Functional and Morphological Changes Associated with Burst Wave Lithotripsy-Treated Pig Kidneys

Abstract

Purpose:

Burst wave lithotripsy (BWL) is a new technique for comminution of urinary stones. This technology is noninvasive, has a low positive pressure magnitude, and is thought to produce minor amounts of renal injury. However, little is known about the functional changes related to BWL treatment. In this study, we sought to determine if clinical BWL exposure produces a functional or morphological change in the kidney.

Materials and Methods:

Twelve female pigs were prepared for renal clearance assessment and served as either sham time controls (6) or were treated with BWL (6). In the treated group, 1 kidney in each pig was exposed to 18,000 pulses at 10 pulses/s with 20 cycles/pulse. Pressure levels related to each pulse were 12 and −7 MPa. Inulin (glomerular filtration rate, GFR) and para-aminohippuric acid (effective renal plasma flow, eRPF) clearance was measured before and 1 hour after treatment. Lesion size analysis was performed to assess the volume of hemorrhagic tissue injury created by each treatment (% FRV).

Results:

No visible gross hematuria was observed in any of the collected urine samples of the treated kidneys. BWL exposure also did not lead to a change in GFR or eRPF after treatment, nor did it cause a measurable amount of hemorrhage in the tissue.

Conclusion:

Using the clinical treatment parameters employed in this study, BWL did not cause an acute change in renal function or a hemorrhagic lesion.

Introduction

Burst wave lithotripsy (BWL) is an emerging technology used to comminute urinary stones. BWL employs multicycle sinusoidal bursts of focused ultrasound to create stress in a calculus, leading to the formation of cracks in the stone material and eventual fragmentation.¹ This technology is transcutaneous and noninvasive, similar to shockwave lithotripsy (SWL), but has a positive focal pressure amplitude considerably lower than SWL-generated shock waves.² Thus, it is hoped that BWL will produce significantly less injury, when compared with SWL, while still maintaining adequate stone comminuting proficiency.

Previous studies have demonstrated that BWL settings shown to fracture human stones can produce small focal hemorrhages in the renal epithelium without noticeable damage to the renal parenchyma.³ However, more extensive damage of kidney tissue may occur if BWL exposure parameters are altered, which result in sustained cavitation (detected as dynamic hyperechogenicity) in the urine-collecting space or the parenchyma.^4,5

Currently, little is known about the functional consequences of BWL treatment and whether changes in renal function occur even if little morphological damage is observed in a BWL-exposed kidney.

Materials and Methods

Animal procedures

The surgical and animal treatment protocols used to assess renal function and morphology changes in this study were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine (approval# 21060).

The design of the experiment and all surgical procedures employed during the experiment followed methods used in previously published studies.^2,6,7 Twelve female pigs, weighing 32–42 kg each (Oak Hill Genetics, Edwin, IL), were anesthetized with a combination of ketamine and xylazine, intubated, and then maintained on isoflurane anesthesia. Both flanks of each animal were shaved to remove hair and the pigs were placed on a surgical table in a supine position. Respiration was spontaneous.

A catheter was placed in an ear vein for infusion of isotonic saline at a rate of 1% body weight per hour and for infusion of inulin and para-aminohippuric acid. Next, catheters were placed in a femoral artery, for blood pressure and heart rhythm monitoring (EMKA Technologies, Sterling, VA) and blood sampling, and in both ureters for timed urine collection. After the inulin and para-aminohippuric acid (PAH) reached a steady state, blood and urine samples were collected from the pig both before (for baseline) and 1 hour after BWL treatment.

After baseline clearance collection was completed, each pig was placed on its left side. Next, Scan ultrasound gel (centrifuged to remove bubbles; Parker Laboratories, Fairfield, NJ) was layered onto the flank and a water tub was secured in place and then filled with degassed water until the tub membrane touched the ultrasound gel layer coupling the tub to the pig. When this was completed, the BWL treatment head and ultrasound imaging probe were both immersed in degassed water (Figs. 1 and 2).

FIG. 1.

Photograph looking down into a tub from above with a BWL treatment head and ultrasound probe in place. Both the BWL treatment head and ultrasound probe are immersed in degassed water. The plastic tub holds a 1-mil-thick plastic membrane in position and helps contain water. The right flank of the pig is indicated in the picture. The ultrasound gel layer is sandwiched between the membrane and flank of the pig (BWL = BWL treatment head and U = ultrasound probe; the arrow indicates the level of degassed water). BWL = burst wave lithotripsy.

FIG. 2.

Photograph showing the side view of the frame that is holding the tub. Also shown is the scaffolding holding the BWL treatment head and ultrasound probe. The plastic tub holds a 1-mil-thick plastic membrane in position and helps contain water. The right flank of the pig is indicated in the picture. The ultrasound gel layer (which cannot be seen here) is sandwiched between the membrane and flank of the pig.

Ultrasound imaging was then used to locate the right kidney of each animal. From there, a lower pole calix of the right kidney was selected and aligned with the focus of the BWL (Fig. 3). Each targeted kidney was either assigned as a sham control, which was observed by ultrasound for 30 minutes, or assigned to receive treatment with BWL using the parameters of 18,000 pulses at a pulse repetition rate of 10 pulses/s with 20 cycles/pulse and a positive pressure amplitude of 12 MPa and negative pressure amplitude of −7 MPa.

FIG. 3.

Image of the ultrasound machine screen showing the right kidney. After the right kidney is located, a lower pole calix of that kidney is aligned with the focus of the BWL head before treatment begins (arrows indicate the outline of the kidney on the ultrasound screen).

Following completion of BWL treatment or the sham control period, the pigs were again placed supine and collection of blood and urine for renal clearance was repeated 1 hour after treatment. Please see the Supplementary Data section for additional details on preparation of the animals and on the BWL equipment.

Lesion analysis

At the end of the experiment, the kidneys were perfusion-fixed in situ with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.4). After the kidneys were removed, they were submerged in fresh fixative for subsequent determination of lesion size. The treated kidneys were analyzed for morphological injury using a process similar to the technique of Handa et al where MRI scans of the kidneys were obtained and analyzed for hemorrhage in the tissue (Fig. 4).⁸

FIG. 4.

Single MRI scan of the T2-weighted signal of a longitudinal section taken from a BWL-treated kidney. The lower pole is at the bottom of the image. Note the lack of dark regions, sites of hemorrhage, located in the kidney parenchyma of the lower pole. Please compare with images from the study by Handa et al⁸ (C = cortex that is dark gray and P = papilla that is light gray).

The images were then used to determine lesion volume and expressed as a percent of the FRV of renal parenchyma damaged for each kidney. Mean lesion size ± SEM was calculated for the different groups, and a one-way ANOVA with Dunnett's method for post hoc comparisons was used to compare the lesion sizes between the sham control, BWL-treated animals, and SWL-treated animals (included for comparison purposes), with BWL-treated animals as the reference group.

Two-sided p-values <0.05 were considered significant.

Renal clearance analysis

Renal function was determined by colorimetric assays of the collected blood and urine samples. The measurements were used to determine the renal clearance of inulin, which was used to estimate the glomerular filtration rate (GFR), and the clearance of PAH, which was used to estimate effective renal plasma flow (eRPF). Mean values of GFR and eRPF ± SEM were calculated, and a paired t-test was used to compare the clearance values between baseline and 1 hour post-treatment for each group.

In addition, two-sample independent t-tests were used to compare the sham group and BWL-treated group with respect to the change in function from baseline. Two-sided p-values <0.05 were considered significant. Data were then expressed as the average percent change from baseline measurements for each animal and include values for 95% confidence intervals for those measurements.

Results

Pig body weights and kidney weights were similar between the groups (mean ± SEM, with body weights of 37.1 ± 1.1 and 34.9 ± 0.8 kg in sham BWL and BWL-treated groups, respectively, and kidney weights of 107 ± 4 and 113 ± 6 g). We did not observe gross hematuria in any of the urine collected from the right kidneys during the BWL treatment or sham control period. In addition, when we reviewed the recorded blood pressure tracings for each animal, we did not detect any change in heart rhythm (premature contractions or delayed beats) during the 30-minute-long BWL treatment period.

For the kidney itself, baseline renal function values for both GFR and eRPF were similar between the sham control and treated groups. The baseline GFR averaged 38 ± 6 and 39 ± 4 mL/min for the sham control and BWL-treated animals, while baseline eRPF averaged 126 ± 10 and 122 ± 9 mL/min for the sham control and BWL-treated animals, respectively. BWL treatment did not appear to alter renal function.

Both GFR and eRPF were not significantly changed in the sham group or in the BWL-treated group 1 hour after treatment when compared with baseline values or when compared between groups (Fig. 5). When lesions were examined, it was determined that BWL treatment, at the parameters used in this study, did not produce a renal lesion (0.022 ± 0.008% FRV) significantly different from that measured in sham control animals (0.028 ± 0.008% FRV) (Fig. 6).

FIG. 5.

Bar graph of the percent change in GFR and eRPF (from baseline) measured after either sham control treatment or BWL treatment. Also included on the graph are values for 95% confidence intervals. For BWL-treated animals, GFR and eRPF did not change significantly from baseline values in either group and the post-treatment values were not significantly different from post-treatment sham values. N = 6 in each group. GFR = glomerular filtration rate; eRPF = effective renal plasma flow. Color images are available online.

FIG. 6.

Bar graph of hemorrhagic lesion sizes from the sham control and BWL-treated groups. The value for the SWL group (far right, Dornier Compact S, 2500 shock waves, power level = 5, at 120 shock waves/minute) is included for comparison purposes and comes from a previously published study using pigs of the same size.² No significant difference was found between the sham control or BWL-treated groups. The lesion produced by SWL is significantly larger than that produced by BWL. N = 5 in the sham control group and N = 6 in both the BWL and SWL groups. *Significantly different from the BWL-treated lesion. SWL = shockwave lithotripsy; NS = not significant. Color images are available online.

Discussion

In this study, we did not observe significant hemorrhagic injury to any kidney treated with BWL using treatment parameters known to fracture stones in animals or in humans.^3,9,10 This finding is consistent with our observation about the lack of visible gross hematuria in any of the urine collected from the treated kidneys since blood from parenchymal hemorrhage normally finds its way into and down the tubules, resulting in urine stained with blood.

In addition to the lack of hemorrhagic injury, this BWL treatment did not cause enough injury to alter renal function. This observation is particularly interesting because numerous studies have shown that other extracorporeal stone removal procedures can cause significant decline in renal function of 50% or more after treatment.^11–15

What could account for the lack of injury associated with this BWL treatment? To answer this question, it may be helpful to compare BWL with the other acoustic-based stone removal therapy, SWL. While BWL and SWL are similar, in that they are both transcutaneous and noninvasive, these techniques diverge significantly when it comes to the total energy delivered to the kidney, the magnitude of positive pressure produced, and the cavitation induced by each type of pulse.

For the BWL treatment described in this study, the pulse energy (for a 5-mm diameter beam) summed over the entire treatment totaled 313 J. For a typical SWL treatment of 2500 SWs at PL = 5 (Dornier Compact S, Dornier Medical Systems, Kennesaw, GA),² the pulse energy (for a 5-mm diameter beam) summed over the entire treatment totals 48 J. One would predict that since BWL delivers more total energy to a kidney over the same approximate time period, BWL would produce a greater hemorrhagic injury than that observed for SWL.

However, hemorrhagic injury for BWL was not observed to be any more than that seen in sham control kidneys (Fig. 6) and it measured much less than that produced in SWL-treated kidneys when using pigs undergoing comparable treatment protocols and with comparable sized kidneys (SWL hemorrhagic lesion measuring 1.55% FRV, included for comparison purposes in Fig. 6).² This observation suggests that factors other than total energy delivered are responsible for the lack of injury observed with BWL treatment.

The magnitude of positive pressure produced during treatment also differs between BWL and SWL pulses. For the BWL treatment described in this study, the pulses had a positive pressure amplitude of 12 MPa. For a typical SWL treatment protocol that mimics a clinical treatment using a Dornier Compact S and produces significant hemorrhagic injury, the pulses have a positive pressure amplitude of ≈52–55 MPa or more.² In addition, SWL pulse waveforms exhibit a steep positive pressure spike of a true shock front.

The observation that SWL produces higher positive pressure and also produces kidney injury may suggest that the pressure amplitude or the presence of a shock front in the pulse plays a decisive role in determining the extent of injury in a kidney. However, a study done by Evan et al suggests that the relationship between pulse pressure and injury is more complicated than what it first appears to be.¹⁶ In that study, kidney damage was significantly reduced when a pressure-release reflector was used to alter the pressure profile of the SWL pulse.

In a normal SWL pulse waveform, the positive pressure spike is followed by a negative pressure trough. However, the pressure-release reflector had a pressure waveform where the negative pressure trough preceded the positive pressure spike. This waveform reversal process did not alter the pressure of the pulse as the measured pulse waveforms maintained the same approximate amplitude for the positive and negative pressure phases.

What did change, however, was that cavitation bubble development was stifled compared with a normal SWL pulse. Moreover, this reduction in cavitation correlated with the almost complete elimination of injury. These observations suggest that while pulse pressure may be lower in BWL, tissue injury in the kidney may be driven more by cavitation activity than by pulse pressure.

Pulse-induced cavitation bubble activity is also different between SWL and BWL. In SWL, the long negative pressure tail of each SW can excite large cavitation bubbles that grow by rectified diffusion,¹⁷ dissolve slowly,¹⁸ and proliferate into dense clouds over successive pulses.¹⁹ On the other hand, BWL pulses lack a similar long negative pressure tail, and this results in smaller bubbles that undergo much less rectified diffusion.²⁰

Consequently, these bubbles tend to dissolve between pulses rather than proliferate into sustained cavitation clouds that have been linked to injury.⁴ This reduction in bubble cloud activity in BWL may explain the lack of injury noted in our present study despite the increase in the rate of delivery of acoustic energy into the kidney. Interestingly, some other investigators have noted tissue hemorrhage in pigs and in patients corresponding to areas where cavitation was observed during a BWL treatment period, and this has led to the recommendation that BWL treatment be temporarily paused if cavitation activity is detected.^4,10

Finally, the animals used in this study were around 36 kg (80 lbs.) in weight and had kidneys that averaged 10.1 cm by 5.7 cm. The lack of injury and lack of change in function in the kidneys used in these experiments imply that treating patients with kidneys down to the size treated here should be safe when using the same BWL parameters as applied during this study.

Conclusions

In conclusion, in this study, we have shown that BWL treatment did not cause a change in renal function, nor did it cause a hemorrhagic lesion, when using a porcine model to detect injury to renal tissue. In addition to these findings, we also did not observe changes in the heart rhythm during treatment or the presence of blood in urine from BWL-exposed kidneys.

Abbreviations Used

BWL

burst wave lithotripsy

eRPF

effective renal plasma flow

FRV

functional renal volume

GFR

glomerular filtration rate

MRI

magnetic resonance imaging

PAH

para-aminohippuric acid

PL

power level

SEM

standard error of the mean

SWL

shockwave lithotripsy

Authors' Contributions

B.A.C. was involved in conceptualization (equal); investigation (lead); methodology (equal); project administration (equal); supervision (lead); visualization (lead); writing—original draft (lead); and writing—review and editing (equal). T.G. was involved in investigation (supporting) and writing—review and editing (equal). Z.L. was involved in formal analysis (equal) and writing—review and editing (equal). J.E.L. was involved in conceptualization (equal); funding acquisition (equal); visualization (supporting); and writing—review and editing (equal). W.K. was involved in methodology (equal); resources; and writing—review and editing (equal). J.C.W. was involved in conceptualization (equal); formal analysis (equal); funding acquisition (equal); investigation (supporting); methodology (equal); project administration (equal); supervision (supporting); visualization (supporting); writing—original draft (supporting); and writing—review and editing (equal).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This project was supported by a grant from the National Institutes of Health (P01-DK43881).

Supplementary Material

Supplementary Data