Epoxyeicosatrienoic acid administration or soluble epoxide hydrolase inhibition attenuates renal fibrogenesis in obstructive nephropathy.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites with biological effects, including antiapoptotic, anti-inflammatory, and antifibrotic functions. Soluble epoxide hydrolase (sEH)-mediated hydrolysis of EETs to dihydroxyeicosatrienoic acids (DHETs) attenuates these effects. Recent studies have demonstrated that inhibition of sEH prevents renal tubulointerstitial fibrosis and inflammation in the chronic kidney disease model. Given the pathophysiological role of the EET pathway in chronic kidney disease, we investigated if administration of EET regioisomers and/or sEH inhibition will promote antifibrotic and renoprotective effects in renal fibrosis following unilateral ureteral obstruction (UUO). EETs administration abolished tubulointerstitial fibrogenesis, as demonstrated by reduced fibroblast activation and collagen deposition after UUO. The inflammatory response was prevented as demonstrated by decreased neutrophil and macrophage infiltration and expression of cytokines in EET-administered UUO kidneys. EET administration and/or sEH inhibition significantly reduced M1 macrophage markers, whereas M2 macrophage markers were highly upregulated. Furthermore, UUO-induced oxidative stress, tubular injury, and apoptosis were all downregulated following EET administration. Combined EET administration and sEH inhibition, however, had no additive effect in attenuating inflammation and renal interstitial fibrogenesis after UUO. Taken together, our findings provide a mechanistic understanding of how EETs prevent kidney fibrogenesis during obstructive nephropathy and suggest EET treatment as a potential therapeutic strategy to treat fibrotic diseases.NEW & NOTEWORTHY Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent antihypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered renoprotective. We found that EET administration and/or soluble epoxide hydrolase inhibition significantly attenuates oxidative stress, renal cell death, inflammation, macrophage differentiation, and fibrogenesis following unilateral ureteral obstruction. Our findings provide a mechanistic understanding of how EETs prevent kidney fibrogenesis during obstructive nephropathy and suggest that EET treatment may be a potential therapeutic strategy to treat fibrotic diseases.

Hepatic and proximal tubule angiotensinogen play distinct roles in kidney dysfunction, glomerular and tubular injury, and fibrosis progression.

Components of the renin-angiotensin system, including angiotensinogen (AGT), are critical contributors to chronic kidney disease (CKD) development and progression. However, the specific role of tissue-derived AGTs in CKD has not been fully understood. To define the contribution of liver versus kidney AGT in the CKD development, we performed 5/6 nephrectomy (Nx), an established CKD model, in wild-type (WT), proximal tubule (PT)- or liver-specific AGT knockout (KO) mice. Nx significantly elevated intrarenal AGT expression and elevated blood pressure (BP) in WT mice. The increase of intrarenal AGT protein was completely blocked in liver-specific AGT KO mice with BP reduction, suggesting a crucial role for liver AGT in BP regulation during CKD. Nx-induced glomerular and kidney injury and dysfunction, as well as fibrosis, were all attenuated to a greater extent in liver-specific AGT KO mice compared with PT-specific AGT KO and WT mice. However, the suppression of interstitial fibrosis in PT- and liver-specific AGT KO mouse kidneys was comparable. Our findings demonstrate that liver AGT acts as a critical contributor in driving glomerular and tubular injury, renal dysfunction, and fibrosis progression, whereas the role of PT AGT was limited to interstitial fibrosis progression in chronic renal insufficiency. Our results provide new insights for the development of tissue-targeted renin-angiotensin system intervention in the treatment of CKD.NEW & NOTEWORTHY Chronic kidney disease (CKD) is a major unmet medical need with no effective treatment. Current findings demonstrate that hepatic and proximal tubule angiotensinogen have distinct roles in tubular and glomerular injury, fibrogenesis, and renal dysfunction during CKD development. As renin-angiotensin system components, including angiotensinogen, are important targets for treating CKD in the clinic, the results from our study may be applied to developing better tissue-targeted treatment strategies for CKD and other fibroproliferative diseases.

Directed co-evolution of interacting protein-peptide pairs by compartmentalized two-hybrid replication (C2HR).

Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein-peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein-peptide pair. Escherichia coli cells co-expressing protein-peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein-peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher-SpyTag pair and co-selecting for functionally interacting variants.

Planar chromatography and immunodetection of hydrocarbons on polyvinylidene difluoride membranes.

Fast, cheap, and simple separation of lipids and hydrocarbons can currently be achieved using thin-layer chromatography. Here, we describe an alternative planar chromatographic method using polyvinylidene difluoride membranes as the stationary phase. The procedure has the same advantages of thin-layer chromatography over other expensive and time-consuming techniques, such as high-performance liquid chromatography or gas chromatography. Polyvinylidene difluoride membranes, however, also provide an immediate support for analyte development via immunodetection, are easy to manipulate, and potentially increase the performance of other detection methods. We show that polyvinylidene difluoride membranes are compatible with a variety of solvents that can migrate by capillarity and redistribute analytes between the membrane and the solvent according to their relative affinities, providing a chromatographic separation. We directly test the developed membranes by immunoblotting using anti-squalene antibodies that cross-react with acyclic isoprenoids. Separations of crude oils and plant extracts under different solvent conditions show the potential to resolve hydrocarbon group types and also to provide characteristic fingerprints of plant pigments and squalene degradation products. Polyvinylidene difluoride membranes prove useful as a stationary phase for planar chromatography and for the subsequent immunodetection of the separated compounds, providing a new and simple chromatographic technique to analyze lipids and hydrocarbons.

Structural evidence for the roles of divalent cations in actin polymerization and activation of ATP hydrolysis.

The structure of the actin filament is known at a resolution that has allowed the architecture of protein components to be unambiguously assigned. However, fully understanding the chemistry of the system requires higher resolution to identify the ions and water molecules involved in polymerization and ATP hydrolysis. Here, we find experimental evidence for the association of cations with the surfaces of G-actin in a 2.0-Šresolution X-ray structure of actin bound to a Cordon-Bleu WH2 motif and in previously determined high-resolution X-ray structures. Three of four reoccurring divalent cation sites were stable during molecular dynamics (MD) simulations of the filament, suggesting that these sites may play a functional role in stabilizing the filament. We modeled the water coordination at the ATP-bound Mg2+, which also proved to be stable during the MD simulations. Using this model of the filament with a hydrated ATP-bound Mg2+, we compared the cumulative probability of an activated hydrolytic water molecule approaching the γ-phosphorous of ATP, in comparison with G-actin, in the MD simulations. The cumulative probability increased in F-actin in line with the activation of actin's ATPase activity on polymerization. However, inclusion of the cations in the filament lowered cumulative probability, suggesting the rate of hydrolysis may be linked to filament flexibility. Together, these data extend the possible roles of Mg2+ in polymerization and the mechanism of polymerization-induced activation of actin's ATPase activity.

Proximal tubule cyclophilin D regulates fatty acid oxidation in cisplatin-induced acute kidney injury.

Regardless of the etiology, acute kidney injury involves aspects of mitochondrial dysfunction and ATP depletion. Fatty acid oxidation is the preferred energy source of the kidney and is inhibited during acute kidney injury. A pivotal role for the mitochondrial matrix protein, cyclophilin D in regulating overall cell metabolism is being unraveled. We hypothesize that mitochondrial interaction of proximal tubule cyclophilin D and the transcription factor PPARα modulate fatty acid beta-oxidation in cisplatin-induced acute kidney injury. Cisplatin injury resulted in histological and functional damage in the kidney with downregulation of fatty acid oxidation genes and increase of intrarenal lipid accumulation. However, proximal tubule-specific deletion of cyclophilin D protected the kidneys from the aforementioned effects. Mitochondrial translocation of PPARα, its binding to cyclophilin D, and sequestration led to inhibition of its nuclear translocation and transcription of PPARα-regulated fatty acid oxidation genes during cisplatin-induced acute kidney injury. Genetic or pharmacological inhibition of cyclophilin D preserved nuclear expression and transcriptional activity of PPARα and prevented the impairment of fatty acid oxidation and intracellular lipid accumulation. Docking analysis identified potential binding sites between PPARα and cyclophilin D. Thus, our results indicate that proximal tubule cyclophilin D elicits impaired mitochondrial fatty acid oxidation via mitochondrial interaction between cyclophilin D and PPARα. Hence, targeting their interaction may be a potential therapeutic strategy to prevent energy depletion, lipotoxicity and cell death in cisplatin-induced acute kidney injury.

Incorporation of Putative Helix-Breaking Amino Acids in the Design of Novel Stapled Peptides: Exploring Biophysical and Cellular Permeability Properties.

Stapled α-helical peptides represent an emerging superclass of macrocyclic molecules with drug-like properties, including high-affinity target binding, protease resistance, and membrane permeability. As a model system for probing the chemical space available for optimizing these properties, we focused on dual Mdm2/MdmX antagonist stapled peptides related to the p53 N-terminus. Specifically, we first generated a library of ATSP-7041 (Chang et al., 2013) analogs iteratively modified by L-Ala and D-amino acids. Single L-Ala substitutions beyond the Mdm2/(X) binding interfacial residues (i.e., Phe3, Trp7, and Cba10) had minimal effects on target binding, α-helical content, and cellular activity. Similar binding affinities and cellular activities were noted at non-interfacial positions when the template residues were substituted with their d-amino acid counterparts, despite the fact that d-amino acid residues typically 'break' right-handed α-helices. d-amino acid substitutions at the interfacial residues Phe3 and Cba10 resulted in the expected decreases in binding affinity and cellular activity. Surprisingly, substitution at the remaining interfacial position with its d-amino acid equivalent (i.e., Trp7 to d-Trp7) was fully tolerated, both in terms of its binding affinity and cellular activity. An X-ray structure of the d-Trp7-modified peptide was determined and revealed that the indole side chain was able to interact optimally with its Mdm2 binding site by a slight global re-orientation of the stapled peptide. To further investigate the comparative effects of d-amino acid substitutions we used linear analogs of ATSP-7041, where we replaced the stapling amino acids by Aib (i.e., R84 to Aib4 and S511 to Aib11) to retain the helix-inducing properties of α-methylation. The resultant analog sequence Ac-Leu-Thr-Phe-Aib-Glu-Tyr-Trp-Gln-Leu-Cba-Aib-Ser-Ala-Ala-NH2 exhibited high-affinity target binding (Mdm2 Kd = 43 nM) and significant α-helicity in circular dichroism studies. Relative to this linear ATSP-7041 analog, several d-amino acid substitutions at Mdm2(X) non-binding residues (e.g., d-Glu5, d-Gln8, and d-Leu9) demonstrated decreased binding and α-helicity. Importantly, circular dichroism (CD) spectroscopy showed that although helicity was indeed disrupted by d-amino acids in linear versions of our template sequence, stapled molecules tolerated these residues well. Further studies on stapled peptides incorporating N-methylated amino acids, l-Pro, or Gly substitutions showed that despite some positional dependence, these helix-breaking residues were also generally tolerated in terms of secondary structure, binding affinity, and cellular activity. Overall, macrocyclization by hydrocarbon stapling appears to overcome the destabilization of α-helicity by helix breaking residues and, in the specific case of d-Trp7-modification, a highly potent ATSP-7041 analog (Mdm2 Kd = 30 nM; cellular EC50 = 600 nM) was identified. Our findings provide incentive for future studies to expand the chemical diversity of macrocyclic α-helical peptides (e.g., d-amino acid modifications) to explore their biophysical properties and cellular permeability. Indeed, using the library of 50 peptides generated in this study, a good correlation between cellular permeability and lipophilicity was observed.

Macrocyclic α helical peptide therapeutic modality: A perspective of learnings and challenges.

Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development.

Clinical Outcome and Biological Predictors of Relapse After Nephrectomy Only for Very Low-risk Wilms Tumor: A Report From Children's Oncology Group AREN0532.

OBJECTIVE: To determine if observation alone after nephrectomy in very low-risk Wilms tumor (defined as stage I favorable histology Wilms tumors with nephrectomy weight <550g and age at diagnosis <2 years) results in satisfactory event-free survival and overall survival, and to correlate relapse with biomarkers. PATIENTS AND METHODS: The AREN0532 study enrolled patients with very low-risk Wilms tumor confirmed by central review of pathology, diagnostic imaging, and surgical reports. After nephrectomy, patients were followed without adjuvant chemotherapy. Evaluable tumors were analyzed for WT1mutation, 1p and 16q copy loss, 1q copy gain, and 11p15 imprinting. The study was powered to detect a reduction in 4-year EFS from 87% to 75% and overall survival from 95% to 88%. RESULTS: A total of 116 eligible patients enrolled with a median follow up of 80 months (range: 5-97 months). Twelve patients relapsed. Estimated 4-year event-free survival was 89.7% (95% confidence interval 84.1-95.2%) and overall survival was 100%. First sites of relapse were lung (n = 5), tumor bed (n = 4), and abdomen (n = 2), with one metachronous tumor in the contralateral kidney (n = 1) at a median time of 4.3 months for those who relapsed (range 2.3-44 months). The presence of intralobar (P = 0.46) or perilobar rests (P = 1.0) were not associated with relapse (P = 0.16). 1q gain, 1p and 16q loss, and WT1 mutation status were not associated with relapse. 11p15 methylation status was associated relapse (20% relapse with loss of heterozygosity, 25% with loss of imprinting, and 3.3% relapse with retention of the normal imprinting (P = 0.011)). CONCLUSIONS: Most patients meeting very low-risk criteria can be safely managed by nephrectomy alone with resultant reduced exposure to chemotherapy. Expansion of an observation alone strategy for low-risk Wilms tumor incorporating both clinical features and biomarkers should be considered.

Patterns of Performance of Oncologic Surgery by North American Pediatric Urologists: A Report from the Pediatric Urologic Oncology Working Group of the Society for Pediatric Urology.

PURPOSE: Objective data on patterns of oncology practice among pediatric urologists are lacking. We reviewed surgical case logs submitted to the American Board of Urology by those self-reporting as pediatric urologists. We hypothesized that logs would reveal a low oncology volume (fewer than 5 cases) and identify orchiectomy as the most common oncology cases, and that less than 25% of logs would show nephrectomy for renal tumor. MATERIALS AND METHODS: Case logs submitted for American Board of Urology certification, recertification or pediatric subspecialty certification were reviewed and standardized to represent 12-month practice. Data were collected on pediatric oncologic surgeries as noted by procedure codes linked with oncologic diagnosis codes for patients up to age 30 years. RESULTS: We identified 281 case logs meeting study criteria. A total of 364 oncology cases were logged and 131 logs (46.6%) listed at least 1 oncology case, while 150 (53.4%) contained no oncology cases. The 75th, 90th and 95th percentiles of oncology volume were represented by reporting 2, 3 and 4 cases, respectively. A total of 13 logs (4.6%) accounted for more than a third of all oncology cases (35.9%). The most frequent oncology case logged was orchiectomy, which was documented in 83 logs (29.5%). On Poisson regression surgeon variables associated with higher oncology volume included male gender (IRR 2.8, 95% CI 2.1-3.9), 2010 log year (IRR 2.4, 95% CI 1.3-4.4), 2015 log year (IRR 3.7, 95% CI 2.1-6.4) and nonpediatric subspecialty certification log (IRR 1.6, 95% CI 1.2-2.3). CONCLUSIONS: Few pediatric urologists perform a high volume of oncologic surgeries based on surgical case logs submitted to the American Board of Urology. A small cohort of pediatric urologists logged the majority of such cases.

A Society for Pediatric Urology Workforce Survey on the Current Perceptions of Oncology Care by Pediatric Urologists: A Report from the Pediatric Urologic Oncology Working Group of the Society for Pediatric Urology.

PURPOSE: Data are lacking on the current perception of oncology care among pediatric urologists. Thus, we developed, pilot tested and administered a survey on this topic to SPU (Society for Pediatric Urology) members. MATERIALS AND METHODS: Approval for this proposal was granted by SPU leadership prior to developing or distributing the survey instrument. The survey was developed and pilot tested by the PUOWG (Pediatric Urologic Oncology Working Group). Response data were collected and descriptive statistics were used for analysis. Logistic regression analysis was performed to correlate surgeon reported factors with higher volumes of reported oncology surgery. RESULTS: A total of 426 surveys were distributed via email to SPU members and 212 individual surveys (49.8%) were returned with the background/introduction section completed. Of these surveys 200 (94.3%) were completed by practicing pediatric urologists. Overall, 155 respondents (77.5%) reported performing 5 or fewer oncology related surgeries per year and 74.9% reported that less than 25% of renal tumor surgery at their institution was performed through the pediatric urology service. On multivariate analysis the self-reported factors significantly associated with increased oncology surgical volume (more than 5 cases per year) were greater than 50% attendance at institutional tumor board meetings (OR 4.8, 95% CI 1.4-16.9) and practicing at a hospital with a higher volume of renal tumor surgery (OR 2.6, 95% CI 1.2-5.8). CONCLUSIONS: Few surveyed pediatric urologists reported performing a high volume of oncology surgery. Respondents expressed interest in ways to increase pediatric urology involvement in oncology care, including opportunities for increased education. Self-reported factors that correlated with higher volume were regular attendance at the institutional pediatric tumor board and practice at a higher volume institution.

Surgical protocol violations in children with renal tumors provides an opportunity to improve pediatric cancer care: a report from the Children's Oncology Group.

BACKGROUND: The purpose of this study was to evaluate the frequency and characteristics of surgical protocol violations (SPVs) among children undergoing surgery for renal tumors who were enrolled on the Children's Oncology Group (COG) renal tumor biology and classification study AREN03B2. METHODS: AREN03B2 was opened in February 2006, and as on March 31, 2013, there were 3,664 eligible patients. The surgical review forms for 3,536 patients with unilateral disease were centrally reviewed for SPVs. The frequency, type, number of violations, institutional prevalence, and quartiles for SPVs were assessed. RESULTS: Of the 3,536 patients, there were a total of 505 with at least one SPV (564 total SPVs reported), for an overall incidence of 14.28%. The types of SPVs included a lack of lymph node sampling in 365 (64.7%), avoidable spill in 61 (10.8%), biopsy immediately before nephrectomy in 89 (15.8%), an incorrect abdominal incision in 32 (5.7%), and unnecessary resection of organs in 17 (3.0%). The SPVs occurred in 163 of 215 participating institutions (75.8%). For centers with at least one SPV, the mean number of SPVs reported was 3.10 ± 2.39 (mean ± standard deviation). The incidence of protocol violation per institution ranged from 0 to 67%. Centers with an average of ≤1 case/year had an incidence of SPVs of 12.2 ± 3.8%, those with an average of >1 to <4 cases/year had an incidence of SPVs of 16.4 ± 3.6%, and those with an average of ≥4 cases/year had an incidence of SPVs of 12.6 ± 5.5% (P > 0.05). CONCLUSIONS: SPVs that potentially result in additional exposure to chemotherapy and radiation therapy are not uncommon in children undergoing resection of renal malignancies.

Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma.

The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability.

Treatment with the immunomodulator FTY720 (fingolimod) significantly reduces renal inflammation in murine unilateral ureteral obstruction.

PURPOSE: The S1P signaling pathway represents an important potential target for the modulation of tissue inflammation/injury. The immunomodulator FTY720, also known as fingolimod, is a potent agonist for multiple S1P receptors that was approved by the Food and Drug Administration to treat multiple sclerosis. We examined the therapeutic role of FTY720 for renal injury secondary to unilateral ureteral obstruction. MATERIALS AND METHODS: CB57BL/6 mice underwent a sham procedure or unilateral ureteral obstruction and were treated with FTY720 by gavage for 1, 3 and 5 days. Control groups received vehicle. Ligated and unligated renal tissue was examined for histopathological changes, inflammatory and fibrotic markers, TGF-ß1, α-SMA, and macrophage infiltration by Western blot and immunohistochemistry. Proinflammatory and profibrotic cytokines were profiled by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Pathological evaluation revealed that FTY720 treatment resulted in a significant reduction in inflammatory infiltration in obstructed kidneys compared to controls. Immunohistochemical and Western blot showed that TGF-ß1 and α-SMA protein levels were similarly decreased, as was macrophage infiltration into the renal interstitial space, compared to untreated mice. In agreement with these observations quantitative reverse transcriptase-polymerase chain reaction revealed that inflammatory and fibrotic cytokines (MCP-1, IL-1ß, CXCL1, TNF-α and TGF-ß1) were also significantly decreased in the FTY720 group. CONCLUSIONS: This study suggests that in a murine ureteral obstruction model FTY720 significantly inhibited the production of inflammatory cytokines and factors regulating interstitial fibrosis and extracellular matrix accumulation. These findings were associated with decreased evidence of renal injury on pathological examination, suggesting that FTY720 or related compounds may be valuable modulators of obstruction induced renal injury.

Contemporary disparities in progression to orchiopexy for cryptorchidism as reported in the Pediatric Health Information System (PHIS) database.

INTRODUCTION: When evaluating the timeliness of orchiopexy for cryptorchidism, health disparities are apparent among Hispanic and African American males and those with public insurance. Since the publication of these data, the COVID-19 pandemic has stressed our healthcare system and significantly affected the provision of pediatric urology care. OBJECTIVE: We sought to assess if certain groups were disproportionately affected in progression to orchiopexy after the diagnosis of cryptorchidism during and after the pandemic in US freestanding children's hospitals. STUDY DESIGN: Using the PHIS database, pediatric patients ≤5 years who underwent orchiopexy between January 2018 and December 2022 were retrospectively analyzed. Exclusion criteria included prematurity, retractile testes, and testicular torsion. Primary outcomes were age at orchiopexy and the proportion of individuals undergoing timely orchiopexy for cryptorchidism. RESULTS: Over the study period 3140 patients ≤5 years old underwent orchiopexy for cryptorchidism. Non-Hispanic Blacks and Hispanics were significantly less likely to have timely orchiopexy and underwent orchiopexy 2.13 and 3.60 months later compared to whites (p < 0.01). As compared to pre-COVID-19, during the pandemic the proportion of patients who had timely surgery was higher and the median age was significantly lower (p = 0.01 and p < 0.01, respectively) in white patients only. Over the study period, patients with public insurance were less likely to have timely orchiopexy and underwent orchiopexy 2.94 months later (p < 0.01) than patients with private insurance. Compared to during the pandemic, post-pandemic a significantly lower proportion of publicly insured patients have since undergone timely orchiopexy (p = 0.04). Patients in the West were less likely to have timely orchiopexy and had a higher age at time of orchiopexy (p < 0.01) than other regions. However, in the West during the pandemic, the proportion of children who had timely surgery was higher compared to pre-and post-COVID-19 (p < 0.01). DISCUSSION: Overall, regardless of insurance status, race, or location, a significant proportion of patients did not undergo timely orchiopexy. During the pandemic white patients had a lower median age and an increased proportion underwent timely orchiopexy, despite the number of orchiopexies remaining constant. Disparities in the post-COVID-19 era have been further exacerbated for publicly insured patients, who a significantly lower proportion of have since undergone timely orchiopexy. Specific efforts are required across the United States to increase timely orchiopexy for all boys. CONCLUSIONS: Progression to timely orchiopexy remains low for all boys in the era surrounding COVID-19; certain groups appear to be more adversely affected.

Inter-disciplinary provider development of an online, interactive adolescent varicocele decision aid prototype.

BACKGROUND: Decision-making regarding varicocele management can be a complex process for patients and families. However, to date, no studies have presented ways to mitigate the decisional conflict surrounding varicoceles. OBJECTIVE: To facilitate a discussion among physicians in order to develop a framework of the decision-making process regarding adolescent varicocele management, which will inform the development of the first online, interactive decision aid. MATERIALS AND METHODS: Semi-structured interviews with pediatric urologists and interventional radiologists were conducted to discuss their rationale for varicocele decision-making. Interviews were audio recorded, transcribed, and coded. Key themes were identified, grouped, and then qualitatively analyzed using thematic analysis. Utilizing the common themes identified and the Ottawa Decision Support Framework, a decision aid prototype was developed and transformed into a user-friendly website: varicoceledecisionaid.com. RESULTS: Pediatric urologists (n = 10) and interventional radiologists (n = 2) were interviewed. Key themes identified included: (1) definition/epidemiology; (2) observation as an appropriate management choice; (3) reasons to recommend repair; (4) types of repair; (5) reasons to recommend one repair over another; (6) shared decision-making; and (7) appropriate counseling. With this insight, a varicocele decision aid prototype was developed that engages patients and parents in the decision-making process. DISCUSSION AND CONCLUSIONS: This is the first interactive and easily accessible varicocele decision aid prototype developed by inter-disciplinary physicians for patients. This tool aids in decision-making surrounding varicocele surgery. It can be used before or after consultation to help families understand more about varicoceles and their repair, and why intervention may or may not be offered. It also considers a patient and family's personal values. Future studies will incorporate the patient and family perspective into the decision-making aid as well as implement and test the usability of this decision aid prototype in practice and in the wider urologic community.

Does KUB play a role in the diagnosis of bladder bowel dysfunction?

INTRODUCTION: Kidney ureter bladder radiography (KUB) is widely used for the evaluation of constipation in children with bladder and bowel dysfunction (BBD); however, there is varying evidence to support its routine diagnostic use. One drawback to KUB is radiation exposure. The dangers of radiation in children are well-documented, and per As Low As Reasonably Achievable, non-beneficial radiation should be avoided. This risk is especially high in children who undergo repeated imaging in the follow up of constipation treatment. OBJECTIVE: We sought to assess the utility of KUB in diagnosing children with BBD by comparing it to four diagnostic tests and/or validated instruments: the Dysfunctional Voiding Symptom Score (DVSS), Rome IV criteria, rectal diameter on ultrasound (RD), and the Bristol Stool Form Score (BSFS). STUDY DESIGN: We prospectively enrolled a cohort of patients presenting to an academic pediatric urology practice with symptoms of BBD. Severity of stool burden on KUB (mild, moderate, or severe), RD on ultrasound (≥3.4 cm), DVSS, Rome IV, and BSFS were obtained for each patient. All imaging was interpreted by a pediatric radiologist and pediatric urologist. Primary outcomes were the association between the four diagnostic tests and KUB stool burden. Bivariate analysis of all individual variables versus KUB was performed, as well as multivariate regressions to determine if multiple measures were predictive of KUB stool burden when combined. RESULTS: Between October 2020 and May 2022, 50 patients were enrolled. All children were under the age of 18, with a median age of 8 years (IQR 3-13). 38 % were male. Median BMI-for-age-percentile was 80.8 (IQR 50.3-98.3). When comparing individual variables to KUB in bivariate analyses, it was found that RD on ultrasound is predictive of significant stool burden on KUB (p = 0.03). No other individual variables were predictive. In the multivariate analyses, no combination of tests was found to be predictive of KUB. DISCUSSION: We compared the effectiveness of four commonly used diagnostic tests in children with BBD to validate the use of KUB. In conclusion, our results support the use of RD on ultrasound as a non-radiating alternative to KUB to assess stool burden. Data also suggest that KUB for fecal load does not correlate with urinary (DVSS) or bowel (Rome IV, BSFS) symptoms in BBD, and that symptoms scores should still be used independently for diagnosis and monitoring of treatment response. CONCLUSION: In conclusion, KUB has a limited role in the diagnosis of BBD.

The need for additional procedures in patients undergoing proximal hypospadias repairs as reported in the pediatric health information system database.

PURPOSE: Using administrative data from freestanding pediatric hospitals in the United States, we characterized the frequency and type of additional procedures required in patients undergoing proximal hypospadias repair in a larger cohort than in published case series across multiple surgeons and institutions. MATERIALS AND METHODS: A search of the Pediatric Health Information System (PHIS) database by CPT code between January 1, 2005 and June 30, 2010 identified patients undergoing 1 or 2-stage repair for proximal hypospadias. Patient records with inconsistent coding or the suggestion of an alternate pathological condition were excluded from study. A forward query to June 30, 2011 identified additional hypospadias related interventions by CPT codes. RESULTS: We identified 1,679 patients from a total of 37 hospitals. Potential followup was 1 to 6.5 years. One-stage repair was performed in 85.7% of patients at a median age of 10 months. In patients undergoing 2-stage repair the median age at initial repair was 10 months and the median interval between stages was 6 months. Of all patients 26.2% required 1 or more additional interventions beyond definitive repair. Of all additional interventions 84.0% were open, 7.2% were endoscopic treatment for stricture, 0.4% were combined endoscopic and open interventions, and 8.4% were endoscopic evaluation. The median interval from definitive repair to the first intervention was 9 months. CONCLUSIONS: These data indicate that more than a quarter of patients who underwent proximal hypospadias repair at pediatric hospitals required additional intervention(s) after what was thought to be definitive repair. These data help create a broader context in a contemporary cohort of patients treated with proximal hypospadias repair.

FTY720 inhibits tumor growth and enhances the tumor-suppressive effect of topotecan in neuroblastoma by interfering with the sphingolipid signaling pathway.

BACKGROUND: Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. Poor outcomes for children with advanced disease underscore the need for novel therapeutic strategies. FTY720, an immunomodulating drug approved for multiple sclerosis, has been investigated in oncology with promising preclinical activities. To date, its effect in NB has not been explored. Herein we describe our preclinical experience with FTY720, alone or in combination with topotecan, and its putative mechanism of action in NB. PROCEDURE: MTT assay was performed to assess the effect of FTY720 on cell viability. A NB xenograft model was employed to assess the efficacy of FTY720 on tumor growth. Quantitative real-time PCR and Western blot were employed to determine changes of mRNA and protein expression, respectively. Liquid chromatography/tandem mass spectrometry was used to measure sphingolipid levels. RESULTS: FTY720, but not FTY720-P induced NB cell death. FTY720 inhibited the growth of NB xenografts and enhanced the tumor-suppressive effect of topotecan both in vitro and in vivo. FTY720 significantly inhibited sphingosine kinase 2 (SphK2) mRNA and protein expression in NB cells. Pro-apoptotic sphingosine levels were increased in NB cells and NB xenografts treated with FTY720. FTY720-induced cell death was caspase-independent and involved the dephosphorylation of Akt and BAD at Ser136. CONCLUSIONS: Our data demonstrate that FTY720 has potent preclinical anti-cancer activity in NB. Its unique death signaling mechanism, interference with the sphingolipid pathway, acts cooperatively with that of topotecan, suggesting that FTY720 related molecules may be useful in NB treatment.

Oncology: the role of partial nephrectomy in Wilms tumor.

Wilms tumor represents the most common pediatric renal malignancy and the fourth most common childhood cancer overall. Overall survival from Wilms tumor has increased to over 90 % secondary to multidisciplinary therapy and multi-institutional cooperative group trials. Recent therapeutic focus has shifted to reduction in treatment morbidity and renal preservation while maintaining the high survival rates. Partial nephrectomy is an integral component of the multimodal treatment protocols for Wilms tumor patients with bilateral disease, solitary kidney, or predisposing syndromes. Recent consideration has been given to utilization of nephron sparing surgery (NSS) in carefully selected patients with nonsyndromic unilateral Wilms tumor. While long-term, prospective data in this subgroup of patients is not yet available, case series demonstrate comparable oncologic outcomes after partial versus radical nephrectomy. The relative rarity of Wilms tumor, especially those amenable to upfront partial nephrectomy, presents a challenge to conducting controlled trials.