Strategies for improved shock wave lithotripsy.

Research in lithotripsy that started with the effort to characterize acute shock wave damage to the kidney has led to advances on several fronts, including discovery of strategies that have improved clinical treatment. It is appreciated now that shock wave trauma is primarily a vascular lesion, that injury is dose dependent, and that hemorrhage can be severe and can lead to a permanent loss of functional renal mass. Studies of the renal functional response to lithotripsy have shown that shock wave treatment triggers vasoconstriction in the kidney. This finding has been turned to advantage, and it is now known that when treatment is begun using low amplitude pulses, subsequent high amplitude shock waves are far less damaging. Thus, when shock waves are delivered judiciously, treatment can have a protective effect. The finding that cavitation is a key mechanism in vessel rupture has led to the development of novel experimental methods of shock wave delivery that can suppress bubble expansion and minimize tissue damage. Progress has also been made in understanding the physical mechanisms involved in stone comminution, and it is seen that the forces generated by cavitation, shear stress and circumferential squeezing act synergistically to fragment stones. Recent work suggests that a broad focal zone may be an advantage, allowing stones to be broken with lower amplitude pulses. Cavitation has been shown to play a critical role in reducing stone fragments to a size that can be voided. Cavitation is also the factor that limits the rate at which treatment can be performed, as stones break significantly better at slow rate than at fast ratean observation from basic research that is now appreciated in clinical practice. The current environment in lithotripsy research is encouraging. There is great interest in developing new technology, and in finding ways to improve how lithotripsy is performed.

Effect of radiographic contrast material exposure on spiral CT attenuation of renal calculi.

RATIONALE AND OBJECTIVES: The authors performed this study to determine whether exposure of renal calculi to radiographic contrast material has an effect on the attenuation values at computed tomography (CT) performed with varying collimation widths. MATERIALS AND METHODS: Renal calculi (23 stones of various composition) were scanned with 1-, 3-, and 10-mm collimation. Stones were then exposed to a solution of radiographic contrast material for 5 minutes, washed with water, and rescanned 36 hours later. The reproducibility of the CT attenuation measurements on different days was evaluated by obtaining measurements in a subset of 16 renal stones on 4 different days. RESULTS: There was no statistically significant change in attenuation after contrast material exposure at narrow collimation. At wider collimation, statistically significant increases were noted in both attenuation and standard deviation. A small amount of variability between readings was noted on different days, with a minimal increase in attenuation each day. Correlation between readings remained very high. CONCLUSION: Exposure of stones to a radiographic contrast material had a statistically significant effect on CT attenuation values only at wide collimation. This may be related to technical factors including volume averaging. Absence of an effect at narrow collimation suggests that the attenuation values of renal stones do not significantly change after exposure to contrast material.

Correction of helical CT attenuation values with wide beam collimation: in vitro test with urinary calculi.

RATIONALE AND OBJECTIVES: Urinary calculi are now commonly detected with helical computed tomography (CT), and it has been proposed that stone composition can be determined from CT attenuation values. However, typical scans are made with a beam collimation of 5 mm or more, resulting in volume averaging and reduction in accuracy of attenuation measurement. The authors tested a model for correction of errors in attenuation values, even at section widths larger than the width of the object. MATERIALS AND METHODS: Human urinary stones were scanned with helical CT at different beam collimation widths. A computer model was used to predict the effect of beam width and stone size on accuracy of measured attenuation. RESULTS: At 3-mm collimation, the model corrected the attenuation readings with an underestimation of 12% +/- 1 (compared with values at 1-mm collimation; 127 stones; diameters of 1.7-11.3 mm). With attenuation measured at 10-mm collimation, the model underestimated the true value by 34% +/- 3 (103 stones), with a significant negative correlation with stone diameter on magnitude of error (diameters of 3.0-11.3 mm). Correlation of data from patient scans with subsequent in vitro scanning of the same stones confirmed the validity of the model, but corrected in vivo scans consistently yielded lower values for the stones than in vitro. CONCLUSION: Volume averaging effects on attenuation in helical CT are predictable in vitro for urinary calculi--and presumably for other roughly spherical structures--as long as section width does not excessively exceed the diameter of the structure.

Mechanical haemolysis in shock wave lithotripsy (SWL): II. In vitro cell lysis due to shear.

In this work we report injury to isolated red blood cells (RBCs) due to focused shock waves in a cavitation-free environment. The lithotripter-generated shock wave was refocused by a parabolic reflector. This refocused wave field had a tighter focus (smaller beam width and a higher amplitude) than the lithotripter wave field, as characterized by a membrane hydrophone. Cavitation was eliminated by applying overpressure to the fluid. A novel passive cavitation detector (HP-PCD) operating at high overpressure (up to 7 MPa) was used to measure acoustic emission due to bubble activity. The typical 'double-bang' emission measured in the lithotripter free-field was replaced by a continuum of weak signals when the fluid was enclosed in a pressure chamber. No acoustic emissions were measured above an overpressure of 5.5 MPa. Aluminium foils were used to study shock wave damage and had distinct deformation features corresponding to exposure conditions, i.e. pitting and denting accompanied by wrinkling. Pitting was eliminated by high overpressure and so was due to cavitation bubble collapse, whereas denting and wrinkling were caused by the reflected shock wave refocused by the parabolic reflector. RBCs suspended in phosphate-buffered saline (PBS) were exposed to the reflected wave field from a parabolic reflector and also from a flat reflector. Exposure to the wave field from the parabolic reflector increased haemolysis four-fold compared with untreated controls and was twice that of cell lysis with the flat reflector. Recently we analysed deformation and rupture of RBCs when subjected to a flow field set up by a focused shock. The cell lysis results presented here are in qualitative agreement with our theoretical prediction that haemolysis is directly related to the gradient of shock strength and validates shearing as a cell lysis mechanism in SWL.

Serum mitochondrial aspartate transaminase activity after isoflurane or halothane anaesthesia.

We examined the effect of halothane or isoflurane anaesthesia on hepatic function in 30 ASA I-III patients aged 18-70 yr undergoing lumbar discectomy. Hepatic function was assessed before anaesthesia, at the end of surgery, and at 3, 6, 24 and 48 h after surgery using routine enzyme tests of hepatic function and mitochondrial aspartate transaminase (mAST) activity. Although serum mAST activities increased after surgery in both groups of patients, these increases were statistically significantly greater in the group that received halothane. The groups were similar with regard to other tests of hepatic function. Calculation of the ratio of serum enzyme activities compared to baseline values suggested that mAST is a sensitive marker of anaesthetic-induced hepatic injury.

Helical CT of urinary calculi: effect of stone composition, stone size, and scan collimation.

OBJECTIVE: Helical CT has become the preferred methodology for identifying urinary calculi. However, the ability to predict stone composition, which influences patient treatment, depends on the accurate measurement of the radiographic attenuation of stones. We studied the effects of stone composition, stone size, and scan collimation width on the measurement of attenuation in vitro. MATERIALS AND METHODS: One hundred twenty-seven human urinary calculi of known composition and size were scanned at 120 kVp, 240 mA, and a 1:1 pitch at different collimations. A model, based on the physics of helical CT, was used to predict the effect of scan collimation width and stone size on measured attenuation. RESULTS: At a 1-mm collimation, stone groups could be differentiated by attenuation: the attenuation of uric acid was less than that of cystine or struvite, which overlapped; these were less than the attenuation of calcium oxalate monohydrate, which was in turn lower than that of brushite and hydroxyapatite, which overlapped and showed the highest values. At a wider collimation, attenuation was lower and the ability to differentiate stone composition was lost. Attenuation also decreased with smaller stones. At a 10-mm collimation, some uric acid stones (

Calcium stone fragility is predicted by helical CT attenuation values.

BACKGROUND AND PURPOSE: Helical CT has become the preferred method for imaging urinary calculi, and so it would be useful if data from helical CT could also be used to predict the number of shockwaves (SWs) needed to break a given stone. METHODS AND MATERIALS: We measured the number of SWs required to comminute calcium stones in vitro. RESULTS: The SW requirement correlated with stone size (volume, weight, diameter) and with helical CT attenuation values when the scans were performed at 3-mm collimation. When CT scans were performed at 1-mm collimation, the number of SWs needed for comminution did not correlate with helical CT attenuation values. This result indicates that the correlation with 3-mm scans was attributable to volume-averaging effects, in which smaller stones yield smaller attenuation values. That is, attenuation values from helical CT at larger beam collimation widths contain information about stone size that can be exploited to predict the fragility of calcium stones. We observed that for calcium stones, the number of SWs to comminution was generally less than half the stone CT attenuation value in Hounsfield units. This "half-attenuation rule" predicted the number of SWs needed to complete fragmentation for 95% of calcium stones (24/24 calcium oxalate monohydrate, 13/13 hydroxyapatite, 8/10 brushite stones). CONCLUSION: This in vitro study suggests that it may be possible to predict effective SW dose using helical CT prior to lithotripsy.

Effect of macroscopic air bubbles on cell lysis by shock wave lithotripsy in vitro.

In studies of cells or stones in vitro, the material to be exposed to shock waves (SWs) is commonly contained in plastic vials. It is difficult to remove all air bubbles from such vials. Because SWs reflect at an air-fluid interface, and because existing gas bubbles can serve as nuclei for cavitation events, we sought to determine in our system whether the inclusion of small, visible bubbles in the specimen vial has an effect on SW-induced cell lysis. We found that even small bubbles led to increased lysis of red blood cells (1- to 3-mm diameter bubbles, 9.8+/-0.5% lysis, n = 7; no bubbles, 4.4+/-0.8%, n = 4), and that the degree of lysis increased with bubble size. Damage could not be reduced by centrifuging the cells to the opposite end of the vial, away from the bubble. B-scan ultrasound imaging of blood in polypropylene pipette bulbs showed that, with each SW, bubbles were recruited from the air interface, mixing throughout the fluid volume, and these appeared to serve as nuclei for increased echogenicity during impact by subsequent SWs; thus, bubble effects in vials could involve the proliferation of cavitation nuclei from existing bubbles. Whereas injury to red blood cells was greatly increased by the presence of bubbles in vials, lytic injury to cultured epithelial cells (LLC-PK1, which have a more complex cytoarchitecture than red blood cells) was not increased by the presence of small air bubbles. This suggests different susceptibility to SW damage for different types of cells. Thus, the presence of even a small air bubble can increase SW-induced cell damage, perhaps by increasing the number of cavitation nuclei throughout the vial, but this effect is variable with cell type.

Cell damage by lithotripter shock waves at high pressure to preclude cavitation.

Acoustic cavitation has been implicated as a cause of cell damage by lithotripter shock waves, particularly under in vitro conditions. When red blood cells were exposed to shock waves (from an electrohydraulic lithotripter) while under high hydrostatic pressure (> 80 atm), cell lysis was dramatically reduced over that seen at atmospheric pressure, which is consistent with damage due to acoustic cavitation. However, even at > 120 atm of pressure, lysis was still 97% above that of cells not exposed to shock waves, revealing significant damage that apparently was due to mechanisms other than cavitation. Hydrostatic pressure alone did not cause cell lysis, and shock-wave-dependent damage occurred when the cells were in fluid suspension, or when they were centrifuged to the end of the vial. Shock-wave damage at high pressure increased with increasing shock-wave number, and was seen at 24 and 20 kV, but not at 16 kV. This shock-wave damage at high pressure makes up a noteworthy portion of the total cell injury seen at atmospheric pressure (about 10% at 24 kV), suggesting significant noncavitational injury to cells in vitro. Because cavitation occurs far more readily in vitro than in vivo, the noncavitational damage seen in the present study could represent a substantial portion of cell injury seen in vivo with shock-wave lithotripsy.

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury.

In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na+K+ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 microM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na+K+ATPase activity in whole cells, Na+K+ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na+K+ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na+K+ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na+K+ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na+K+ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.

Hyperosmotic stress up-regulates amino acid transport in vascular endothelial cells.

Cultured vascular endothelial cells take up L-proline by sodium-dependent transport. Cells incubated in medium made hyperosmotic by addition of sucrose showed a dose-dependent increase in Na+/proline cotransport. Studies with alpha-(methylamino)isobutyric acid revealed that the up-regulation was specific for amino acid transport system A. Up-regulation was blocked by actinomycin D and cycloheximide, indicating roles for gene transcription and protein synthesis. Up-regulation was maximum after five to six hours of hyperosmotic treatment, but returned to control levels when osmotic stress was maintained for 24 hours. The decline at 24 hours was accompanied by a significant increase in Na+/gamma-aminobutyric acid cotransport. The activity of this system, which also transports betaine, remained unchanged after just five hours of hyperosmotic stress. Inclusion of betaine in the hyperosmotic medium reduced up-regulation of system A. Na/Pi cotransport also was up-regulated by five hours of hyperosmotic stress. Up-regulation of system A, but not Na/Pi cotransport, was detected in isolated membrane fractions indicating that increased activity of this membrane transport system may be one mechanism by which vascular endothelial cells accumulate amino acids. The amino acids may act as organic osmolytes to help maintain normal cell volume during the early phase of hyperosmotic stress.

Quantitation of shock wave cavitation damage in vitro.

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.

The effect of polypropylene vials on lithotripter shock waves.

In studies to understand the mechanisms responsible for shock wave lithotripsy (SWL) cell injury, we observed that shock waves (SWs) are influenced by the shape of the specimen vial. Lytic injury to kidney cells treated in a Dornier HM3 lithotripter was higher (p < 0.0001) when SWs entered the vial through the flat end (cap end) compared to the round end. Measurements of the acoustic field within polypropylene vials were carried out using both lithotripter SWs and pulsed ultrasound (US) in the megahertz frequency range. We compared pressure amplitudes inside the round and flat vials and found significant differences. When SWs entered through the round end, the average peak positive pressure was 40% of free-field pressure, due mostly to a dramatic reduction in pressure off axis. The average peak pressure inside the flat vial was twice that of the round vial. Experiments with US demonstrated that sound field focusing was induced by the curved interface of the round vial. Ray analysis for the round vial indicates the presence of "hot spots" on axis and "cold spots" off axis, in qualitative agreement with pressure profiles. We conclude that the shape of the specimen vial is an important factor that should be considered in model systems of SWL cell injury.

Apical membrane permeability of MDCK cells.

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.

Hypertonic activation and recovery of system A amino acid transport in renal MDCK cells.

Amino compounds are abundant within the renal inner medulla, but their possible role during hypertonic stress is not clear. Renal epithelial Madin-Darby canine kidney cells were used to examine the osmoregulation of system A transport, a major Na(+)-dependent process for neutral amino acid transport. System A activity was markedly increased after 6 h of hypertonic challenge, and intracellular alanine content increased more than twofold. The activation of system A was reversed after 24 h of hypertonic challenge. This downregulation was accompanied by the activation of betaine transport, as measured by gamma-aminobutyric acid uptake. Extracellular betaine prevented the early activation of system A. The hypertonic activation of system A was blocked by actinomycin D and cycloheximide. When cells were returned to isotonic medium after hypertonic activation, the recovery of system A transport also was partially inhibited by actinomycin D and puromycin. The results are consistent with the possibility that hypertonicity, by disrupting a repressor protein, leads to increased synthesis of a system A-related protein. The isotonic recovery may require synthesis of new repressor proteins.

The effect of lethal acid stress on Na+/H+ exchanger isoforms in cultured inner medullary collecting duct cells: deletion of NHE-2 and over expression of NHE-1.

Cultured inner medullary collecting duct (mIMCD-3) cells express Na+/H+ exchanger isoforms NHE-2 and NHE-1 (Soleimani et al. (1994) J. Biol. Chem. 269, 27973-27978). In the present studies we examined the effect of lethal acid stress on Na+/H+ exchanger activity and isoform expression in mIMCD-3 cells. mIMCD-3 cells were incubated for 10 min with 20 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0) for 120 min. Thereafter, cells were recovered and grown in normal culture media. The surviving clones were isolated and subjected to two additional cycles of acid stress. A mutant clone was isolated and characterized for Na+/H+ exchange activity and isoform expression. The mutant mIMCD-3 clone demonstrated significant over-expression of Na+/H+ exchange activity as assessed by acid-stimulated 22Na influx (11.56 nmol/mg protein in mutant vs. 4.06 nmol/mg in parent cells, P < 0.001, n = 4) and sodium-dependent pHi recovery from an acid load (0.55 pH/min in mutant vs. 0.28 pH/min in parent cells, P < 0.01, n = 6). A dose-response inhibition of the exchanger showed that the mutant cells were very sensitive to dimethylamiloride (IC50 158 nM in mutant vs. 889 nM in parent mIMCD-3 cells, P < 0.001). To compare the Na+/H+ exchanger isoforms in mutant and parent mIMCD-3 cells, poly(A)+ RNA was isolated from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA. The expression of NHE-1 mRNA was increased by approximately 100% in mutant cells. The NHE-2 mRNA, on the other hand, was found to be absent in mutant mIMCD-3 cells. Examination of the regulatory mechanisms of the Na+/H+ exchanger isoforms in parent mIMCD-3 cells, which express NHE-2 and NHE-1, and mutant mIMCD-3 cells, which only express NHE-1, would be helpful in elucidating the roles of NHE-2 and NHE-1 in inner medullary collecting duct cells.

An in vitro test of the cell stretch-proliferation hypothesis of renal cyst enlargement.

In renal cystic disease, fluid accumulation within cyst lumens might stretch cyst walls and in this way stimulate cell proliferation. To test this idea, the effects of mechanical stretch on Madin-Darby canine kidney cells grown as cysts in a hydrated collagen gel or as monolayers on collagen-coated Flexcell membranes were examined. The percentage of cells synthesizing DNA (labeling index) was determined by measuring bromodeoxyuridine incorporation and counting cell numbers. The distension of single cysts for 1 h by the intraluminal injection of saline failed to produce a significant increase in labeling index. The exposure of cysts for 2.5 h to 1 mM dibutyryl cAMP + 0.1 mM isobutylmethylxanthine led to a 37% increase in luminal surface area (due to stimulated fluid secretion) and a 30% increase in labeling index. The stretch (25%) of Madin-Darby canine kidney monolayers approximately doubled the labeling index between 12 and 24 h after starting the stretch. After 48 h, the cell population density was significantly increased (P < 0.001), from 41.9 +/- 0.2 (SE; N = 12) to 48.2 +/- 0.5 (N = 12) cells/10,000 microns2. The labeling index increased linearly with applied stretch, from 7.2 +/- 0.3% (N = 36) with no stretch to 16.2 +/- 1.0% (N = 6) with 30% stretch. Stretch had to be maintained for 8 h or more to produce an increase in labeling index at 18 h. No evidence was obtained for the release of a diffusible growth factor by stretched monolayers. The increase in labeling index induced by stretch was unaffected by 50 microM gadolinium, a stretch-activated channel blocker, but was abolished by 5 micrograms/mL cytochalasin B, an actin microfilament-disrupting agent. It was concluded that prolonged stretch stimulates renal epithelial cells to synthesize DNA. This supports the idea that increased wall tension in renal cysts may stimulate cell proliferation and, thereby, may contribute to cyst enlargement.

Parathyroid hormone inhibits plasma membrane Pi transport without changing endocytic activity in opossum kidney cells.

Parathyroid hormone (PTH) inhibits Na(+)-dependent Pi uptake in renal epithelial cells from opossum kidney (OK). This requires an intact endocytic pathway, suggesting that one action of PTH may be to promote endocytic removal of Na+/Pi cotransporters from the cell membrane. The present study tested if PTH, at a dose that inhibited membrane Pi transport, also produced an increase in endocytic activity. Pi transport was measured in isolated plasma membrane vesicles. Endocytosis was measured by allowing cells to take up horseradish peroxidase (HRP) followed by assay of triton-sensitive (latent) HRP activity in subcellular fractions isolated by density gradient centrifugation. Incubation of OK cells with 10(-7) M PTH for 3 h decreased Na+/Pi cotransport by membrane vesicles to 328 +/- 54 pmol/mg/min compared to 448 +/- 67 pmol/mg/min (mean +/- S.E., P < 0.03) in controls. Latent HRP content of endosomal fractions was dependent on the time and temperature used to load cells with HRP and on the concentration of HRP. However, incubation of OK cells with 10(-7) M PTH for either 1 or 3 h produced no change in latent HRP activity. Thus the action of PTH on the Na+/Pi cotransporter in the plasma membrane of OK cells does not require a change in the rate of endocytosis.