COVID-19 in pediatric kidney transplantation: a follow-up report of the Improving Renal Outcomes Collaborative.

BACKGROUND: We report follow-up data from an ongoing prospective cohort study of COVID-19 in pediatric kidney transplantation through the Improving Renal Outcomes Collaborative (IROC). METHODS: Patient-level data from the IROC registry were combined with testing, indication, and outcomes data collected to describe the epidemiology of COVID testing, treatment, and clinical outcomes; determine the incidence of a positive COVID-19 test; describe rates of COVID-19 testing; and assess for clinical predictors of a positive COVID-19 test. RESULTS: From September 2020 to February 2021, 21 centers that care for 2690 patients submitted data from 648 COVID-19 tests on 465 patients. Most patients required supportive care only and were treated as outpatients, 16% experienced inpatient care, and 5% experienced intensive care. Allograft complications were rare, with acute kidney injury most common (7%). There was 1 case of respiratory failure and 1 death attributed to COVID-19. Twelve centers that care for 1730 patients submitted complete testing data on 351 patients. The incidence of COVID-19 among patients at these centers was 4%, whereas the incidence among tested patients was 19%. Risk factors to predict a positive COVID-19 test included age > 12 years, symptoms consistent with COVID-19, and close contact with a confirmed case of COVID-19. CONCLUSIONS: Despite the increase in testing and positive tests over this study period, the incidence of allograft loss or death related to COVID-19 remained extremely low, with allograft loss or death each occurring in < 1% of COVID-19-positive patients and in less than < 0.1% of all transplant patients within the IROC cohort. A higher resolution version of the Graphical abstract is available as Supplementary information.

COVID-19 in pediatric kidney transplantation: The Improving Renal Outcomes Collaborative.

There are limited data on the impact of COVID-19 in children with a kidney transplant (KT). We conducted a prospective cohort study through the Improving Renal Outcomes Collaborative (IROC) to collect clinical outcome data about COVID-19 in pediatric KT patients. Twenty-two IROC centers that care for 2732 patients submitted testing and outcomes data for 281 patients tested for SARS-CoV-2 by PCR. Testing indications included symptoms and/or potential exposures to COVID-19 (N = 134, 47.7%) and/or testing per hospital policy (N = 154, 54.8%). Overall, 24 (8.5%) patients tested positive, of which 15 (63%) were symptomatic. Of the COVID-19-positive patients, 16 were managed as outpatients, six received non-ICU inpatient care and two were admitted to the ICU. There were no episodes of respiratory failure, allograft loss, or death associated with COVID-19. To estimate incidence, subanalysis was performed for 13 centers that care for 1686 patients that submitted all negative and positive COVID-19 results. Of the 229 tested patients at these 13 centers, 10 (5 asymptomatic) patients tested positive, yielding an overall incidence of 0.6% and an incidence among tested patients of 4.4%. Pediatric KT patients in the United States had a low estimated incidence of COVID-19 disease and excellent short-term outcomes.

Conversion to permanent vascular access is associated with improved markers of hemodialysis efficacy in children: Pediatric nephrology research consortium study.

BACKGROUND AND OBJECTIVES: Arteriovenous fistulae (AVF) and grafts (AVG) are preferred permanent vascular access (PVA) for chronic hemodialysis (HD) patients. Our objective was to examine the change in markers of HD efficacy after successful establishment of a PVA among children who started HD with a tunneled cuffed catheter (TCC). MATERIALS AND METHODS: Retrospective chart reviews were completed on patients from 20 pediatric dialysis centers. All patients used TCC prior to AVF/AVG, and each patient acted as his/her own control. Data on markers of HD efficacy (single-pool Kt/V, urea reduction ratio (URR), serum albumin and hematocrit (Hct)) were collected at the creation of AVF/AVG and for 2 years thereafter. Statistical methods included hypothesis testing and statistical modeling after adjusting for relevant demographic variables. RESULTS: First PVA was created in 98 individual children: 87 (89%) were AVF and 11 (11%) were AVG. The mean TCC vintage prior to AVF/AVG was 10.4 ± 17.3 months. At 1-year follow-up, Kt/V improved by 0.15 ± 0.06 (p = 0.02) and URR improved by 4.54 ± 1.17% (p < 0.0001). Furthermore, PVA was associated with improved serum albumin by 0.31 ± 0.07 g/dL (p < 0.0001) and Hct by 2.80 ± 0.65% (p < 0.0001) at 1 year. These HD efficacy markers remained statistically significant at 2nd-year follow-up. These observations were further supported by the adjusted models. Conversion to AVF was associated with statistically significant improvement in all four markers of HD efficacy at 1-year follow-up. This trend was not demonstrated for subjects who were converted to AVG. CONCLUSION: Switching to PVA was associated with improved markers of HD efficacy, single-pool Kt/V, URR, serum albumin, and Hct. This improvement was mostly demonstrated at 1 year and maintained for the 2nd year. The potential differential impact of the type of PVA on the trajectory of markers of HD efficacy should be further investigated.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.

Plasma pseudouridine levels reflect body size in children on hemodialysis.

BACKGROUND: Dialysis in children as well as adults is prescribed to achieve a target spKt/Vurea, where Vurea is the volume of distribution of urea. Waste solute production may however be more closely correlated with body surface area (BSA) than Vurea which rises in proportion with body weight. Plasma levels of waste solutes may thus be higher in smaller patients when targeting spKt/Vurea since they have higher BSA relative to body weight. This study measured levels of pseudouridine (PU), a novel marker solute whose production is closely proportional to BSA, to test whether prescription of dialysis to a target spKt/Vurea results in higher plasma levels of PU in smaller children. METHODS: PU and urea nitrogen (ureaN) were measured in plasma and dialysate at the midweek hemodialysis session in 20 pediatric patients, with BSA ranging from 0.65-1.87m2. Mathematical modeling was employed to estimate solute production rates and average plasma solute levels. RESULTS: The dialytic clearance (Kd) of PU was proportional to that of ureaN (average KdPU/KdUreaN 0.69 ± 0.13, r2 0.84, p < 0.001). Production of PU rose in proportion with BSA (r2 0.57, p < 0.001). The pretreatment plasma level of PU was significantly higher in smaller children (r2 0.20, p = 0.051) while the pretreatment level of ureaN did not vary with size. CONCLUSIONS: Prescribing dialysis based on urea kinetics may leave uremic solutes at higher levels in small children. Measurement of a solute produced proportional to BSA may provide a better index of dialysis adequacy than measurement of urea.

Predictors of time to first cannulation for arteriovenous fistula in pediatric hemodialysis patients: Midwest Pediatric Nephrology Consortium study.

BACKGROUND: Permanent vascular access (PVA) is preferred for long-term hemodialysis. Arteriovenous fistulae (AVF) have the best patency and the lowest complication rates compared to arteriovenous grafts (AVG) and tunneled cuffed catheters (TCC). However, AVF need time to mature. This study aimed to investigate predictors of time to first cannulation for AVF in pediatric hemodialysis patients. METHODS: Data on first AVF and AVG of patients at 20 pediatric dialysis centers were collected retrospectively, including demographics, clinical information, dialysis markers, and surgical data. Statistical modeling was used to investigate predictors of outcome. RESULTS: First PVA was created in 117 children: 103 (88%) AVF and 14 (12%) AVG. Mean age at AVF creation was 15.0 ± 3.3 years. AVF successfully matured in 89 children (86.4%), and mean time to first cannulation was 3.6 ± 2.5 months. In a multivariable regression model, study center, age, duration of non-permanent vascular access (NPVA), and Kt/V at AVF creation predicted time to first cannulation, with study center as the strongest predictor (p < 0.01). Time to first cannulation decreased with increasing age (p = 0.03) and with increasing Kt/V (p = 0.01), and increased with duration of NPVA (p = 0.03). Secondary failure occurred in 10 AVF (11.8%). Time to first cannulation did not predict secondary failure (p = 0.29), but longer time to first cannulation tended towards longer secondary patency (p = 0.06). CONCLUSIONS: Study center is the strongest predictor of time to first cannulation for AVF and deserves further investigation. Time to first cannulation is significantly shorter in older children, with more efficient dialysis treatments, and increases with longer NPVA duration.

Apical Shear Stress Enhanced Organic Cation Transport in Human OCT2/MATE1-Transfected Madin-Darby Canine Kidney Cells Involves Ciliary Sensing.

Active transport by renal proximal tubules plays a significant role in drug disposition. During drug development, estimates of renal excretion are essential to dose determination. Kidney bioreactors that reproduce physiologic cues in the kidney, such as flow-induced shear stress, may better predict in vivo drug behavior than do current in vitro models. In this study, we investigated the role of shear stress on active transport of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) by Madin-Darby canine kidney cells exogenously expressing the human organic cation transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1). Cells cultured in a parallel plate under continuous media perfusion formed a tight monolayer with a high barrier to inulin. In response to increasing levels of shear stress (0.2-2 dynes/cm2), cells showed a corresponding increase in transport of ASP+, reaching a maximal 4.2-fold increase at 2 dynes/cm2 compared with cells cultured under static conditions. This transport was inhibited with imipramine, indicating active transport was present under shear stress conditions. Cells exposed to shear stress of 2 dynes/cm2 also showed an increase in RNA expression of both transfected human and endogenous OCT2 (3.7- and 2.0-fold, respectively). Removal of cilia by ammonium sulfate eliminated the effects of shear on ASP+ transport at 0.5 dynes/cm2 with no effect on ASP+ transport under static conditions. These results indicate that shear stress affects active transport of organic cations in renal tubular epithelial cells in a cilia-dependent manner.

Correction to: Predictors of patency for arteriovenous fistulae and grafts in pediatric hemodialysis patients.

The original version of this article unfortunately contained a mistake. The name of Vimal Chadha was presented incorrectly. The corrected author list is given above.

Predictors of patency for arteriovenous fistulae and grafts in pediatric hemodialysis patients.

BACKGROUND: Hemodialysis (HD) guidelines recommend permanent vascular access (PVA) in children unlikely to receive kidney transplant within 1 year of starting HD. We aimed to determine predictors of primary and secondary patency of PVA in pediatric HD patients. METHODS: Retrospective chart reviews were performed for first PVAs in 20 participating centers. Variables collected included patient demographics, complications, interventions, and final outcome. RESULTS: There were 103 arterio-venous fistulae (AVF) and 14 AV grafts (AVG). AVF demonstrated superior primary (p = 0.0391) and secondary patency (p = 0.0227) compared to AVG. Primary failure occurred in 16 PVA (13.6%) and secondary failure in 14 PVA (12.2%). AVF were more likely to have primary failure (odds ratio (OR) = 2.10) and AVG had more secondary failure (OR = 3.33). No demographic, clinical, or laboratory variable predicted primary failure of PVA. Anatomical location of PVA was predictive of secondary failure, with radial having the lowest risk compared to brachial (OR = 12.425) or femoral PVA (OR = 118.618). Intervention-free survival was predictive of secondary patency for all PVA (p = 0.0252) and directly correlated with overall survival of AVF (p = 0.0197) but not AVG. Study center demonstrated statistically significant effect only on intervention-free AVF survival (p = 0.0082), but not number of complications or interventions, or outcomes. CONCLUSIONS: In this multi-center pediatric HD cohort, AVF demonstrated primary and secondary patency advantages over AVG. Radial PVA was least likely to develop secondary failure. Intervention-free survival was the only predictor of secondary patency for AVF and directly correlated with overall access survival. The study center effect on intervention-free survival of AVF deserves further investigation.

Positive Cumulative Fluid Balance Is Associated With Mortality in Pediatric Acute Respiratory Distress Syndrome in the Setting of Acute Kidney Injury.

OBJECTIVES: As acute kidney injury and elevated cumulative fluid balance commonly co-occur in pediatric acute respiratory distress syndrome, we aimed to identify risk factors for their development and evaluate their independent relationships with mortality. We hypothesized that acute kidney injury and elevated cumulative fluid balance would be associated with markers of inflammation and that children with elevated cumulative fluid balance and concomitant acute kidney injury would have worse outcomes than other children. DESIGN: Prospective observational study using the pediatric Risk, Injury, Failure, Loss, End-Stage acute kidney injury classification. SETTING: Five academic PICUs. PATIENTS: Two-hundred sixty patients 1 month to 18 years old meeting the Berlin definition of acute respiratory distress syndrome between 2008 and 2014. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: PICU mortality was 13% (34/260). Relative to survivors, nonsurvivors had greater cumulative fluid balance on day 3 of acute respiratory distress syndrome (+90.1 mL/kg; interquartile range 26.6-161.7 vs +44.9 mL/kg; interquartile range 10.0-111.3; p = 0.008) and also had higher prevalence of acute kidney injury on day 3 of acute respiratory distress syndrome (50% vs 23%; p = 0.001). On stratified analysis, greater cumulative fluid balance on day 3 of acute respiratory distress syndrome was associated with mortality among patients with concomitant acute kidney injury (+111.5 mL/kg for nonsurvivors; interquartile range 82.6-236.8 vs +58.5 mL/kg for survivors; interquartile range 0.9-176.2; p = 0.041) but not among patients without acute kidney injury (p = 0.308). The presence of acute kidney injury on acute respiratory distress syndrome day 3 was associated with mortality among patients with positive cumulative fluid balance (29.1% vs 10.4% mortality; p = 0.001) but not among patients with even or negative cumulative fluid balance (p = 0.430). Day 1 plasma interleukin-6 levels were associated with the development of day 3 positive cumulative fluid balance, day 3 acute kidney injury, and PICU mortality and the association between elevated day 1 interleukin-6 and PICU mortality was partially mediated by the interval development of day 3 positive cumulative fluid balance and day 3 acute kidney injury (p < 0.001). CONCLUSIONS: In pediatric acute respiratory distress syndrome, elevated cumulative fluid balance on day 3 of acute respiratory distress syndrome is associated with mortality specifically in patients with concomitant acute kidney injury. Plasma interleukin-6 levels are associated with the development of positive cumulative fluid balance and acute kidney injury, suggesting a potential mechanism by which inflammation might predispose to mortality.

De novo lumen formation and elongation in the developing nephron: a central role for afadin in apical polarity.

A fundamental process in biology is the de novo formation and morphogenesis of polarized tubules. Although these processes are essential for the formation of multiple metazoan organ systems, little is known about the molecular mechanisms that regulate them. In this study, we have characterized several steps in tubule formation and morphogenesis using the mouse kidney as a model system. We report that kidney mesenchymal cells contain discrete Par3-expressing membrane microdomains that become restricted to an apical domain, coinciding with lumen formation. Once lumen formation has been initiated, elongation occurs by simultaneous extension and additional de novo lumen generation. We demonstrate that lumen formation and elongation require afadin, a nectin adaptor protein implicated in adherens junction formation. Mice that lack afadin in nephron precursors show evidence of Par3-expressing membrane microdomains, but fail to develop normal apical-basal polarity and generate a continuous lumen. Absence of afadin led to delayed and diminished integration of nectin complexes and failure to recruit R-cadherin. Furthermore, we demonstrate that afadin is required for Par complex formation. Together, these results suggest that afadin acts upstream of the Par complex to regulate the integration and/or coalescence of membrane microdomains, thereby establishing apical-basal polarity and lumen formation/elongation during kidney tubulogenesis.

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis.

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.

Acute changes in blood pressure in patients with neuroblastoma treated with ¹³¹I-metaiodobenzylguanidine (MIBG).

BACKGROUND: Iodine-131-metaiodobenzylguanidine ((131)I-MIBG) provides targeted radiotherapy for children with neuroblastoma. The aim of our study was to evaluate systematically the acute effects of (131)I-MIBG on blood pressure in patients with neuroblastoma and to identify possible predictors of hypertension. PROCEDURE: We conducted a retrospective chart review of neuroblastoma patients who were treated with (131)I-MIBG between January 1, 1999 and June 1, 2012 at the University of California, San Francisco. Clinical data for 172 patients with neuroblastoma, receiving 218 administrations of (131)I-MIBG, were collected. The primary endpoint was development of systolic blood pressure above the 95th percentile for age. Logistic regression with generalized estimating equations to account for multiple administrations in some subjects was used to identify bivariate and multivariate predictors of hypertension. RESULTS: Of the 218 administrations of (131)I-MIBG, 112 (51.3%) were associated with at least one episode of systolic hypertension during or after the (131)I-MIBG infusion. The majority of these acute elevations in blood pressure resolved within 48 hours of the infusion. Only six administrations in five patients required nifedipine administration to lower blood pressure. Younger age (P = 0.012), lower eGFR (P = 0.047), and elevated blood pressure measurements immediately before infusion began (P = 0.010) were all independently associated with risk of treatment-associated hypertension. CONCLUSIONS: Acute elevations in blood pressure are common after therapeutic doses of (131) I-MIBG. Elevations in blood pressure typically occur only within the first 48 hours after (131)I-MIBG administration. Blood pressure monitoring during this period of risk is recommended.

Feasibility of an implantable bioreactor for renal cell therapy using silicon nanopore membranes.

The definitive treatment for end-stage renal disease is kidney transplantation, which remains limited by organ availability and post-transplant complications. Alternatively, an implantable bioartificial kidney could address both problems while enhancing the quality and length of patient life. An implantable bioartificial kidney requires a bioreactor containing renal cells to replicate key native cell functions, such as water and solute reabsorption, and metabolic and endocrinologic functions. Here, we report a proof-of-concept implantable bioreactor containing silicon nanopore membranes to offer a level of immunoprotection to human renal epithelial cells. After implantation into pigs without systemic anticoagulation or immunosuppression therapy for 7 days, we show that cells maintain >90% viability and functionality, with normal or elevated transporter gene expression and vitamin D activation. Despite implantation into a xenograft model, we find that cells exhibit minimal damage, and recipient cytokine levels are not suggestive of hyperacute rejection. These initial data confirm the potential feasibility of an implantable bioreactor for renal cell therapy utilizing silicon nanopore membranes.

Persistent Increase in Serum Ferritin Levels despite Converting to Permanent Vascular Access in Pediatric Hemodialysis Patients: Pediatric Nephrology Research Consortium Study.

Our objective was to examine serum ferritin trends after conversion to permanent vascular access (PVA) among children who started hemodialysis (HD) using tunneled cuffed catheters (TCC). Retrospective chart reviews were completed on 98 subjects from 20 pediatric HD centers. Serum ferritin levels were collected at the creation of PVA and for two years thereafter. There were 11 (11%) arteriovenous grafts (AVG) and 87 (89%) arteriovenous fistulae (AVF). Their mean TCC use was 10.4 ± 17.3 months. Serum ferritin at PVA creation was elevated at 562.64 ± 492.34 ng/mL, increased to 753.84 ± 561.54 ng/mL (p = < 0.001) in the first year and remained at 759.60 ± 528.11 ng/mL in the second year (p = 0.004). The serum ferritin levels did not show a statistically significant linear association with respective serum hematocrit values. In a multiple linear regression model, there were three predictors of serum ferritin during the first year of follow-up: steroid-resistant nephrotic syndrome as primary etiology (p = 0.035), being from a center that enrolled >10 cases (p = 0.049) and baseline serum ferritin level (p = 0.017). Increasing serum ferritin after conversion to PVA is concerning. This increase is not associated with serum hematocrit trends. Future studies should investigate the correlation of serum transferrin saturation and ferritin levels in pediatric HD patients.

Urologic Considerations in Pediatric Chronic Kidney Disease.

Common causes of pediatric ESRD are distinct from those seen in the adult population. In the pediatric population, the most common are congenital anomalies of the kidney and urinary tract (CAKUT), affecting approximately 30% of children with CKD. These structural anomalies often require coordinated care with the pediatric urology team to address voiding issues, bladder involvement, and the potential need for surgical intervention. For pediatric nephrologists and urologists, common CAKUT that are encountered include antenatal hydronephrosis, obstructive uropathies (eg, posterior urethral valves), and vesicoureteral reflux. As more pediatric patients with CAKUT, CKD, and ESRD transition to adult care, it is important for receiving adult nephrologists to understand the clinical presentation, natural history, and prognosis for these diagnoses. This review outlines the diagnosis and potential interventions for these conditions, including strategies to address bladder dysfunction that is often seen in children with CAKUT. A discussion of these management decisions (including surgical intervention) for CAKUT, which are quite common to pediatric nephrology and urology practices, may provide unique learning opportunities for adult nephrologists who lack familiarity with these pediatric conditions.

Association of Plasma Uremic Solute Levels with Residual Kidney Function in Children on Peritoneal Dialysis.

BACKGROUND AND OBJECTIVES: Residual native kidney function confers health benefits in patients on dialysis. It can facilitate control of extracellular volume and inorganic ion concentrations. Residual kidney function can also limit the accumulation of uremic solutes. This study assessed whether lower plasma concentrations of uremic solutes were associated with residual kidney function in pediatric patients on peritoneal dialysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Samples were analyzed from 29 pediatric patients on peritoneal dialysis, including 13 without residual kidney function and ten with residual kidney function. Metabolomic analysis by untargeted mass spectrometry compared plasma solute levels in patients with and without residual kidney function. Dialytic and residual clearances of selected solutes were also measured by assays using chemical standards. RESULTS: Metabolomic analysis showed that plasma levels of 256 uremic solutes in patients with residual kidney function averaged 64% (interquartile range, 51%-81%) of the values in patients without residual kidney function who had similar total Kt/Vurea. The plasma levels were significantly lower for 59 of the 256 solutes in the patients with residual kidney function and significantly higher for none. Assays using chemical standards showed that residual kidney function provides a higher portion of the total clearance for nonurea solutes than it does for urea. CONCLUSIONS: Concentrations of many uremic solutes are lower in patients on peritoneal dialysis with residual kidney function than in those without residual kidney function receiving similar treatment as assessed by Kt/Vurea.

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity.

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

Simple Rules Determine Distinct Patterns of Branching Morphogenesis.

Many metazoan organs are comprised of branching trees of epithelial tubes; how patterning occurs in these trees is a fundamental problem of development. Commonly, branch tips fill the volume of the organ approximately uniformly, e.g., in mammalian lung, airway branch tips are dispersed roughly uniformly throughout the volume of the lung. In contrast, in the developing metanephric kidney, the tips of the ureteric bud tree are located close to the outer surface of the kidney rather than filling the kidney. Here, we describe a simple alteration in the branching rules that accounts for the difference between the kidney pattern that leads to tips near the organ surface versus previously known patterns that lead to the branch tips being dispersed throughout the organ. We further use a simple toy model to deduce from first principles how this rule change accounts for the differences in the two types of trees.

Application of physiological shear stress to renal tubular epithelial cells.

Renal tubular epithelial cells are consistently exposed to flow of glomerular filtrate that creates fluid shear stress at the apical cell surface. This biophysical stimulus regulates several critical renal epithelial cell functions, including transport, protein uptake, and barrier function. Defining the in vivo mechanical conditions in the kidney tubule is important for accurately recapitulating these conditions in vitro. Here we provide a summary of the fluid flow conditions in the kidney and how this translates into different levels of fluid shear stress down the length of the nephron. A detailed method is provided for measuring fluid flow in the proximal tubule by intravital microscopy. Devices to mimic in vivo fluid shear stress for in vitro studies are discussed, and we present two methods for culture and analysis of renal tubule epithelial cells exposed physiological levels of fluid shear stress. The first is a microfluidic device that permits application of controlled shear stress to cells cultured on porous membranes. The second is culture of renal tubule cells on an orbital shaker. Each method has advantages and disadvantages that should be considered in the context of the specific experimental objectives.