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Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Ed Carstensen has made many contributions to biomedical ultrasound but among those that are becoming more and more relevant to current clinical practice are those that determine the conditions under which cavitation is induced in vivo. For many years, it was assumed that the medical ultrasound devices were unable to induce cavitation in living tissue because either the acoustic conditions were not sufficient or the nucleation sites that are required were too small. With the advent of lithotripters and high‐intensity focused ultrasound (HIFU) devices, cavitation generation in vivo is commonplace. Our current research at the University of Washington has focused on the role that cavitation plays in stone comminution and tissue damage during lithotripsy, as well as the enhancement or reduction of desirable coagulative necrosis during HIFU application. During HIFU application, we find enhanced heating that results from nonlinear acoustic wave propagation (a key Carstensen contribution) leads to vapor bubble formation. This presentation will review our recent studies in this area. [Work supported in part by the NIH under CA109557 and DK43881, and the NSBRI SMS00402 under NCC9‐58‐50.]

This study examined whether organic anion secretion contributes to fluid accumulation in cysts in polycystic kidney disease. Clearance and micropuncture studies were done on young (7 to 16 wk old), mostly male, heterozygous Han:SPRD cystic rats and healthy control littermate rats. Heterozygous Han:SPRD rats manifest a slowly progressive autosomal dominant polycystic kidney disease that closely resembles the human disorder. Left kidney GFR (polyfructosan clearance), in microl/min per 100 g body wt, averaged 331 +/- 36 (SD) in seven healthy rats and 278 +/- 75 in seven cystic rats. The maximal rate of p-aminohippurate (PAH) secretion, in micromol/min per 100 g body wt, averaged 0.94 +/- 0.24 in healthy rats and 0.83 +/- 0.11 in cystic rats. In these young rats, there were no significant differences in GFR or the maximal rate of PAH secretion despite the presence of cystic disease. Using fluorescence microscopy, it was found that 27 of 29 proximal cysts secreted sulfonefluorescein, an organic anion transported by the PAH system. Transmission electron micrographs of superficial cysts that had secreted sulfonefluorescein demonstrated the presence of both normal-appearing and poorly differentiated proximal tubule cells. Segments of superficial proximal convoluted tubules or cysts, isolated by upstream and downstream wax blocks, failed to accumulate fluid when PAH was infused intravenously. With the stationary microperfusion technique, PAH secretion by both normal and cystic nephrons was demonstrated. It is concluded that most proximal cystic epithelia retain the ability to secrete organic anions. Secretion of organic anions, however, does not appear to contribute in any substantial way to fluid accumulation in cysts in the rat kidney.

Acoustic cavitation damage was quantitated using aluminum foil targets placed within 2-mL polypropylene cryovials. The vials contained various media tested for their potential to support cavitation and were exposed to shock waves using an unmodified Dornier HM3 lithotripter. Foil damage, expressed in terms of a “damage index”, was measured from digitized light microscopy images by quantitating the spread of gray-scale histograms. Target sensitivity was demonstrated by reproducible dose-response curves over the range (1–200 shock waves) commonly used for in vitro cell injury studies. Increased shock wave repetition rate reduced the damage index. Untreated foils showed a very low damage index (0.001% ± 0.001%), while treated foils submerged in Ringer buffer yielded significant damage (2.2% ± 0.3%, p < 0.001). Degassing the buffer reduced damage to 0.3% ± 0.1% (p < 0.001). Foils submerged in castor oil showed virtually no damage. These results implicate acoustic cavitation in target damage. Targets immersed in biological fluids (blood and urine) had significantly less damage than in Ringer. The effect of degassing was also evaluated in a red blood cell lysis assay. Hemoglobin release in degassed preparations was significantly reduced compared to nondegassed controls (p < 0.001) and correlated with reduced foil damage index in cell-free vials. These findings characterize a sensitive method to quantitate acoustic cavitation and implicate a role for cavitation in shock wave lithotripsy-induced cell lysis.

Therapeutic ultrasound has an increasing number of applications in urology, including shockwave lithotripsy, stone propulsion, tissue ablation, and hemostasis. However, the threshold of renal injury using ultrasound is unknown. The goal of this study was to determine kidney injury thresholds for a range of intensities between diagnostic and ablative therapeutic ultrasound. A 2 MHz annular array generating spatial peak pulse average intensities (ISPPA) up to 30,000 W/cm2 in water was placed on the surface of in vivo porcine kidneys and focused on the adjacent parenchyma. Treatments consisted of pulses of 100 μs duration triggered every 3 ms for 10 min at various intensities. The perfusion-fixed tissue was scored by three blinded independent experts. Above a threshold of 20,000 W/cm2, the majority of injury observed included emulsification, necrosis, and hemorrhage. Below this threshold, almost all injury presented as focal cell and tubular swelling and/or degeneration. These findings provide evidence for a wide range of potentially therapeutic ultrasound intensities that has a low probability of causing injury. While this study did not examine all combinations of treatment parameters of therapeutic ultrasound, tissue injury appears dose-dependent. [Work supported by NIH DK43881, DK092197, and NSBRI through NASA NCC 9-58.]

The osmotic water permeability (Pf) and permeability to nonelectrolytes were determined for the apical membrane of clonal strain Madin-Darby canine kidney (MDCK) C12 cells cultured as cysts with the apical membrane facing the surrounding medium. Pf and solute permeabilities were calculated from the rate of volume change of cysts by digitizing images at 1-s intervals after instantaneous osmotic challenge. Image measurement was fully automated with the use of a program that separated the image of the cyst from the background by using adaptive intensity thresholding and shape analysis. Pf, calculated by curve fitting to the volume loss data, averaged 2.4 +/- 0.1 micron/s and was increased by addition of amphotericin B. The energy of activation for Pf was high (16.3 kcal/mol), and forskolin (50 microM) had no effect on Pf. Two populations of MDCK cysts were studied: those with two to three cells and those that appeared to be composed of only one cell. The Pf of multicell cysts was the same as single cell cysts, suggesting that paracellular water flow is not significant. Solute permeability was measured using paired osmotic challenges (sucrose and test solute) on the same cyst. Urea permeability was not different from zero, whereas the permeabilities of acetamide and formamide were consistent with their relative oil-water partition coefficients. Our data are similar to values from studies on the permeability properties of vesicles of water-tight epithelial apical membrane. The combination of the unique model of MDCK apical-out cysts and fully automated data analysis enabled determination of apical membrane permeability in intact epithelial cells with high precision.

The utility of cross-linked hemoglobin (Hgb) solutions has been predicted to be limited in part by potential renal toxicity and consequent renal dysfunction (Hess et al. 1989, Keipert and Chang 1987, Messmer 1978, Snyder et al. 1987). However, this concern has been based upon models and assumptions that may not be entirely pertinent since direct studies have not as yet been performed in both euvolemic and hypovolemic models pertinent to the utility of red cell substitutes.

A case is presented for the important role of cavitation in stone comminution and tissue injury in shock wave lithotripsy (SWL). Confocal hydrophones and a coincidence algorithm were used to detect cavitation in kidney parenchyma. Elevated hydrostatic pressure dissolved cavitation nuclei and suppressed cell injury and stone comminution in vitro. A low‐insertion‐loss, thin, mylar film nearly eliminated stone erosion and crack formation only when in direct contact with the stone. This result indicates not only that cavitation is important in both cracking and erosion but also that bubbles act at the surface. Time inversion of the shock wave by use of a pressure‐release reflector reduced the calculated pressure at bubble collapse and the measured depth of bubble‐induced pits in aluminum. Correspondingly tissue injury in vivo was nearly eliminated. Cavitation was localized and intensified by the use of synchronously triggered, facing lithotripters. This dual pulse lithotripter enhanced comminution at its focus and reduced lysis in surrounding blood samples. The enhancement of comminution was lost when stones were placed in glycerol, which retarded bubble implosion. Thus, cavitation is important in comminution and injury and can be controlled to optimize efficacy and safety. [Work supported by NIH DK43381, DK55674, and FIRCA.]

The growth of renal cysts leads to morbidity, contributes to mortality, and is a lesson in applied physiology. The fact that chemical, electrical, osmotic, and hydrostatic driving forces determine cyst volumes is inferred from observations that cyst fluids vary in their concentrations of osmotically active substances. Most have concentrations of sodium-based salts that are lower and nonsodium-based salts that are higher than those present in normal plasma. Nonsodium solutes include osmolytes that normally are present in higher concentrations inside of cells, including potassium, amino acids, and so-called idiogenic osmoles. The basic process of cyst growth therefore involves 1) the osmotic equilibration of water across cysts walls that have variable permeability characteristics and 2) the replacement of sodium salts with other osmotically active solutes in cyst fluids. Cyst volume is governed by the amounts and kinds of osmolytes that enter and become entrapped in lumina. Proliferation and necrosis of mural cells are events that are fundamental to the growth of renal cysts.

Recently, a system that uses focused ultrasound to expel renal stone fragments from the kidney by radiation force was developed by Shah etal. [Urol. Res. 38, 491–495 (2010)]. A worst-case treatment protocol using this system would require a total exposure time of 10 min, with a spatial peak pulse average intensity (I_SPPA) of 3600 W/cm2 in water (16 MPa peak positive pressure) and a 3% duty cycle. As the system operates above the FDA limits for diagnostic ultrasound, our goal is to verify the safety of the system by determining the threshold for renal injury. A 2-MHz annular array generating I_SPPA up to 29 000 W/cm2 in water was placed on the surface of in vivo porcine kidneys and focused in the proximal parenchyma. Exposures of 10 min duration with varying I_SPPAs and duty cycles were repeated at least 6 times. Mechanical tissue damage and cell viability were evaluated histologically using H&E, PAS, and NADH diaphorase stains. The proportion of samples showing injury was plotted versus duty cycle and I_SPPA. The results indicate that the system to expel renal stones operates below the threshold for kidney injury. [Work supported by NIH DK043881, DK086371, DK092197, and NSBRI through NASA NCC 9–58.]

Although lithotripters can differ substantially in acoustic output, renal response to SWs has been thoroughly characterized for only one type of lithotripter (moderate‐pressure ∼40 MPa, intermediate‐focal‐width 8–9 mm, Dornier‐HM3). Therefore, we assessed injury using a high‐pressure (∼85 MPa), narrow‐focal‐width (3–4 mm) device (Storz‐SLX). SWs were administered to the lower renal pole of pigs (15 kg) (PL‐9, 2000 or 4000 SWs at 120 SWs/min; or 2000SWs at 60 SW/min) followed by serial sectioning to quantify lesion. Tissue damage with the SLX showed an abrupt transition between injured and unaffected tissue. Core of lesion was typically devoid of recognizable structure, with renal tubules, glomeruli, vessels essentially eliminated. The lesion extended from cortex to medulla, and in some kidneys homogenized tissue could be tracked across full thickness of the kidney. In comparison, the lesion using HM3 was not as severe with only focal spots of hemorrhage and no areas of complete tissue destruction. Doubling the SW‐dose with SLX did not increase lesion volume as occurred with HM3. Also, slowing the SW‐rate of the SLX to 60 SW/min did not have a protective effect as observed with other lithotripters. These findings suggest that lithotripters having different acoustic characteristics are capable of producing different forms of tissue damage. [Work supported by NIH‐DK43881.]

Ultrasonically activated microbubbles are promising candidates for targeted therapeutic applications such as drug and gene delivery. Understanding vascular permeability is thus important. In this study, ultra high‐speed microphotography was used to directly visualize the dynamics of microbubbles in microvessels, under the excitation of ultrasound pulses with a duration time of about 2 μs, a center frequency of 1 MHz, and peak negative pressures (PNPs) between 0.8 and 7.2 MPa. Correlated vascular permeability was examined by imaging extravasation of bubbles from the vessels and by looking at the treated region histologically and under transmission electron microscopy. Local vessel distention and invagination caused by microbubble oscillations and liquid jet impinging on the vessel wall all appear to contribute to enhanced permeability for microvessels with diameters 4 MPa. Vascular permeation was also observed in larger vessels. However, in these cases vessel invagination was greater than distention in more than 80% of the cases and liquid jets always directed away from the vessel wall. We thus hypothesize that the dominant mechanisms for microbubble‐induced permeation of smaller microvessels are different from larger microvessels. [Work supported by NIH EB000350 and AR053652.]

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.

reply : We thank Dr. Miner for his thoughtful comments ([3][1]) on our study ([7][2]). He is right that the entire middle region of the glomerular capillary wall (GCW) in the amphibian kidney probably should not be called glomerular basement membrane (GBM), although we and others have done so ([4][

Pretreating the kidney with low‐energy shock waves (SWs) before administering a clinical dose of high‐energy SWs has been found to greatly decrease the hemorrhagic lesion that normally results from high‐energy shock wave lithotripsy (SWL) treatment alone. We tested the hypothesis that this novel pretreatment SWL protocol results in a greater and/or earlier renal vasoconstriction than clinical SWL alone, which could potentially explain the reduced bleeding in the kidney. Anesthetized pigs were treated with a clinical dose of SWs (2000 SWs, 24 kV, 120 SWs/min) or the pretreatment protocol (500 SWs, 12 kV, 120 SWs/min + 3‐min pause + 2000 SWs, 24 kV, 120 SWs/min) using the HM3 lithotripter. Sonography (color and pulsed ultrasound) was used to locate a resistance artery within the SW‐treated pole of the kidney and to take resistance index (RI) measurements from the blood vessel to estimate the resistance/impedance to blood flow. The results demonstrated that RI increased (renal vasoconstriction) earlier and g...

Shock wave lithotripsy (SWL) is known to induce oxidative stress and a rapid inflammatory response in renal tissue. The purpose of this study was to determine the location and quantify markers for oxidative stress and inflammation in a porcine model of acute SWL‐induced renal injury. The lower pole calyx of left kidneys of female pigs received 2000 shock waves at 24 kV/2 Hz from a Dornier HM3 lithotripter and subsequently monitored for 4 h. Heme oxygenase‐1 protein (HO‐1) was measured in renal microsomes by Western blot. Renal medulla HO‐1/β‐actin ratios were 1.186 ± 0.48 for treated pole (F2), 0.293 ± 0.23 for treated kidney upper pole, and 0.122 ± 0.075 for contralateral kidney (n=6). Thus, we observed an eightfold induction of HO‐1 in renal medulla at F2 compared to the upper pole medulla of the treated kidney and the contralateral kidney medulla. In contrast, renal cortex at F2 did not show a similar HO‐1 induction. The highly localized HO‐1 induction in SWL‐treated renal medulla parallels our previou...

The renal papilla is particularly susceptible to injury by shock waves (SWs). Since the papilla is vital for urinary pH regulation, we hypothesized that shock wave lithotripsy (SWL)‐induced damage of papillae will alter the pH of the urine. Experiments were conducted in four female adult Gottingen minipigs. The HM3 lithotripter delivered a total of 8000 SWs (24 kV, 120 SWs/min) among all papillae of one kidney, while the untreated, opposite kidney was used as control. The pigs were allowed to recover and 5 weeks later they were anesthetized. Serial urine collections were obtained from the SW‐treated and untreated kidney. Urine was collected under mineral oil and immediately tested for its pH. Both kidneys were then perfusion fixed for histological analysis. Urinary pH was 0.17 units greater (P

The morphological structure of individual vascular smooth muscle cells from intestinal venules was evaluated with a combination of quantitative scanning (SEM) and transmission (TEM) electron microscopy techniques. In addition, growth of individual venular smooth muscle cells and of the overall vessel wall was compared from measurements of these variables during the rapid juvenile growth spurt from ages 4 to 6 and 10 to 12 weeks in Wistar-Kyoto rats. SEM revealed that smooth muscle cells of intestinal venules in weanling rats are very long (379 +/- 91 [SD] microns) and wide (6.0 +/- 1.3 microns) and very little further cell enlargement occurs during rapid juvenile growth. TEM studies indicated that passive inner vessel diameter and total muscle layer cross-sectional area of both the largest and intermediate diameter venules of young rats, as well as the percentage of the total wall area as muscle tissue in each venule type, did not significantly increase during body growth. These observations indicate that both the intestinal venules and their smooth muscle cells reach mature dimensions at a very early stage of life. Comparison of intestinal vascular smooth muscle cell dimensions indicates that venular smooth muscle cells are much larger in both cell length and volume than comparable arteriolar smooth muscle cells.

Both clinical and experimental reports clearly show that shock wave lithotripsy (SWL) causes acute renal effects in a majority, if not all, treated kidneys. SWL-induced acute renal damage may result in severe injury to the nephron, microvasculature, and the surrounding interstitium. In addition, at least three chronic adverse effects have been identified when shock waves are administered at a therapeutic dose. These include (1) an accelerated rise in arterial blood pressure, (2) a decrease in renal function, and (3) an increased rate of stone recurrence. The clinical and experimental data that document tissue injury as a result of shock wave treatment are compelling, but have not allowed us to determine the factors responsible for the adverse acute side effects or to identify conditions that may predispose a patient to serious long-term health problems. Thus, there is an urgent need for incisive, fundamental experimental studies to establish the safe limits for shock wave delivery. To accomplish this goal, animal experimentation is required so that the time course and severity of acute and chronic alterations can be followed in a model that closely mimics human renal structure and functions. The minipig provides this model.

Kidney injury in lithotripsy can lead to long‐term adverse effects, so minimizing injury is beneficial. We have found that injury is significantly reduced when shock wave (SW) administration is halted briefly‐ 3‐minutes‐early in treatment. Previous studies have shown that SWs stimulate renal blood vessels to constrict. Therefore, we tested the idea that vasoconstriction mediates SW‐induced protection of the kidney. Doppler ultrasound was used to measure resistive index (RI), a ratio of systolic and diastolic velocities, indicating vasoconstriction. RI was determined for single intralobar arteries in targeted porcine kidneys treated by standard versus protection protocols (2,000SW, uninterrupted vs 500SW‐3‐min pause‐2,000SW) using a Dornier‐HM3 lithotripter (2Hz, 24kV). Significant differences in RI from baseline within a group and between groups at various time points were determined using mixed‐effect models for repeated measures with Holm's step‐down method for multiple comparison adjustment. All animal...

The spontaneously hypertensive rat (SHR) exhibits increased renal sympathetic nerve activity and neurotransmitter levels compared with the control Wistar-Kyoto rat (WKY). These renal nerve abnormalities have been implicated as the cause of hypertension in the SHR. The aims of the present study were to characterize the ontogeny of renal sympathetic innervation in SHR in order to determine any functional implications. Glyoxylic acid histofluorescent and radio-enzymatic norepinephrine assays demonstrated an accelerated development of renal innervation in newborn and 1-, 2-, 3- and 6-week-old SHR compared with WKY. Sympathetic nervous system function was blocked in developing male SHR by treating pups from days 0 to 14 with: (1) guanethidine, (2) combined alpha- and beta-receptor antagonists (prazosin and timolol), or (3) vehicle (5% sucrose). Blood pressure (mean), renal function (plasma creatinine) and histologic renal damage were assessed at 42 weeks of age. Although the blood pressure of the drug-treated rats remained elevated, renal damage was reduced and renal function was improved compared with control (sucrose-treated) SHR. The data demonstrate that the SHR kidney develops a precocious sympathetic innervation and that inhibition of the development of sympathetic function ameliorates renal damage independently of systemic hypertension.

A clinical dose of SWL consistently and specifically disrupts the cellular layers of renal veins and arteries, first in the medulla, but progressing into the cortex with an associated inflammatory response. The resulting hemorrhage within the focal zone produces regions of intraparenchymal and subcapsular hematomas of varying size. Renal blood flow and glomerular filtration rate fall acutely by 50% in the treated kidney and by 20% in the untreated kidney. The sites of acute injury form scars with a permanent loss of renal function and have been associated with apatite/brushite stone formation and hypertension. Our team has shown that the size of the SWL‐induced lesion can be dramatically reduced by lowering the kV level, applying fewer shock waves, slowing the shock wave rate, and by pretreating the kidney with 100–500 shock waves at 12 kV prior to standard SWL. The mechanism of tissue injury caused by SWL has not been defined; however, protocols that reduce cavitation activity also reduce the size of the...