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Transcription factor over-expression-based cellular conversion methods often endure low conversion efficiency. Here the authors show how to increase conversion efficiency by combining a computational method for prioritizing more efficient TF combinations with a transposon-based genomic integration system for delivery.

Vaidyanathan et al. present a heuristic algorithm for the selection of fluorescent reporters in the context of single-cell analysis. They present a tool to enable biologists to design multi-colour fluorophore panels based on specific equipment’s configurations. The authors demonstrate the efficacy of their algorithm by comparing computational predictions with experimental observations.

Abstract The fast‐paced field of synthetic biology is fundamentally changing the global biosecurity framework. Current biosecurity regulations and strategies are based on previous governance paradigms for pathogen‐oriented security, recombinant DNA research, and broader concerns related to genetically modified organisms (GMOs). Many scholarly discussions and biosecurity practitioners are therefore concerned that synthetic biology outpaces established biosafety and biosecurity measures to prevent deliberate and malicious or inadvertent and accidental misuse of synthetic biology's processes or products. This commentary proposes three strategies to improve biosecurity: Security must be treated as an investment in the future applicability of the technology; social scientists and policy makers should be engaged early in technology development and forecasting; and coordination among global stakeholders is necessary to ensure acceptable levels of risk.

Summary: Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.

Introduction: About 1 in 11 Americans will experience a kidney stone, but underlying causes remain obscure. The objective of the present study was to separate idiopathic calcium oxalate stone formers by whether or not they showed positive evidence of forming a stone on Randall's plaque (RP). Materials and Methods: In patients undergoing either percutaneous or ureteroscopic procedures for kidney stone removal, all stone material was extracted and analyzed using micro-CT imaging to identify those attached to RP. Twenty-four-hour urine samples were collected weeks after the stone removal procedure and patients were off of medications that would affect urine composition. The endoscopic video was analyzed for papillary pathology (RP, pitting, plugging, dilated ducts, and loss of papillary shape) by an observer blinded to the data on stone type. The percent papillary area occupied by RP and ductal plugging was quantified using image analysis software. Results: Patients having even one stone on RP ( N  = 36) did not differ from non-RP patients ( N  = 37) in age, sex, BMI, or other clinical characteristics. Compared with the non-RP group, RP stone formers had more numerous, but smaller, stones, more abundant papillary RP formation, and fewer ductal plugs, both by quantitative measurement of surface area (on average, three times more plaque area, but only 41% as much plug area as in non-RP patients) and by semiquantitative visual grading. Serum and blood values did not differ between RP and non-RP stone formers by any measure. Conclusions: Growth of many small stones on plaque seems the pathogenetic scheme for the RP stone-forming phenotype, whereas the non-RP phenotype stone pathogenesis pathway is less obvious. Higher papillary plugging in non-RP patients suggests that plugs play a role in stone formation and that these patients have a greater degree of papillary damage. Underlying mechanisms that create these distinctive phenotypes are presently unknown.

Chronic kidney disease (CKD) leads to musculoskeletal impairments that are impacted by muscle metabolism. We tested the hypothesis that 10-weeks of voluntary wheel running can improve skeletal muscle mitochondria activity and function in a rat model of CKD. Groups included (n = 12-14/group): (1) normal littermates (NL); (2) CKD, and; (3) CKD-10 weeks of voluntary wheel running (CKD-W). At 35-weeks old the following assays were performed in the soleus and extensor digitorum longus (EDL): targeted metabolomics, mitochondrial respiration, and protein expression. Amino acid-related compounds were reduced in CKD muscle and not restored by physical activity. Mitochondrial respiration in the CKD soleus was increased compared to NL, but not impacted by physical activity. The EDL respiration was not different between NL and CKD, but increased in CKD-wheel rats compared to CKD and NL groups. Our results demonstrate that the soleus may be more susceptible to CKD-induced changes of mitochondrial complex content and respiration, while in the EDL, these alterations were in response the physiological load induced by mild physical activity. Future studies should focus on therapies to improve mitochondrial function in both types of muscle to determine if such treatments can improve the ability to adapt to physical activity in CKD.

Kidney stones frequently develop as an overgrowth on Randall's plaque (RP) which is formed in the papillary interstitium. The organic composition of RP is distinct from stone matrix in that RP contains fibrillar collagen; RP in tissue has also been shown to have two proteins that are also found in stones, but otherwise the molecular constituents of RP are unstudied. We hypothesized that RP contains unique organic molecules that can be differentiated from the stone overgrowth by fluorescence. To test this, we used micro-CT-guided polishing to expose the interior of kidney stones for multimodal imaging with multiphoton, confocal and infrared microscopy. We detected a blue autofluorescence signature unique to RP, the specificity of which was also confirmed in papillary tissue from patients with stone disease. High-resolution mineral mapping of the stone also showed a transition from the apatite within RP to the calcium oxalate in the overgrowth, demonstrating the molecular and spatial transition from the tissue to the urine. This work provides a systematic and practical approach to uncover specific fluorescence signatures which correlate with mineral type, verifies previous observations regarding mineral overgrowth onto RP and identifies a novel autofluorescence signature of RP demonstrating RP's unique molecular composition.

The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2-null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2-null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.

Objectives: To describe the papillary pathology found in uric acid (UA) stone formers, and to investigate the mineral form of tissue deposits. Materials and Methods: We studied eight UA stone formers treated with percutaneous nephrolithotomy. Papillae were imaged intraoperatively using digital endoscopy, and cortical and papillary biopsies were taken. Biopsies were analyzed by light microscopy, micro-CT, and microinfrared spectroscopy. Results: As expected, urine pH was generally low. UA supersaturation exceeded one in all but one case, compatible with the stone material. By intraoperative imaging, the renal papillae displayed a heterogeneous mixture of plaque and plugging, ranging from normal to severe. All patients had mineral in ducts of Bellini and inner medullary collecting ducts, mainly apatite with lesser amounts of urate and/or calcium oxalate in some specimens. Papillary and cortical interstitial tissue injury was modest despite the tubule plugging. No instance was found of a stone growing attached to either plaque or plugs. Conclusions: UA stone formers resemble those with ileostomy in having rather low urine pH while forming tubule plugs that contain crystals that can only form at pH values above those of their bulk urine. This discrepancy between tissue mineral deposits and stone type suggests that local tubular pH exceeds that of the bulk urine, perhaps because of localized tubule injury. The manner in which UA stones form and the discordance between tubule crystals and stone type remain open research questions.

Introduction: We have previously found that papillary histopathology differs greatly between calcium oxalate and brushite stone formers (SF); the latter have much more papillary mineral deposition, tubular cell injury, and tissue fibrosis., Methods: In this study, we applied unbiased orthogonal omics approaches on biopsied renal papillae and extracted stones from patients with brushite or calcium oxalate (CaOx) stones. Our goal was to discover stone type-specific molecular signatures to advance our understanding of the underlying pathogenesis., Results: Brushite SF did not differ from CaOx SF with respect to metabolic risk factors for stones but did exhibit increased tubule plugging in their papillae. Brushite SF had upregulation of inflammatory pathways in papillary tissue and increased neutrophil markers in stone matrix compared with those with CaOx stones. Large-scale 3-dimensional tissue cytometry on renal papillary biopsies showed an increase in the number and density of neutrophils in the papillae of patients with brushite versus CaOx, thereby linking the observed inflammatory signatures to the neutrophils in the tissue. To explain how neutrophil proteins appear in the stone matrix, we measured neutrophil extracellular trap (NET) formation-NETosis-and found it significantly increased in the papillae of patients with brushite stones compared with CaOx stones., Conclusion: We show that increased neutrophil infiltration and NETosis is an unrecognized factor that differentiates brushite and CaOx SF and may explain the markedly increased scarring and inflammation seen in the papillae of patients with brushite stones. Given the increasing prevalence of brushite stones, the role of neutrophil activation in brushite stone formation requires further study.

Introduction: There is evidence that patients with a history of ileostomies, who produce acidic urine and form uric acid or calcium oxalate stones, may plug some collecting ducts with calcium phosphate (CaP) and urate crystals. This is a paradoxical finding as such minerals should not form at an acid pH. One possible explanation is the presence of acidification defects due to focal damage to inner medullary collecting duct and Bellini duct (BD) cells. We sought to further investigate this hypothesis through direct measurement of ductal pH in dilated BDs in patients with ileostomies undergoing percutaneous nephrolithotomy (PCNL) for stone removal. Methods: After obtaining institutional review board approval, we used a fiber-optic pH microsensor with a 140-μm-diameter tip to measure intraluminal pH from the bladder, saline irrigant, and dilated BDs of patients undergoing PCNL. Results: Measurements were taken from three patients meeting inclusion criteria. Measured pH of bladder urine ranged from 4.97 to 5.58 and pH of saline irrigant used during surgery ranged from 5.17 to 5.75. BD measurements were achieved in 11 different BDs. Mean intraductal BD pH was more than 1 unit higher than bulk urine (6.43 ± 0.22 vs 5.31 ± 0.22, p  < 0.01). Conclusions: This is the first evidence for focal acidification defects within injured/dilated BDs of human kidneys producing highly acidic bulk phase urine. These results may help explain the paradoxical finding of CaP and urate plugs in dilated ducts of patients with stone-forming diseases characterized by highly acidic urine.

Randall's plaque, an attachment site over which calcium oxalate stones form, begins in the basement membranes of thin limbs of the loop of Henle. The mechanism of its formation is unknown. Possibly, enhanced delivery of calcium out of the proximal tubule, found in many stone formers, increases reabsorption of calcium from the thick ascending limb into the interstitium around descending vasa recta, which convey that calcium into the deep medulla, and raises supersaturations near thin limbs ("vas washdown"). According to this hypothesis, plaque should form preferentially on ascending thin limbs, which do not reabsorb water. We stained serial sections of papillary biopsies from stone-forming patients for aquaporin 1 (which is found in the descending thin limb) and the kidney-specific chloride channel ClC-Ka (which is found in the ascending thin limb). Plaque (which is detected using Yasue stain) colocalized with ClC-Ka, but not with aquaporin 1 (χ 2  = 464, P < 0.001). We conclude that plaque forms preferentially in the basement membranes of ascending thin limbs, fulfilling a critical prediction of the vas washdown theory of plaque pathogenesis. The clinical implication is that treatments such as a low-sodium diet or thiazide diuretics that raise proximal tubule calcium reabsorption may reduce formation of plaque as well as calcium kidney stones.

Objective: To determine if an innovative extracorporeal electrohydraulic shock wave (SW) device (sparker array [SPA]) can effectively fracture artificial stones in vitro and in vivo, and if SPA treatment produces a renal lesion in our pig model of lithotripsy injury. Results of these experiments will be used to help evaluate the suitability of this device as a clinical lithotripter., Materials and Methods: Ultracal-30 artificial stones were placed in a holder at the focus of the SPA and treated with 600 SWs (21.6 kV, 60 shocks/min). Stone fragments were collected, dried, and weighed to determine stone breakage. In vivo stone breakage entailed implanting stones into pigs. These stones were treated with 600 or 1200 SWs and the fragments were collected for analysis. Lesion analysis consisted of treating the left kidney of pigs with 1200 or 2400 SWs and quantitating the hemorrhagic lesion., Results: In vitro, 71% ± 2% of each artificial stone was fractured to <2 mm in size. In vivo stone breakage averaged 63%. Renal injury analysis revealed that only 1 of 7 kidneys showed evidence of hemorrhagic injury in the treated area., Conclusion: The SPA consistently comminuted artificial stones demonstrating its ability to fracture stones like other lithotripters. Also, the SPA caused little to no renal injury at the settings used in this study. These findings suggest further research is warranted to determine the potential of this device as a clinical lithotripter., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Mechanisms of early stone retention in the kidney are under studied and poorly understood. To date attachment via Randall's plaque is the only widely accepted theory in this regard, which is best described in idiopathic calcium oxalate stone formers. Brushite stone formers are known to have distinct papillary morphology relative to calcium oxalate stone formers. As such we sought to determine whether stone attachment mechanisms in such patients may be similarly unique., Materials and Methods: Patients undergoing percutaneous and or ureteroscopic procedures for stone removal consented to endoscopic renal papillary examination and individual stone collection. Each removed stone was processed using micro computerized tomography to assess the 3-dimensional microstructure and the minerals contained, and search for common structural features indicative of novel mechanisms of early growth and attachment to renal tissue., Results: A total of 25 intact brushite stones were removed from 8 patients and analyzed. Video confirmed attachment of 13 of the 25 stones with the remainder believed to have been accidently dislodged during the procedure. Microscopic examination by light and computerized tomography failed to show evidence of Randall's plaque associated with any stone containing brushite. Conversely each brushite stone demonstrated microstructural evidence of having grown attached to a ductal plug formed of apatite., Conclusions: Three-dimensional analysis of small brushite stones suggests overgrowth on ductal apatite plugs as a mechanism of early stone growth and retention. Such findings represent what is to our knowledge the initial supporting evidence for a novel mechanism of stone formation which has previously been hypothesized but never verified., (Copyright © 2018 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

In this paper, an extracorporeal shock wave source composed of small ellipsoidal sparker units is described. The sparker units were arranged in an array designed to produce a coherent shock wave of sufficient strength to fracture kidney stones. The objective of this paper was to measure the acoustical output of this array of 18 individual sparker units and compare this array to commercial lithotripters. Representative waveforms acquired with a fiber-optic probe hydrophone at the geometric focus of the sparker array indicated that the sparker array produces a shock wave (P + ∼40-47 MPa, P - ∼2.5-5.0 MPa) similar to shock waves produced by a Dornier HM-3 or Dornier Compact S. The sparker array's pressure field map also appeared similar to the measurements from a HM-3 and Compact S. Compared to the HM-3, the electrohydraulic technology of the sparker array produced a more consistent SW pulse (shot-to-shot positive pressure value standard deviation of ±4.7 MPa vs ±3.3 MPa).

Purpose: Pretreating a pig kidney with 500 low-energy shock waves (SWs) before delivering a clinical dose of SWs (2000 SWs, 24 kV, 120 SWs/min) has been shown to significantly reduce the size of the hemorrhagic lesion produced in that treated kidney, compared with a protocol without pretreatment. However, since the time available for patient care is limited, we wanted to determine if fewer pretreatment SWs could be used in this protocol. As such, we tested if pretreating with 300 SWs can initiate the same reduction in renal lesion size as has been observed with 500 SWs., Materials and Methods: Fifteen female farm pigs were placed in an unmodified Dornier HM-3 lithotripter, where the left kidney of each animal was targeted for lithotripsy treatment. The kidneys received 300 SWs at 12 kV (120 SWs/min) followed immediately by 2000 SWs at 24 kV (120 SWs/min) focused on the lower pole. These kidneys were compared with kidneys given a clinical dose of SWs with 500 SW pretreatment, and without pretreatment. Renal function was measured both before and after SW exposure, and lesion size analysis was performed to assess the volume of hemorrhagic tissue injury (% functional renal volume, FRV) created by the 300 SW pretreatment regimen., Results: Glomerular filtration rate fell significantly in the 300 SW pretreatment group by 1 hour after lithotripsy treatment. For most animals, low-energy pretreatment with 300 SWs significantly reduced the size of the hemorrhagic injury (to 0.8% ± 0.4%FRV) compared with the injury produced by a typical clinical dose of SWs., Conclusions: The results suggest that 300 pretreatment SWs in a voltage ramping treatment regimen can initiate a protective response in the majority of treated kidneys and significantly reduce tissue injury in our model of lithotripsy injury., Competing Interests: Author Disclosure Statement No competing financial interests exist.

Nephrolithiasis, or stones, is one of the oldest urological diseases, with descriptions and treatment strategies dating back to ancient times. Despite the enormous number of patients affected by stones, a surprising lack of conceptual understanding of many aspects of this disease still exists. This lack of understanding includes mechanisms of stone formation and retention, the clinical relevance of different stone compositions and that of formation patterns and associated pathological features to the overall course of the condition. Fortunately, a number of new tools are available to assist in answering such questions. New renal endoscopes enable kidney visualization in much higher definition than was previously possible, while micro-CT imaging is the optimal technique for assessment of stone microstructure and mineral composition in a nondestructive fashion. Together, these tools have the potential to provide novel insights into the aetiology of stone formation that might unlock new prevention and treatment strategies, and enable more effective management of patients with nephrolithiasis.

The most common presentation of nephrolithiasis is idiopathic calcium stones in patients without systemic disease. Most stones are primarily composed of calcium oxalate and form on a base of interstitial apatite deposits, known as Randall's plaque. By contrast some stones are composed largely of calcium phosphate, as either hydroxyapatite or brushite (calcium monohydrogen phosphate), and are usually accompanied by deposits of calcium phosphate in the Bellini ducts. These deposits result in local tissue damage and might serve as a site of mineral overgrowth. Stone formation is driven by supersaturation of urine with calcium oxalate and brushite. The level of supersaturation is related to fluid intake as well as to the levels of urinary citrate and calcium. Risk of stone formation is increased when urine citrate excretion is <400 mg per day, and treatment with potassium citrate has been used to prevent stones. Urine calcium levels >200 mg per day also increase stone risk and often result in negative calcium balance. Reduced renal calcium reabsorption has a role in idiopathic hypercalciuria. Low sodium diets and thiazide-type diuretics lower urine calcium levels and potentially reduce the risk of stone recurrence and bone disease.

We describe the in vivo use of an optic-chemo microsensor to measure intraluminal papillary duct urine pH in a large mammal. Fiber-optic pH microsensors have a tip diameter of 140-µm that allows insertion into papillary Bellini ducts to measure tubule urine proton concentration. Anesthetized adult pigs underwent percutaneous nephrolithotomy to access the lower pole of the urinary collecting system. A flexible nephroscope was advanced towards an upper pole papilla with the fiber-optic microsensor contained within the working channel. The microsensor was then carefully inserted into Bellini ducts to measure tubule urine pH in real time. We successfully recorded tubule urine pH values in five papillary ducts from three pigs (1 farm pig and 2 metabolic syndrome Ossabaw pigs). Our results demonstrate that optical microsensor technology can be used to measure intraluminal urine pH in real time in a living large mammal. This opens the possibility for application of this optical pH sensing technology in nephrolithiasis.

Background: Kidney stone matrix protein composition is an important yet poorly understood aspect of nephrolithiasis. We hypothesized that this proteome is considerably more complex than previous reports have indicated and that comprehensive proteomic profiling of the kidney stone matrix may demonstrate relevant constitutive differences between stones. We have analyzed the matrices of two unique human calcium oxalate stones (CaOx-Ia and CaOx-Id) using a simple but effective chaotropic reducing solution for extraction/solubilization combined with label-free quantitative mass spectrometry to generate a comprehensive profile of their proteomes, including physicochemical and bioinformatic analysis.`., Results: We identified and quantified 1,059 unique protein database entries in the two human kidney stone samples, revealing a more complex proteome than previously reported. Protein composition reflects a common range of proteins related to immune response, inflammation, injury, and tissue repair, along with a more diverse set of proteins unique to each stone., Conclusion: The use of a simple chaotropic reducing solution and moderate sonication for extraction and solubilization of kidney stone powders combined with label-free quantitative mass spectrometry has yielded the most comprehensive list to date of the proteins that constitute the human kidney stone proteome.

Introduction and Objective: Studies in patients and experimental animals have shown that percutaneous nephrolithotomy (PCNL) can acutely impair glomerular filtration and renal perfusion, but the factors contributing to this decline in renal function are unknown. The present study assessed the contribution of needle puncture of the kidney vs dilation of the needle tract to the acute decline in renal hemodynamic and tubular transport function associated with PCNL surgery., Materials and Methods: Acute experiments were performed in three groups of anesthetized adult farm pigs: sham-percutaneous access (PERC), that is, no surgical procedure (n = 7); a single-needle stick to access the renal collecting system (n = 8); expansion of the single-needle access tract with a 30F NephroMax balloon dilator and insertion of a nephrostomy sheath (n = 10). The glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and renal extraction of para-amino hippurate (EPAH, estimates tubular organic anion transporter [OAT] activity) were assessed before and 1 to 4.5 hours after sham-PERC or PERC surgical procedures., Results: Overall, GFR responses were similar in all three groups. Sham-treated PERC pigs showed no significant change in ERPF over the experimental observation period, whereas a single-needle stick to access the renal collecting system resulted in renal vasoconstriction (∼30% reduction in ERPF, p < 0.05). Dilation of the single-needle access tract to create the nephrostomy did not lead to a further decline in ERPF. PERC surgical procedure-mediated renal vasoconstriction was most evident at the 1-hour posttreatment time point. A reduction in EPAH was only observed in pig kidneys with a nephrostomy., Conclusions: Needle puncture of the kidney for percutaneous access to the renal collecting system is the major driving force for the renal vasoconstriction observed after PCNL surgery, whereas creation of the nephrostomy appears to be largely responsible for decreasing tubular OAT activity.

Background and Purpose: The appearance of the renal papillae in patients with nephrolithiasis can be quite variable and can range from entirely healthy to markedly diseased. The implications of such findings remain unknown. One potential reason is the lack of a standardized system to describe such features. We propose a novel grading scale to describe papillary appearance at the time of renal endoscopy., Methods: Comprehensive endoscopic renal assessment and mapping were performed on more than 300 patients with nephrolithiasis. Recurring abnormal papillary characteristics were identified and quantified based on degree of severity., Results: Four unique papillary features were chosen for inclusion in the PPLA scoring system- ductal Plugging, Pitting, Loss of contour, and Amount of Randall's plaque. Unique scores are calculated for individual papillae based on reference examples., Conclusions: The description and study of renal papillary appearance in stone formers have considerable potential as both a clinical and research tool; however, a standardized grading system is necessary before using it for these purposes.

Introduction: An overlooked finding at the time of renal endoscopy for patients with nephrolithiasis is the appearance of the renal papillae. Recent work has demonstrated that it is possible to distinguish specific stone-forming phenotypes by endoscopic patterns of papillary appearance alone. 1-4 These variable expressions are likely to have clinical significance; yet, the ability to pursue such research efforts remains limited by the lack of a standardized system to describe these findings. Herein, we describe a novel grading system designed to standardize and simplify the description of renal papillary appearance in stone formers at the time of endoscopy. Materials and Methods: Since 1999, 342 patients have been prospectively enrolled and given consent to be part of an NIH funded project studying the pathogenesis of stone formation at a single institution (Methodist Hospital, Indiana University Health). Patients have been treated and studied using both percutaneous and retrograde ureteroscopic approaches. Digital scopes are utilized when feasible along with fluoroscopy to map the affected renal unit(s), and stones are removed and analyzed individually when possible. 5 Results: Four recurring abnormal papillary features were identified based upon the collective knowledge and expertise of the primary research team. Each variable was then quantitated based on the severity in appearance. Three features believed to be associated with papillary injury include ductal plugging, pitting, and loss of papillary contour. Ductal plugging is evident as either suburothelial deposits of yellow mineral or as dilated ducts of Bellini, presumably left behind after a plug has passed. These two subfeatures are considered the same for the purposes of grading. Pitting reflects crater-like erosion of the papillary surface. Loss of contour reflects global depression of the papilla relative to the surrounding tissue. Upon papillary inspection, each papilla receives a numerical grade from 0 to 2 for each of these measured domains. The three scores are then added together to create a sum total score regarding the degree of papillary injury ranging from 0 to 6. The fourth feature, the amount of Randall's plaque, is evident as white deposits along the papillary surface. It is not known to cause papillary injury 6 and, as such, is designated with an alphabetical subscore (a-c) rather than a number. Each papilla then receives a final unique score incorporating both the sum numerical and alphabetical grade. Reference examples are shown in the accompanying video. Conclusions: The creation of a standardized system to describe the papillary appearance in stone formers has considerable clinical and academic utility. On a clinical level, it could be applied as a tool to document intraoperative findings and determine changes in papillary appearance over time in recurrent stone formers. It also has the potential to distinguish high-risk patients with more pressing needs of metabolic evaluations, medical therapy, and surveillance imaging. As a research tool, it would help create a common language to describe papillary appearance and improve collaboration between researchers. It also might allow surgeons to better correlate endoscopic findings to pathological findings and clinical outcomes such as stone analysis, associated metabolic diseases, risk of progressive renal injury, and stone recurrence. No competing financial interests exist. Accompanying manuscript submitted to Journal of Endourology (END-2015-0298; in review). Runtime of video: 5 mins 37 secs., (Copyright 2015, Mary Ann Liebert, Inc.)

Nephrolithiasis is a highly prevalent disorder affecting approximately one in eleven people and is associated with multiple complications including hypertension, cardiovascular disease, and chronic kidney disease. Significant epidemiologic associations with chronic kidney disease and ESRD have been noted and are reviewed herein, but debate persists in the literature as to whether kidney stone formation is a pathogenic process contributing to kidney disease. Corroborating evidence supporting the presence of kidney disease in stone formers includes the variability of renal function by stone type, the positive association of stone size with renal dysfunction, the presence of markers of renal injury in the urine of even asymptomatic stone formers, and direct evidence of renal tissue injury on histopathology. Proposed pathogenic mechanisms include recurrent obstruction and comorbid conditions such as recurrent urinary tract infections and structural abnormalities. Recent work evaluating the renal histopathology of different groups of stone formers adds further granularity, suggesting variability in mechanisms of renal injury by stone type and confirming the pathogenic effects of crystal formation. Genetic abnormalities leading to stone formation including cystinuria and primary hyperoxaluria, among others, contribute to the burden of disease in the stone-forming population.

Purpose: Nephrocalcinosis is commonly present in primary hyperparathyroidism, distal renal tubular acidosis and medullary sponge kidney disease. To our knowledge it has not been studied in patients with calcium phosphate stones who do not have systemic disease., Materials and Methods: We studied patients undergoing percutaneous nephrolithotomy who had calcium phosphate or calcium oxalate stones and did not have hyperparathyroidism, distal renal tubular acidosis or medullary sponge kidney disease. On postoperative day 1 all patients underwent noncontrast computerized tomography. If there were no residual calcifications, the patient was categorized as not having nephrocalcinosis. If there were residual calcifications, the patient underwent secondary percutaneous nephrolithotomy. If the calcifications were found to be stones, the patient was categorized as not having nephrocalcinosis. If the calcifications were not stones, the patient was categorized as having nephrocalcinosis. Patients were grouped based on the type of stones that formed, including hydroxyapatite, brushite and idiopathic calcium oxalate. The extent of nephrocalcinosis was quantified as 0--absent nephrocalcinosis to 3--extensive nephrocalcinosis. Patients with residual calcifications on postoperative day 1 noncontrast computerized tomography who did not undergo secondary percutaneous nephrolithotomy were excluded from analysis. The presence or absence of nephrocalcinosis was correlated with metabolic studies., Results: A total of 67 patients were studied, including 14 with hydroxyapatite, 19 with brushite and 34 with idiopathic calcium oxalate calculi. Nephrocalcinosis was present in 10 of 14 (71.4%), 11 of 19 (57.9%) and 6 of 34 patients (17.6%) in the hydroxyapatite, brushite and idiopathic calcium oxalate groups, respectively (chi-square p = 0.01). The mean extent of nephrocalcinosis per group was 1.98, 1.32 and 0.18 for hydroxyapatite, brushite and idiopathic calcium oxalate, respectively (p ≤0.001). The presence of nephrocalcinosis positively correlated with urine calcium excretion (mean ± SD 287.39 ± 112.49 vs 223.68 ± 100.67 mg per day, p = 0.03)., Conclusions: Patients without systemic disease who form hydroxyapatite and brushite stones commonly have coexistent nephrocalcinosis. Nephrocalcinosis can occur in calcium oxalate stone formers but the quantity and frequency of nephrocalcinosis in this group are dramatically less., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Idiopathic hypercalciuria (IH) is a common familial trait among patients with calcium nephrolithiasis. Previously, we have demonstrated that hypercalciuria is primarily due to reduced renal proximal and distal tubule calcium reabsorption. Here, using measurements of the clearances of sodium, calcium, and endogenous lithium taken from the General Clinical Research Center, we test the hypothesis that patterns of segmental nephron tubule calcium reabsorption differ between the sexes in IH and normal subjects. When the sexes are compared, we reconfirm the reduced proximal and distal calcium reabsorption. In IH women, distal nephron calcium reabsorption is decreased compared to normal women. In IH men, proximal tubule calcium reabsorption falls significantly, with a more modest reduction in distal calcium reabsorption compared to normal men. Additionally, we demonstrate that male IH patients have lower systolic blood pressures than normal males. We conclude that women and men differ in the way they produce the hypercalciuria of IH, with females reducing distal reabsorption and males primarily reducing proximal tubule function., (Copyright © 2015 the American Physiological Society.)

Medullary sponge kidney (MSK) is associated with recurrent stone formation, but the clinical phenotype is unclear because patients with other disorders may be incorrectly labeled MSK. We studied 12 patients with histologic findings pathognomonic of MSK. All patients had an endoscopically recognizable pattern of papillary malformation, which may be segmental or diffuse. Affected papillae are enlarged and billowy, due to markedly enlarged inner medullary collecting ducts (IMCD), which contain small, mobile ductal stones. Patients had frequent dilation of Bellini ducts, with occasional mineral plugs. Stones may form over white (Randall's) plaque, but most renal pelvic stones are not attached, and have a similar morphology as ductal stones, which are a mixture of calcium oxalate and apatite. Patients had no abnormalities of urinary acidification or acid excretion; the most frequent metabolic abnormality was idiopathic hypercalciuria. Although both Runx2 and Osterix are expressed in papillae of MSK patients, no mineral deposition was seen at the sites of gene expression, arguing against a role of these genes in this process. Similar studies in idiopathic calcium stone formers showed no expression of these genes at sites of Randall's plaque. The most likely mechanism for stone formation in MSK appears to be crystallization due to urinary stasis in dilated IMCD with subsequent passage of ductal stones into the renal pelvis where they may serve as nuclei for stone formation., (© 2015 Wiley Periodicals, Inc.)

Human stone calcium phosphate (CaP) content correlates with higher urine CaP supersaturation (SS) and urine pH as well as with the number of shock wave lithotripsy (SWL) treatments. SWL does damage medullary collecting ducts and vasa recta, sites for urine pH regulation. We tested the hypothesis that SWL raises urine pH and therefore Cap SS, resulting in CaP nucleation and tubular plugging. The left kidney (T) of nine farm pigs was treated with SWL, and metabolic studies were performed using bilateral ureteral catheters for up to 70 days post-SWL. Some animals were given an NH4Cl load to sort out effects on urine pH of CD injury vs. increased HCO3 (-) delivery. Histopathological studies were performed at the end of the functional studies. The mean pH of the T kidneys exceeded that of the control (C) kidneys by 0.18 units in 14 experiments on 9 pigs. Increased HCO3 (-) delivery to CD is at least partly responsible for the pH difference because NH4Cl acidosis abolished it. The T kidneys excreted more Na, K, HCO3 (-), water, Ca, Mg, and Cl than C kidneys. A single nephron site that could produce losses of all of these is the thick ascending limb. Extensive injury was noted in medullary thick ascending limbs and collecting ducts. Linear bands showing nephron loss and fibrosis were found in the cortex and extended into the medulla. Thus SWL produces tubule cell injury easily observed histopathologically that leads to functional disturbances across a wide range of electrolyte metabolism including higher than control urine pH., (Copyright © 2015 the American Physiological Society.)

Purpose: To determine whether shock wave lithotripsy (SWL) may be a risk factor for renal functional impairment in a swine model of metabolic syndrome (MetS)., Materials and Methods: Nine-month-old female Ossabaw pigs were fed an excess calorie atherogenic diet to induce MetS. At 15 months of age, the MetS pigs were treated with 2000 SWs or an overtreatment dose of 4000 SWs targeted at the upper pole calyx of the left kidney (24 kV at 120 SWs/min using the unmodified Dornier HM3 lithotripter; n=5-6 per treatment group). Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured in conscious pigs before and ∼60 days after SWL to provide a qualitative assessment of how well both kidneys were filtering (glomerular filtration rate [GFR]). Bilateral renal function was assessed at ∼65 days post-SWL in anesthetized pigs with GFR and effective renal plasma flow (ERPF) quantified by the renal clearance of inulin and para-amino hippurate, respectively., Results: Cr and BUN values were within normal limits before SWL and remained unchanged after lithotripsy in both the 2000 SW- and 4000 SW-treated pigs. GFR and ERPF of kidneys treated with SWL at either SW dose were similar to the contralateral nontreated kidney. Chronic histological changes in the SW-treated pole of the kidney included interstitial fibrosis, sclerotic glomeruli, and dilated and atrophic tubules., Conclusions: Our results are consistent with the view that a single SWL session does not result in renal impairment, even in the presence of MetS.

Purpose: We performed a pilot study to assess whether renal shock wave lithotripsy influences metabolic syndrome onset and severity., Materials and Methods: Three-month-old juvenile female Ossabaw miniature pigs were treated with shock wave lithotripsy (2,000 shock waves at 24 kV with 120 shock waves per minute in 2) or sham shock wave lithotripsy (no shock waves in 2). Shock waves were targeted to the upper pole of the left kidney to model treatment that would also expose the pancreatic tail to shock waves. Pigs were then instrumented to directly measure arterial blood pressure via an implanted radiotelemetry device. They later received a hypercaloric atherogenic diet for about 7 months. Metabolic syndrome development was assessed by the intravenous glucose tolerance test., Results: Metabolic syndrome progression and severity were similar in the sham treated and lithotripsy groups. The only exception arterial blood pressure, which remained relatively constant in sham treated pigs but began to increase at about 2 months towards hypertensive levels in lithotripsy treated pigs. Metabolic data on the 2 groups were pooled to provide a more complete assessment of metabolic syndrome development and progression in this juvenile pig model. The intravenous glucose tolerance test revealed substantial insulin resistance with impaired glucose tolerance within 2 months on the hypercaloric atherogenic diet with signs of further metabolic impairment at 7 months., Conclusions: These preliminary results suggest that renal shock wave lithotripsy is not a risk factor for worsening glucose tolerance or diabetes mellitus onset. However, it appears to be a risk factor for early onset hypertension in metabolic syndrome., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Micro-computed tomographic imaging (micro-CT) provides unprecedented information on stone structure and mineral composition. High-resolution micro-CT even allows visualization of the lumens of tubule and/or vessels within Randall's plaque, on stones or in papillary biopsies, thus giving a non-destructive way to study these sites of stone adhesion. This paper also shows an example of a stone growing on a different anchoring mechanism: a mineral plug within the lumen of a Bellini duct (BD plug). Micro-CT shows striking structural differences between stones that have grown on Randall's plaque and those that have grown on BD plugs. Thus, Randall's plaque can be distinguished by micro-CT, and this non-destructive method shows great promise in helping to elucidate the different mechanisms by which small stones are retained in the kidney during the development of nephrolithiasis.

The precise mechanisms of kidney stone formation and growth are not completely known, even though human stone disease appears to be one of the oldest diseases known to medicine. With the advent of the new digital endoscope and detailed renal physiological studies performed on well phenotyped stone formers, substantial advances have been made in our knowledge of the pathogenesis of the most common type of stone former, the idiopathic calcium oxalate stone former as well as nine other stone forming groups. The observations from our group on human stone formers and those of others on model systems have suggested four entirely different pathways for kidney stone formation. Calcium oxalate stone growth over sites of Randall's plaque appear to be the primary mode of stone formation for those patients with hypercalciuria. Overgrowths off the ends of Bellini duct plugs have been noted in most stone phenotypes, do they result in a clinical stone? Micro-lith formation does occur within the lumens of dilated inner medullary collecting ducts of cystinuric stone formers and appear to be confined to this space. Lastly, cystinuric stone formers also have numerous small, oval, smooth yellow appearing calyceal stones suggestive of formation in free solution. The scientific basis for each of these four modes of stone formation are reviewed and used to explore novel research opportunities.

Purpose: We determined whether shock wave lithotripsy of the kidney of pigs with metabolic syndrome would worsen glucose tolerance or increase the risk of diabetes mellitus., Materials and Methods: Nine-month-old female Ossabaw miniature pigs were fed a hypercaloric atherogenic diet to induce metabolic syndrome. At age 15 months the pigs were treated with 2,000 or 4,000 shock waves (24 kV at 120 shock waves per minute) using an unmodified HM3 lithotripter (Dornier MedTech, Kennesaw, Georgia). Shock waves were targeted to the left kidney upper pole calyx to model treatment that would also expose the pancreatic tail to shock waves. The intravenous glucose tolerance test was done in conscious fasting pigs before lithotripsy, and 1 and 2 months after lithotripsy with blood samples taken for glucose and insulin measurement., Results: Pigs fed the hypercaloric atherogenic diet were obese, dyslipidemic, insulin resistant and glucose intolerant, consistent with metabolic syndrome. Assessments of insulin resistance, glucose tolerance and pancreatic β cell function from fasting plasma glucose and insulin levels, and the glucose and insulin response profile to the intravenous glucose tolerance test were similar before and after lithotripsy., Conclusions: The metabolic syndrome status of pigs treated with shock wave lithotripsy was unchanged 2 months after kidney treatment with 2,000 high amplitude shock waves or overtreatment with 4,000 high amplitude shock waves. These findings do not support a single shock wave lithotripsy treatment of the kidney as a risk factor for the onset of diabetes mellitus., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Our previous work has shown that stone formers who form calcium phosphate (CaP) stones that contain any brushite (BRSF) have a distinctive renal histopathology and surgical anatomy when compared with idiopathic calcium oxalate stone formers (ICSF). Here we report on another group of idiopathic CaP stone formers, those forming stone containing primarily hydroxyapatite, in order to clarify in what ways their pathology differs from BRSF and ICSF. Eleven hydroxyapatite stone formers (HASF) (2 males, 9 females) were studied using intra-operative digital photography and biopsy of papillary and cortical regions to measure tissue changes associated with stone formation. Our main finding is that HASF and BRSF differ significantly from each other and that both differ greatly from ICSF. Both BRSF and ICSF patients have significant levels of Randall's plaque compared with HASF. Intra-tubular deposit number is greater in HASF than BRSF and nonexistent in ICSF while deposit size is smaller in HASF than BRSF. Cortical pathology is distinctly greater in BRSF than HASF. Four attached stones were observed in HASF, three in 25 BRSF and 5-10 per ICSF patient. HASF and BRSF differ clinically in that both have higher average urine pH, supersaturation of CaP, and calcium excretion than ICSF. Our work suggests that HASF and BRSF are two distinct and separate diseases and both differ greatly from ICSF., (Copyright © 2014 Wiley Periodicals, Inc.)

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/cm(2) 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/cm(2) was needed before a statistically significant portion of the samples showed injury. This is nearly seven times the 2,400-W/cm(2) 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.

Purpose: Focused ultrasonic propulsion is a new noninvasive technique designed to move kidney stones and stone fragments out of the urinary collecting system. However, to our knowledge the extent of tissue injury associated with this technique is not known. We quantitated the amount of tissue injury produced by focused ultrasonic propulsion under simulated clinical treatment conditions and under conditions of higher power or continuous duty cycles. We compared those results to extracorporeal shock wave lithotripsy injury., Materials and Methods: A human calcium oxalate monohydrate stone and/or nickel beads were implanted by ureteroscopy in 3 kidneys of live pigs weighing 45 to 55 kg and repositioned using focused ultrasonic propulsion. Additional pig kidneys were exposed to extracorporeal shock wave lithotripsy level pulse intensity or continuous ultrasound exposure 10 minutes in duration using an ultrasound probe transcutaneously or on the kidney. These kidneys were compared to 6 treated with an unmodified Dornier HM3 lithotripter (Dornier Medical Systems, Kennesaw, Georgia) using 2,400 shocks at 120 shock waves per minute and 24 kV. Histological analysis was performed to assess the volume of hemorrhagic tissue injury created by each technique according to the percent of functional renal volume., Results: Extracorporeal shock wave lithotripsy produced a mean ± SEM lesion of 1.56% ± 0.45% of functional renal volume. Ultrasonic propulsion produced no detectable lesion with simulated clinical treatment. A lesion of 0.46% ± 0.37% or 1.15% ± 0.49% of functional renal volume was produced when excessive treatment parameters were used with the ultrasound probe placed on the kidney., Conclusions: Focused ultrasonic propulsion produced no detectable morphological injury to the renal parenchyma when using clinical treatment parameters but produced injury comparable in size to that of extracorporeal shock wave lithotripsy when using excessive treatment parameters., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Introduction: Our research group is studying a noninvasive transcutaneous ultrasound device to expel small kidney stones or residual post-treatment stone fragments from the kidney. 1-3 The purpose of this study was to evaluate the efficacy and safety of ultrasonic propulsion in a live porcine model. Materials and Methods: In domestic female swine (50-60 kg), human stones (calcium oxalate monohydrate) and metalized glass beads (2-8 mm) were ureteroscopically implanted. 4 Target stones and beads were placed in the lower half of the kidney and a reference bead was placed in the upper pole. Ultrasonic propulsion was achieved through a single ultrasound system that allowed targeting, stone propulsion, and ultrasound imaging using a Philips HDI C5-2 commercial imaging transducer and a Verasonics diagnostic ultrasound platform. Stone propulsion was achieved through the delivery of 1-second bursts of focused, ultrasound pulses, which consist of 250 finely focused pulses 0.1 milliseconds in duration. Stone propulsion was then observed using fluoroscopy, ultrasound, and visually with the ureteroscope. The kidneys were then perfusion-fixed with glutaraldehyde, embedded in paraffin, sectioned, and stained. Samples were histologically scored for injury by a blinded independent expert. Using the same pulsing scheme, while varying acoustic intensities, an injury threshold and patterns of injury were determined in additional pigs. 5,6 Results: Stones were successfully implanted in 14 kidneys. Overall, 17 of 26 (65)% stones/beads were moved the entire distance to the renal pelvis, ureteropelvic junction (UPJ), or proximal ureter. The average procedure time for successfully repositioned stones was 14.2±7.9 minutes with 23±16 push bursts. No gross or histologic damage was identified from the ultrasound propulsion procedure. Under this pulsing scheme, a maximum exposure of 2400 W/cm 2 was delivered during each treatment. An intensity threshold of 16,620 W/cm 2 was determined at which, above this level, tissue injury consistent with emulsification, necrosis, and hemorrhage appeared to be dose dependent. Conclusions: Ultrasonic propulsion is effective with most stones being relocated to the renal pelvis, UPJ, or proximal ureter in a timely fashion. The procedure appears safe with no evidence of injury. The acoustic intensities delivered at maximum treatment settings are well below the threshold at which injury is observed. The angle and alignment of directional force are the most critical factors determining the efficacy of stone propulsion. We are now pursuing FDA approval for a human feasibility study. No competing financial interests exist. Runtime of video: 5 mins 44 secs Acknowledgments: This work was supported by NIH DK43881, DK092197, NSBRI through NASA NCC 9-58, the Coulter Foundation, and the University of Washington. This material is the result of work supported by resources from the VA Puget Sound Health Care System, Seattle, Washington. We are very grateful for the help of a large team at the University of Washington and the Consortium for Shock Waves in Medicine, which we cannot list in detail., (Copyright 2013, Mary Ann Liebert, Inc.)

The sequence of events by which primary hyperoxaluria type 1 (PH1) causes renal failure is unclear. We hypothesize that proximal tubule (PT) is vulnerable because oxalate secretion raises calcium oxalate (CaOx) supersaturation (SS) there, leading to crystal formation and cellular injury. We studied cortical and papillary biopsies from two PH1 patients with preserved renal function, and seven native kidneys removed from four patients at the time of transplant, after short-term (2) or longer term (2) dialysis. In these patients, and another five PH1 patients without renal failure, we calculated oxalate secretion, and estimated PT CaOx SS. Plasma oxalate was elevated in all PH1 patients and inverse to creatinine clearance. Renal secretion of oxalate was present in all PH1 but rare in controls. PT CaOx SS was >1 in all nonpyridoxine-responsive PH1 before transplant and most marked in patients who developed end stage renal disease (ESRD). PT from PH1 with preserved renal function had birefringent crystals, confirming the presence of CaOx SS, but had no evidence of cortical inflammation or scarring by histopathology or hyaluronan staining. PH1 with short ESRD showed CaOx deposition and hyaluronan staining particularly at the corticomedullary junction in distal PT while cortical collecting ducts were spared. Longer ESRD showed widespread cortical CaOx, and in both groups papillary tissue had marked intratubular CaOx deposits and fibrosis. CaOx SS in PT causes CaOx crystal formation, and CaOx deposition in distal PT appears to be associated with ESRD. Minimizing PT CaOx SS may be important for preserving renal function in PH1.

Purpose: Focused ultrasound has the potential to expel small stones or residual stone fragments from the kidney, or move obstructing stones to a nonobstructing location. We evaluated the efficacy and safety of ultrasonic propulsion in a live porcine model., Materials and Methods: Calcium oxalate monohydrate kidney stones and laboratory model stones (2 to 8 mm) were ureteroscopically implanted in the renal pelvicalyceal system of 12 kidneys in a total of 8 domestic swine. Transcutaneous ultrasonic propulsion was performed using an HDI C5-2 imaging transducer (ATL/Philips, Bothell, Washington) and the Verasonics® diagnostic ultrasound platform. Successful stone relocation was defined as stone movement from the calyx to the renal pelvis, ureteropelvic junction or proximal ureter. Efficacy and procedure time was determined. Three blinded experts evaluated histological injury to the kidney in the control, sham treatment and treatment arms., Results: All 26 stones were observed to move during treatment and 17 (65%) were relocated successfully to the renal pelvis (3), ureteropelvic junction (2) or ureter (12). Average ± SD successful procedure time was 14 ± 8 minutes and a mean of 23 ± 16 ultrasound bursts, each about 1 second in duration, were required. There was no evidence of gross or histological injury to the renal parenchyma in kidneys exposed to 20 bursts (1 second in duration at 33-second intervals) at the same output (2,400 W/cm(2)) used to push stones., Conclusions: Noninvasive transcutaneous ultrasonic propulsion is a safe, effective and time efficient means to relocate calyceal stones to the renal pelvis, ureteropelvic junction or ureter. This technology holds promise as a useful adjunct to surgical management for renal calculi., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Conduct a laboratory evaluation of a novel low-pressure, broad focal zone electrohydraulic lithotripter (TRT LG-380)., Methods: Mapping of the acoustic field of the LG-380, along with a Dornier HM3, a Storz Modulith SLX, and a XiXin CS2012 (XX-ES) lithotripter was performed using a fiberoptic hydrophone. A pig model was used to assess renal response to 3000 shockwaves (SW) administered by a multistep power ramping protocol at 60 SW/min, and when animals were treated at the maximum power setting at 120 SW/min. Injury to the kidney was assessed by quantitation of lesion size and routine measures of renal function., Results: SW amplitudes for the LG-380 ranged from (P(+)/P(-)) 7/-1.8 MPa at PL-1 to 21/-4 MPa at PL-11 while focal width measured ~20 mm, wider than the HM3 (8 mm), SLX (2.6 mm), or XX-ES (18 mm). For the LG-380, there was gradual narrowing of the focal width to ~10 mm after 5000 SWs, but this had negligible effect on breakage of model stones, because stones positioned at the periphery of the focal volume (10 mm off-axis) broke nearly as well as stones at the target point. Kidney injury measured less than 0.1% FRV (functional renal volume) for pigs treated using a gradual power ramping protocol at 60 SW/min and when SWs were delivered at maximum power at 120 SW/min., Conclusions: The LG-380 exhibits the acoustic characteristics of a low-pressure, wide focal zone lithotripter and has the broadest focal width of any lithotripter yet reported. Although there was a gradual narrowing of focal width as the electrode aged, the efficiency of stone breakage was not affected. Because injury to the kidney was minimal when treatment followed either the recommended slow SW-rate multistep ramping protocol or when all SWs were delivered at fast SW-rate using maximum power, this appears to be a relatively safe lithotripter.

The objective of this preliminary study was to examine the spatial correlation between microbubble (MB)-induced vessel wall displacements and resultant microvascular bioeffects. MBs were injected into venules in ex vivo rat mesenteries and insonated by a single short ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 1.5-5.6 MPa. MB and vessel dynamics were observed under ultra-high speed photomicrography. The tissue was examined by histology or transmission electron microscopy for vascular bioeffects. Image registration allowed for spatial correlation of MB-induced vessel wall motion to corresponding vascular bioeffects, if any. In cases in which damage was observed, the vessel wall had been pulled inward by more than 50% of the its initial radius. The observed damage was characterized by the separation of the endothelium from the vessel wall. Although the study is limited to a small number of observations, analytic statistical results suggest that vessel invagination comprises a principal mechanism for bioeffects in venules by microbubbles., (Published by Elsevier Inc.)

Unlabelled: Study Type--Therapy (case series) Level of Evidence 4. What's known on the subject? and What does the study add? Animal studies have shown that one approach to reduce SWL-induced renal injury is to pause treatment for 3-4 min early in the SWL-treatment protocol. However, there is typically no pause in treatment during clinical lithotripsy. We show in a porcine model that a pause in SWL treatment is unnecessary to achieve a reduction in renal injury if treatment is begun at a low power setting that generates low-amplitude SWs, and given continuously for ≈ 4 min before applying higher-amplitude SWs., Objective: • To test the idea that a pause (≈ 3 min) in the delivery of shockwaves (SWs) soon after the initiation of SW lithotripsy (SWL) is unnecessary for achieving a reduction in renal injury, if treatment is begun at a low power setting that generates low-amplitude SWs., Materials and Methods: • Anaesthetised female pigs were assigned to one of three SWL treatment protocols that did not involve a pause in SW delivery of >10 s (2000 SWs at 24 kV; 100 SWs at 12 kV + ≈ 10-s pause + 2000 SWs at 24 kV; 500 SWs at 12 kV + ≈ 10-s pause + 2000 SWs at 24 kV). • All SWs were delivered at 120 SWs/min using an unmodified Dornier HM3 lithotripter. • Renal function was measured before and after SWL. • The kidneys were then processed for quantification of the SWL-induced haemorrhagic lesion. Values for lesion size were compared to previous data collected from pigs in which treatment included a 3-min pause in SW delivery., Results: • All SWL treatment protocols produced a similar degree of vasoconstriction (23-41% reduction in glomerular filtration rate and effective renal plasma flow) in the SW-treated kidney. • The mean renal lesion in pigs treated with 100 low-amplitude SWs delivered before the main dose of 2000 high-amplitude SWs (2.27% functional renal volume [FRV]) was statistically similar to that measured for pigs treated with 2000 SWs all at high-amplitude (3.29% FRV). • However, pigs treated with 500 low-amplitude SWs before the main SW dose had a significantly smaller lesion (0.44% FRV) that was comparable with the lesion in pigs from a previous study in which there was a 3-min pause in treatment separating a smaller initial dose of 100 low-amplitude SWs from the main dose of 2000 high-amplitude SWs (0.46% FRV). The time between the initiation of the low - and high-amplitude SWs was ≈ 4 min for these latter two groups compared with ≈ 1 min when there was negligible pause after the initial 100 low-amplitude SWs in the protocol., Conclusions: • Pig kidneys treated by SWL using a two-step low-to-high power ramping protocol were protected from injury with negligible pause between steps, provided the time between the initiation of low-amplitude SWs and switching to high-amplitude SWs was ≈ 4 min. • Comparison with results from previous studies shows that protection can be achieved using various step-wise treatment scenarios in which either the initial dose of SWs is delivered at low-amplitude for ≈ 4 min, or there is a definitive pause before resuming SW treatment at higher amplitude. • Thus, we conclude that renal protection can be achieved without instituting a pause in SWL treatment. It remains prudent to consider that renal protection depends on the acoustic and temporal properties of SWs administered at the beginning stages of a SWL ramping protocol, and that this may differ according to the lithotripter being used., (© 2012 BJU INTERNATIONAL.)

Unlabelled: What's known on the subject? and What does the study add? Of all the SW lithotriptors manufactured to date, more research studies have been conducted on and more is known about the injury (both description of injury and how to manipulate injury size) produced by the Dornier HM-3 than any other machine. From this information have come suggestions for treatment protocols to reduce shock wave (SW)-induced injury for use in stone clinics. By contrast, much less is known about the injury produced by narrow-focus and high-pressure lithotriptors like the Storz Modulith SLX. In fact, a careful study looking at the morphology of the injury produced by the SLX itself is lacking, as is any study exploring ways to reduce renal injury by manipulating SW delivery variables of this lithotriptor. The present study quantitates the lesion size and describes the morphology of the injury produced by the SLX. In addition, we report that reducing the SW delivery rate, a manoeuvre known to lower injury in the HM-3, does not reduce lesion size in the SLX., Objective: • To assess renal injury in a pig model after treatment with a clinical dose of shock waves using a narrow focal zone (≈3 mm) lithotriptor (Modulith SLX, Karl Storz Lithotripsy)., Materials and Methods: • The left kidney of anaesthetized female pigs were treated with 2000 or 4000 shock waves (SWs) at 120 SWs/min, or 2000 SWs at 60 SWs/min using the Storz SLX. • Measures of renal function (glomerular filtration rate and renal plasma flow) were collected before and 1 h after shock wave lithotripsy (SWL) and the kidneys were harvested for histological analysis and morphometric quantitation of haemorrhage in the renal parenchyma with lesion size expressed as a percentage of functional renal volume (FRV). • A fibre-optic probe hydrophone was used to determine acoustic output and map the focal width of the lithotriptor. • Data for the SLX were compared with data from a previously published study in which pigs of the same age (7-8 weeks) were treated (2000 SWs at 120 or 60 SWs/min) using an unmodified Dornier HM3 lithotriptor., Results: • Treatment with the SLX produced a highly focused lesion running from cortex to medulla and often spanning the full thickness of the kidney. Unlike the diffuse interstitial haemorrhage observed with the HM3, the SLX lesion bore a blood-filled core of near-complete tissue disruption devoid of histologically recognizable kidney structure. • Despite the intensity of tissue destruction at the core of the lesion, measures of lesion size based on macroscopic determination of haemorrhage in the parenchyma were not significantly different from kidneys treated using the HM3 (2000 SWs, 120 SWs/min: SLX, 1.86 ± 0.52% FRV; HM3, 3.93 ± 1.29% FRV). • Doubling the SW dose of the SLX from 2000 to 4000 SWs did not significantly increase lesion size. In addition, slowing the firing rate of the SLX to 60 SWs/min did not reduce the size of the lesion (2.16 ± 0.96% FRV) compared with treatment at 120 SWs/min, as was the case with the HM3 (0.42 ± 0.23% FRV vs 3.93 ± 1.29% FRV). • Renal function fell significantly below baseline in all treated groups but was similar for both lithotriptors. • Focal width of the SLX (≈2.6 mm) was about one-third that of the HM3 (≈8 mm) while peak pressures were higher (SLX at power level 9: P+≈90 MPa, P-≈-12 MPa; HM3 at 24 kV: P+≈46 MPa, P-≈-8 MPa)., Conclusions: • The lesion produced by the SLX (narrow focal width, high acoustic pressure) was a more focused, more intense form of tissue damage than occurs with the HM3. • Slowing the SW rate to 60 SWs/min, a strategy shown to be effective in reducing injury with the HM3, was not protective with the SLX. • These findings suggest that the focal width and acoustic output of a lithotriptor affect the renal response to SWL., (© 2012 BJU INTERNATIONAL.)

The purpose of this study was to determine if pretreatment of porcine kidneys with low-energy shock waves (SWs) prior to delivery of a clinical dose of 2,000 SWs reduces or prevents shock wave lithotripsy (SWL)-induced acute oxidative stress and inflammation in the treated kidney. Pigs (7-8 weeks old) received 2,000 SWs at 24 kV (120 SW/min) with or without pretreatment with 100 SWs at 12 kV/2 Hz to the lower pole calyx of one kidney using the HM3. Four hours post-treatment, selected samples of renal tissue were frozen for analysis of cytokine, interleukin-6 (IL-6), and stress response protein, heme oxygenase-1 (HO-1). Urine samples were taken before and after treatment for analysis of tumor necrosis factor-α (TNF-α). Treatment with 2,000 SWs with or without pretreatment caused a statistically significant elevation of HO-1 and IL-6 in the renal medulla localized to the focal zone of the lithotripter. However, the increase in HO-1 and IL-6 was significantly reduced using the pretreatment protocol compared to no pretreatment. Urinary excretion of TNF-α increased significantly (p < 0.05) from baseline for pigs receiving 2,000 SWs alone; however, this effect was completely abolished with the pretreatment protocol. We conclude that pretreatment of the kidney with a low dose of low-energy SWs prior to delivery of a clinical dose of SWs reduces, but does not completely prevent, SWL-induced acute renal oxidative stress and inflammation.

OBJECTIVE To determine if the magnitude of the acute injury response to shock-wave lithotripsy (SWL) depends on the number of SWs delivered to the kidney, as SWL causes acute renal oxidative stress and inflammation which are most severe in the portion of the kidney within the focal zone of the lithotripter. MATERIALS AND METHODS Pigs (7-8 weeks old) received 500, 1000 or 2000 SWs at 24 kV from a lithotripter to the lower pole calyx of one kidney. At 4 h after treatment the kidneys were removed, and samples of cortex and medulla were frozen for analysis of the cytokine, interleukin-6, and for the stress response protein, heme oxygenase-1 (HO-1). Urine samples taken before and after treatment were analysed for the inflammatory cytokine, tumour necrosis factor-α. For comparison, we included previously published cytokine data from pigs exposed to sham treatment. RESULTS Treatment with either 1000 or 2000 SWs caused a significant induction of HO-1 in the renal medulla within the focal zone of the lithotripter (F2, 1000 SWs, P < 0.05; 2000 SWs, P < 0.001). Interleukin-6 was also significantly elevated in the renal medulla of the pigs that received either 1000 or 2000 SWs (P < 0.05 and <0.001, respectively). Linear dose-response modelling showed a significant correlation between the HO-1 and interleukin-6 responses with SW dose (P < 0.001). Urinary excretion of tumour necrosis factor-α from the lithotripsy-treated kidney increased only for pigs that received 2000 SWs (P < 0.05). CONCLUSION The magnitude of renal oxidative stress and inflammatory response in the medulla increased with the number of SWs. However, it is not known if the HO-1 response is beneficial or deleterious; determining that will inform us whether SWL-induced renal injury can be assessed by quantifying markers of oxidative stress and inflammation., (© 2010 THE AUTHORS. JOURNAL COMPILATION © 2010 BJU INTERNATIONAL.)

Micro-computed tomographic (micro CT) imaging has become an important tool for the study of urinary stones. The method involves the collection of a series of X-ray pictures of the stone as it is rotated, and the internal structure of the stone is computationally reconstructed from these pictures. The entire process takes from 30 min to an hour with present technology. Resulting images of the stone provide unprecedented detail of the mineral composition and its morphological arrangement within the stone. For smaller stones, reconstructions can easily have voxel sizes of <5 μm, making this a truly microscopic view of the stone. The micro CT reconstructions can be viewed with any of a number of existing methods for visualizing the structure of both the surface and internal features of the stone. Because the entire process is non-destructive, traditional analysis methods--such as dissection and spectroscopic examination of portions of the stones--can also be performed. Micro CT adds value to traditional methods by identifying regions of the stone to be analyzed, and also with its ability to scan a cluster of stones or stone fragments at once. Finally, micro CT has become a powerful tool to help investigate events in stone formation that distinguish different kinds of stone disease.

Nephrocalcinosis generally refers to the presence of calcium salts within renal tissue, but this term is also used radiologically in diagnostic imaging in disease states that also produce renal stones, so that it is not always clear whether it is tissue calcifications or urinary calculi that give rise to the characteristic appearance of the kidney on x-ray or computed tomography (CT). Recent advances in endoscopic imaging now allow the visual distinction between stones and papillary nephrocalcinosis, and intrarenal endoscopy can also verify the complete removal of urinary stones, so that subsequent radiographic appearance can be confidently attributed to nephrocalcinosis. This report shows exemplary cases of primary hyperparathyroidism, type I distal renal tubular acidosis, medullary sponge kidney, and common calcium oxalate stone formation. In the first three cases--all being conditions commonly associated with nephrocalcinosis--it is shown that the majority of calcifications seen by radiograph may actually be stones. In common calcium oxalate stones formers, it is shown that Randall's plaque can appear as a small calculus on CT scan, even when calyces are known to be completely clear of stones. In the current era with the use of non-contrast CT for the diagnosis of nephrolithiasis, the finding of calcifications in close association with the renal papillae is common. Distinguishing nephrolithiasis from nephrocalcinosis requires direct visual inspection of the papillae and so the diagnosis of nephrocalcinosis is essentially an endoscopic, not radiologic, diagnosis.