lithotripsy, highlighted by the study of Shah et al. reporting a novel method to non-invasively displace small stones or stone fragments from the calyceal system to the ureteropelvic junction using pulsed ultrasound. The next International Urolithiasis Research Symposium is scheduled for 16–17 June 2011, again in Indianapolis. We look forward to seeing you there.

MDCK cells dissociated from monolayer culture were either dispersed within medium-hydrated collagen gel or seeded atop a collagen substrate which was immediately overlaid with collagen gel. Individual cells exhibited clonal growth in three dimensions to form spherical cysts in which a simple epithelium surrounded a fluid-filled lumen. The cells of MDCK cysts were polarized with apical surface bordering the lumen. MDCK cysts increased in diameter with continued culture. Maximum cyst size was dependent on seeding density and was influenced by medium composition. MDCK cysts could be isolated from the collagen substrate by digestion with collagenase. Also, collagen gel could be dissected from the cyst wall to give unrestricted access to regions of the basolateral cell surface. This novel method of renal cell culture provides a study system to model the influence of the extracellular matrix on kidney epithelial cell structure and function. It also offers an in vitro model of general application to the study of epithelial cyst formation and growth.

A reliable method is described for processing substrate-dependent cells raised on culture-grade plastic for both scanning (SEM) and transmission electron microscopy (TEM). This technique allows collection of specimens for TEM and SEM from the same culture dish or flask. In this way it is possible to study the surface morphology (SEM) and thin section ultrastructure of cells from contiguous regions of a culture. Specific regions of a culture can be selected and processed so that specimens retain orientation throughout mounting or embedment and sectioning. The method is applicable both to confluent cultures as well as isolated colonies.

A simple and convenient method is described to aid the processing of minute tissue specimens and isolated single cells for scanning electron microscopy (SEM). The technique employs an inexpensive, disposable carrier fashioned from common Beem capsules and Nitex nylon screen. This method avoids excessive handling, transfers or centrifugation steps that may damage delicate specimens. Specimens processed by this method show excellent preservation of cell surface fine structure.

This report describes the preparation of medium hydrated collagen gels for use as the physical support material for explant culture. Collagen gels are simple to prepare from rat tail tendon-derived stock collagen. These gels provide a firm, fluid-permeable substrate for submersion or interface culture of organ explants. Collagen gels can be conveniently applied to serum-free medium conditions.

This report describes a method for organ culture of the fetal rat lung at the early preglandular phase of development. When the intact fetal lung is cultured fully submerged in medium it develops as an expanded hollow organ. This pattern of in vitro morphogenesis provides a specimen uniquely suited to analysis of the pulmonary epithelium by scanning electron microscopy. In this system alveolar Type II cells and cell types of the distal pulmonary airways undergo differentiation in serum-free media.

This report describes the assembly and use of several types of filtration devices for use in the preparation of dissociated cells for primary culture. These Nitex-screen filter units are assembled from common, inexpensive laboratory materials. They provide a convenient means of filtering cell suspensions under sterile conditions and are adaptable for large and small volume work.