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Icodextrin has a lower absorption rate, and icodextrin peritoneal dialysate contributes to more water removal than glucose dialysate in patients with high peritoneal permeability. There are limited data on icodextrin dialysate use in children.This study included all pediatric patients who received peritoneal equilibration tests and peritoneal dialysis with icodextrin dialysate at the study center. The factors related to ultrafiltration volume with icodextrin dialysate with long dwell time were statistically analyzed. Then the ultrafiltration volume with icodextrin and medium-concentration glucose dialysate was compared in individual cycles in the same patients.Thirty-six samples were included in the icodextrin group, and nine samples were used to compare the ultrafiltration volume with icodextrin and glucose dialysate. Dwell time, D/P-creatinine, D/DThe ultrafiltration volume with icodextrin dialysate decreases in patients with small stature. Providing sufficient dwell time and volume is important for maximal water removal even in children. Ultrafiltration volume is superior with icodextrin than medium-concentration glucose dialysate for long dwell times. A higher resolution version of the Graphical abstract is available as Supplementary information.

The clinical spectrum of Henoch-Schönlein purpura nephritis (HSPN), now known as IgA vasculitis-associated nephritis (IgAVN), ranges from isolated microscopic haematuria to nephrotic syndrome, progressive glomerulonephritis, and kidney failure. The outcome also varies, and the management of IgAVN is controversial. The presence of nephrotic state at disease onset is thought to be a risk factor of a poor prognosis. However, not all patients with nephrotic state have a poor prognosis, and it is unclear whether they need early treatment.We herein retrospectively examined the clinical course of paediatric IgAVN cases with nephrotic state (serum albumin [sAlb]3.0 g/dL and urine protein-creatinine ratio of2.0 g/ gCr) without kidney injury treated at our hospital between 2010 and 2018.Of the 216 patients with IgAVN identified, 17 met the inclusion criteria. The median follow-up period from disease onset to the last observation was 40.5 months (IQR:31.0-74.2). Eleven patients were male, the median age at onset was 5 years, the minimum serum Alb level was 1.9 g/dL, the maximum proteinuria value was 12.3 g/gCr, and the median minimum eGFR was 86.0 mL/min/1.73 mOur study found that half of paediatric patients with IgAVN with nephrotic state achieved spontaneous resolution without treatment and enjoyed a favourable short-term outcome. Consideration of the duration of nephrotic state and trends in the serum albumin level in children with IgAVN may allow unnecessary kidney biopsies and immunosuppressive therapy to be avoided.

Background In typical cases of Bartter syndrome (BS), assessing response to diuretics (furosemide and thiazide), hereinafter referred to as diuretic loading test, may be used to diagnose the type by detecting which part of the kidney tubule is not functioning correctly. However, the diuretic loading test may not always agree with the results of genetic analyses. Case presentation A 5-year-old boy was admitted due to lower extremity weakness and abnormal gait. He had a recurrent episode of muscle weakness and laboratory results showed severe hypokalemia. The direct genomic sequencing of the case revealed a new mutation in the SLC12A1 gene, which is associated with type I Bartter syndrome. Because there was the difference between the phenotype and genotype, we conducted a diuretic loading test to confirm the diagnosis. However, the results showed a clear increase in urine excretion of Na and Cl. These results were not consistent with typical type I BS, but consistent with the patient’s phenotype. Conclusion The diuretic loading test has limited utility for diagnosis especially in atypical cases. On the other hand, this test, which allows assessment of channel function, is useful for better understanding of the genotype-phenotype correlation.

AIM Accurate and precise estimation of glomerular filtration rate (GFR) is essential in kidney disease. We evaluated the usefulness of the mean of creatinine clearance (CCr ) and urea clearance (CUN ) examined over a 1-h urine collection period (1-h (CCr + CUN )/2) in a retrospective, cross-sectional study across two centres, as a relatively simple method for estimating GFR in children. METHODS Children aged ≤18 years who underwent inulin clearance (CIn ) tests were eligible. Two clearance values were obtained during a 2-h test consisting of two periods of 1 h each. The mean clearance in two periods was defined as 1-h clearance. 1-h (CCr + CUN )/2, 1-h CCr , 1-h CUN and GFR estimated by Cr-based and cystatin C (CysC)-based formulas for Japanese children were compared with CIn . Bland-Altman plots were used to evaluate correlations. The primary outcome measure was the correlation between 1-h (CCr + CUN )/2 and CIn . RESULTS Fifty-three children were analysed. Their median age was 10.9 (interquartile range [IQR] 5.3-14.2) years, and median CIn and 1-h (CCr + CUN )/2 were 77.0 (IQR: 51.5-95.1) and 81.0 (IQR: 64.1-97.7) ml/min/1.73 m2 , respectively. Percentage difference of CIn and 1-h (CCr + CUN )/2 in the Bland-Altman plot was -11.2% (95% confidence interval - 15.3% - -7.1%), with 95% lower and upper limits of agreement of -40.3% and 18.0%, respectively. Thus, 1-h (CCr + CUN )/2 was 1.12 times CIn . CONCLUSION 1 h (CCr + CUN )/2 was almost concordant with CIn . 1-h (CCr + CUN )/2 can estimate GFR accurately and precisely, making it a simple and speedy test for use in clinical practice.

Although the concept of chronic kidney disease (CKD) in children is similar to that in adults, pediatric CKD has some peculiarities, and there is less evidence and many factors that are not clearly understood. The past decade has witnessed several additional registry and cohort studies of pediatric CKD and kidney failure. The most common underlying disease in pediatric CKD and kidney failure is congenital anomalies of the kidney and urinary tract (CAKUT), which is one of the major characteristics of CKD in children. The incidence/prevalence of CKD in children varies worldwide. Hypertension and proteinuria are independent risk factors for CKD progression; other factors that may affect CKD progression are primary disease, age, sex, racial/genetic factors, urological problems, low birth weight, and social background. Many studies based on registry data revealed that the risk factors for mortality among children with kidney failure who are receiving kidney replacement therapy are younger age, female sex, non-White race, non-CAKUT etiologies, anemia, hypoalbuminemia, and high estimated glomerular filtration rate at dialysis initiation. The evidence has contributed to clinical practice. The results of these registry-based studies are expected to lead to new improvements in pediatric CKD care.

Severe congenital anomalies of the kidney and urinary tract (CAKUT) progress to infantile kidney failure with replacement therapy (KFRT). Although prompt and precise prediction of kidney outcomes is important, early predictive factors for its progression remain incompletely defined.This retrospective cohort study included patients with CAKUT treated at 12 centers between 2009 and 2020. Patients with a maximum serum creatinine level ≤ 1.0 mg/dL during the first 3 days, patients who died of respiratory failure during the neonatal period, patients who progressed to KFRT within the first 3 days, and patients lacking sufficient data were excluded.Of 2187 patients with CAKUT, 92 were finally analyzed. Twenty-five patients (27%) progressed to KFRT and 24 (26%) had stage 3-5 chronic kidney disease without replacement therapy during the median observation period of 52.0 (interquartile range, 22.0-87.8) months. Among these, 22 (24%) progressed to infantile KFRT. The kidney survival rate during the infantile period was significantly lower in patients with a maximum serum creatinine level during the first 3 days (Cr-day3-max) ≥ 2.5 mg/dL (21.8%) compared with those with a Cr-day3-max 2.5 mg/dL (95.2%) (log-rank, P 0.001). Multivariate analysis demonstrated Cr-day3-max (P 0.001) and oligohydramnios (P = 0.025) were associated with higher risk of infantile KFRT. Eighty-two patients (89%) were alive at the last follow-up.Neonatal kidney function, including Cr-day3-max, was associated with kidney outcomes in patients with severe CAKUT. Aggressive therapy for severe CAKUT may have good long-term life outcomes through infantile dialysis and kidney transplantation. A higher resolution version of the Graphical abstract is available as Supplementary information.

Background The assessment of kidney size is essential for treating kidney disease. However, there are no reliable and sufficiently robust ultrasonographic reference values or prediction formulas for kidney length in Japanese children, based on a sufficient number of participants. Methods We retrospectively analyzed kidney measurements by ultrasonography in children aged 18 years or younger from eight facilities throughout Japan between January 1991 and September 2018. Detailed reference values were developed by aggregating the left and right kidneys of boys and girls separately. Simple and practical reference values were developed by combining all the data from left and right kidneys and boys and girls. The estimation formulas for the average value and lower limit of the normal range for kidney length were developed based on regression analysis. Results Based on the aggregated kidney length data of 1984 participants (3968 kidneys), detailed reference values and simple reference values for kidney length were determined. From the regression analysis, the formula for calculating the average kidney length was generated as “kidney length (cm) = body height (m) × 5 + 2”, and that for predicting the lower limit of normal kidney length in children under 130 cm was calculated as “lower limit (cm) = 0.85 × [body height (m) × 5 + 2]”. Conclusion Detailed ultrasonographic reference values of kidney length for Japanese children and simple reference values and estimation formulas for daily practice have been established.

Background Hepatocyte nuclear factor 1β(HNF1B), located on chromosome 17q12, causes renal cysts and diabetes syndrome (RCAD). Moreover, various phenotypes related to congenital anomalies of the kidney and urinary tract (CAKUT) or Bartter-like electrolyte abnormalities can be caused by HNF1B variants. In addition, 17q12 deletion syndrome presents with multi-system disorders, as well as RCAD. As HNF1B mutations are associated with different phenotypes and genotype–phenotype relationships remain unclear, here, we extensively studied these mutations in Japan. Methods We performed genetic screening of RCAD, CAKUT, and Bartter-like syndrome cases. Heterozygous variants or whole-gene deletions in HNF1B were detected in 33 cases (19 and 14, respectively). All deletion cases were diagnosed as 17q12 deletion syndrome, confirmed by multiplex ligation probe amplification and/or array comparative genomic hybridization. A retrospective review of clinical data was also conducted. Results Most cases had morphological abnormalities in the renal–urinary tract system. Diabetes developed in 12 cases (38.7%). Hyperuricemia and hypomagnesemia were associated with six (19.3%) and 13 cases (41.9%), respectively. Pancreatic malformations were detected in seven cases (22.6%). Ten patients (32.3%) had liver abnormalities. Estimated glomerular filtration rates were significantly lower in the patients with heterozygous variants compared to those in patients harboring the deletion (median 37.6 vs 58.8 ml/min/1.73 m2; p = 0.0091). Conclusion We present the clinical characteristics of HNF1B-related disorders. To predict renal prognosis and complications, accurate genetic diagnosis is important. Genetic testing for HNF1B mutations should be considered for patients with renal malformations, especially when associated with other organ involvement.

Background Malignancy after kidney transplantation (KT) is one of the most serious post-transplant complications. This study aimed to investigate the incidence, type, and outcomes of malignancy after pediatric KT. Methods We performed a retrospective cohort study on pediatric kidney transplant recipients aged 18 years or younger who received their first transplant between 1975 and 2009. Results Among the 375 children who underwent KT, 212 were male (56.5%) and 163 were female (43.5%) (median age at KT, 9.6 years [interquartile range {IQR}] 5.8–12.9 years). The incidence of malignancy was 5.6% (n = 21). The cumulative incidences of cancer were 0.8%, 2.5%, 2.8%, 4.2%, 5.5%, and 15.6% at 1, 5, 10, 15, 20, and 30 years post-transplantation, respectively. Of 375 patients, 12 (3.2%) had solid cancer and nine (2.4%) had lymphoproliferative malignancy. The median age at the first malignancy was 21.3 years (IQR 11.5–33.3 years). The median times from transplant to diagnosis were 22.3 years (IQR 12.3–26.6 years) for solid cancer and 2.2 years (IQR 0.6–2.8) for lymphoproliferative malignancies. During follow-up, five recipients died due to malignancy. The causes of death were hepatocellular carcinoma in one patient, squamous cell carcinoma in the transplanted kidney in one patient, malignant schwannoma in one patient, and Epstein-Barr virus-related lymphoma in two patients. The mortality rate was 0.79 per 1000 person-years (95% confidence interval 0.38, 1.85). Conclusions Early diagnosis and treatment of malignancies in transplant recipients is an important challenge. Therefore, enhanced surveillance and continued vigilance for malignancy following KT are necessary.

Kidney transplantation, and preferably preemptive transplantation, is widely accepted as the optimal mode of renal replacement therapy (RRT) in all children with end-stage kidney disease (ESKD). For this reason, data on the long-term outcome of dialysis in children are scarce. Yet, as most patients with ESKD have to undergo chronic dialysis while waiting for kidney transplantation, and some patients require dialysis for a long time, it is of imminent importance to evaluate the impact of long-term dialysis on the physical and psychosocial condition and development of children with ESKD. This chapter reviews the potential hazards of long-term dialysis in children based on pathophysiology, adult data and the scarce pediatric epidemiological data that exist on the topic. While mortality in patients with pediatric-onset ESKD during the course of peritoneal dialysis (PD) and hemodialysis (HD) has decreased over the recent decades, mortality risk remains extremely high as compared to the general population. The morbidity associated with long-term dialysis is also examined in this chapter, focusing on cardiovascular disease, chronic kidney disease-mineral bone disorder, psychosocial development, and cognitive functioning. We also pay special attention to one of the most life-threatening late technical complications of PD: encapsulating peritoneal sclerosis (EPS).

No reports describe high-dose chemotherapy (HDCT) with autologous peripheral blood stem cell transplantation (auto-PBSCT) in pediatric patients with neuroblastoma and end-stage renal disease. Here, we report the case of a patient with high-risk neuroblastoma who developed anuria during treatment. HDCT with auto-PBSCT under hemodialysis, with strict attention to the ultrafiltration volume and dose modification of alkylating agents, was performed. Although the first auto-PBSCT led to engraftment failure, the second auto-PBSCT resulted in successful myeloid engraftment 8 months after anuria. This case demonstrated that HDCT with auto-PBSCT can be safely performed in children with renal failure under hemodialysis.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (P(meta)=6.71E-28, OR=1.88) and TNFSF15 (P(meta)=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).

Objectives To investigate the frequency of vesicoureteral reflux, and the relationship of pretransplant decreased bladder capacity and post-transplant vesicoureteral reflux in children undergoing kidney transplantation. Methods A voiding cystourethrography was carried out in 172 pediatric kidney transplantation recipients before, and 4 months after, transplantation to evaluate bladder capacity and vesicoureteral reflux. The correlation of post-transplant vesicoureteral reflux with pretransplant bladder capacity, vesicoureteral reflux in the native kidney and the method of ureteral reimplantation (intravesical/extravesical) was analyzed. Atrophic bladder was defined as having ≤50% functional bladder capacity (age in years + 2) × 25 (mL) or ≤150 mL in patients aged >10 years. Results Bladder capacity increased remarkably after transplantation in both post-transplant vesicoureteral reflux- group (from 180 to 253 mL) and vesicoureteral reflux+ group (from 82 to 171 mL). Voiding cystourethrography showed vesicoureteral reflux in 12 cases of kidney transplantation (7%; grade 1: 2, grade 2: 3, grade 3: 7). Pretransplant atrophic bladder was an independent risk factor of post-transplant vesicoureteral reflux (P = 0.004, hazard ratio 9.5). There was no difference in renal function between the vesicoureteral reflux- group and vesicoureteral reflux+ group at 4 months to 5 years post-transplantation. Conclusions Pretransplant atrophic bladder is a risk factor of post-transplant vesicoureteral reflux in pediatric patients. However, bladder capacity can remarkably increase after transplantation, and kidney function in the post-transplant vesicoureteral reflux+ group is stable.

Aim Recently eculizumab, a monoclonal antibody to C5, was found to improve the disease course of atypical haemolytic uraemic syndrome (aHUS) and has been recommended as the first line treatment by an international consensus guideline. However, several practical issues in the use of eculizumab for the acute phase of aHUS have yet to be resolved. Methods Children who received eculizumab with diagnosis of aHUS between March 2010 and December 2015 at Tokyo Metropolitan Children's Medical Center were enrolled. aHUS was diagnosed according to the haemolytic uraemic syndrome (HUS) criteria after excluding Shiga toxin-inducing Escherichia coli (STEC) -associated HUS and thrombocytopaenic purpura. We retrieved and analyzed data from the electronic medical records at our institution. Results We reviewed four patients with suspected aHUS. Eculizumab was discontinued in one patient in whom STEC-HUS was later diagnosed. Treatment was continued in the remaining three patients without recurrence. Practical issues included difficulty in diagnosing aHUS, particularly in the acute phase, risk of infection by encapsulated organisms, especially Neisseria meningitis, and infusion reaction. In addition to issues relating to the acute phase, discontinuing eculizumab in stable patients in the chronic phase must be considered. Conclusion Eculizumab, the first line treatment for children with aHUS, is usually effective. However, certain problems associated with its use require caution to be exercised. As clinical information on eculizumab are still very limited, and the rationale for its long-term use has yet to be established, physicians are advised to exercise care when using eculizumab to manage aHUS.

In pediatric patients, due to variations in baseline serum creatinine (Cr) reference values, renal dysfunctions sometimes go unnoticed. In addition, renally excreted drugs need dose adjustment while nephrotoxic drugs should be avoided altogether in patients with impaired renal function. However, most physicians are apparently unaware of these facts and may administer these drugs to vulnerable patients. We administered a questionnaire to all physicians and pharmacists specializing in pediatric medical care at six Tokyo metropolitan government-run hospitals in Japan. 276 (59%) of 470 physicians and pharmacists participated. The rate of correct answers given by physicians who were asked to state the serum Cr reference range for 4-year-olds and 8-year-olds was 83 and 74%, respectively. On the other hand, the rate of correct answers given by pharmacists to the same question was only 27 and 24%, respectively. Only about 50% of physicians were aware that histamine H2-receptor antagonists and oseltamivir are renally excreted or that acyclovir and angiotensin II receptor blocker are nephrotoxic. However, most of the pharmacists recognized that histamine H2-receptor antagonists and oseltamivir are renally excreted drugs. For the majority of the investigated drugs, the awareness that we need to reduce dosages for patients with renal dysfunction was insufficient. To ensure safe drug administration, communication between physicians and pharmacists is paramount. There is an urgent need for the creation of a safe drug administration protocol for pediatric patients with renal dysfunction.

Summary Introduction Kidney transplantation (KTx) is the most effective treatment for end-stage renal disease in children. Objectives We aimed to compare the long-term outcomes and surgical complications of the intraperitoneal approach (IPA) and extraperitoneal approach (EPA) for KTx in children weighing Study design We performed a retrospective cohort study on pediatric kidney transplant recipients, weighing Results The median follow-up duration was 14.1 years (interquartile range, 9.0–19.2). Comparing the two groups (IPA group, n = 62; EPA group, n = 38), the median age and body weight were significantly lower in the IPA group (4.2 vs. 4.8 years, P = 0.03; 11.7 vs. 13.0 kg, P Discussion We assessed the long-term outcomes of the surgical approaches used for pediatric patients weighing Conclusion The transplant approach did not influence the long-term outcomes in children weighing Summary Table . Comparison of surgical complications by surgical approach. Variables IPA group (n = 62) EPA group (n = 38) P-value Early surgical complications 15 (24%) 1 (3%) Urinary leak 3 0 Urinary stenosis 1 0 Ileus 2 0 Renal artery thrombosis 1 0 Renal vein thrombosis 1 0 Anastomotic bleeding 1 0 Lymphocele 1 0 Wound infection 1 0 Renal allograft compression syndrome 4 1 Late surgical complications 9 (15%) 1 (3%) 0.08 Ileus 6 0 Bowel obstruction 2 0 Urinary stenosis 0 1 Bladder stone (due to sutures) 1 0 Total 24 (39%) 2 (6%) Surgical reintervention 10 (16%) 1 (3%) 0.05 EPA, extraperitoneal approach; IPA, intraperitoneal approach; KTx, kidney transplantation. Values are presented as numbers (%). Early surgical complications were identified within 30 days post-KTx. Late surgical complications were defined as complications occurring more than 1 month post-KTx.

Null variants in LAMB2 cause Pierson syndrome (PS), a severe congenital nephrotic syndrome with ocular and neurological defects. Patients' kidney specimens show complete negativity for laminin β2 expression on glomerular basement membrane (GBM). In contrast, missense variants outside the laminin N-terminal (LN) domain in LAMB2 lead to milder phenotypes. However, we experienced cases not showing these typical genotype–phenotype correlations. In this paper, we report six PS patients: four with mild phenotypes and two with severe phenotypes. We conducted molecular studies including protein expression and transcript analyses. The results revealed that three of the four cases with milder phenotypes had missense variants located outside the LN domain and one of the two severe PS cases had a homozygous missense variant located in the LN domain; these variant positions could explain their phenotypes. However, one mild case possessed a splicing site variant (c.3797 + 5G>A) that should be associated with a severe phenotype. Upon transcript analysis, this variant generated some differently sized transcripts, including completely normal transcript, which could have conferred the milder phenotype. In one severe case, we detected the single-nucleotide substitution of c.4616G>A located outside the LN domain, which should be associated with a milder phenotype. However, we detected aberrant splicing caused by the creation of a novel splice site by this single-base substitution. These are novel mechanisms leading to an atypical genotype–phenotype correlation. In addition, all four cases with milder phenotypes showed laminin β2 expression on GBM. We identified novel mechanisms leading to atypical genotype–phenotype correlation in PS.

OBJECTIVES To evaluate long-term outcomes and risk factors for graft loss in pediatric kidney transplantation over a 30-year period. METHODS We retrospectively assessed 400 consecutive kidney transplants carried out in 377 children during 1975-2009. Patients were stratified according to the immunosuppressive regimen (era 1: methylprednisolone and azathioprine; era 2: calcineurin inhibitor-based therapy, including methylprednisolone and azathioprine or mizoribine; era 3: basiliximab induction therapy, including calcineurin inhibitors, methylprednisolone and mycophenolate mofetil). RESULTS The median age and bodyweight at transplantation were 9.7 years and 20.6 kg, respectively. In total, 364 (91.0%) children received a living related donor transplantation. The acute rejection rate within 1 year post-transplant decreased significantly from 61.0% in era 1 to 14.5% in era 3 (P

Background Despite advances in non-invasive vascular imaging, detection of renal artery stenosis via catheter angiography is the criterion standard for the diagnosis of renovascular hypertension (RVH). However, because of lack of evidence, the utility of various blood tests and imaging modalities remains unclear. Methods We retrospectively analyzed the utility of blood tests (plasma renin activity [PRA], aldosterone, and renal vein renin [RVR] values) and imaging studies (computed tomography angiography [CTA], kidney ultrasonography [US]) by comparing them with catheter angiography. Ten pediatric patients with RVH at two institutions from January 2008 to December 2017 were recruited. The sensitivities for diagnosing RVH via imaging and blood tests (kidney [US], PRA, and aldosterone) were derived by examining patient records. Furthermore, the sensitivity and specificity of CT angiography were calculated by considering both the affected and non-affected renal arteries of the patients. Results A high sensitivity for diagnosing RVH via kidney US (89%) and PRA (80%) was observed. The sensitivity and specificity of CTA were 100%, each. RVR sampling did not aid in the diagnosis of RVH; only two of six patients with unilateral RVH showed significant laterality of RVR boundary ratios. Renal scintigraphy facilitated detection of a non-functional kidney (split renal function Conclusions RVH in children could be diagnosed utilizing non-invasive blood and imaging tests, without catheter angiography. We recommend kidney length measurement along with measurement of PRA level, as a simple and highly useful screening test, followed by CTA as a diagnostic test.

Histopathological findings can play an important role in the management of atypical haemolytic uraemic syndrome (aHUS). We report a case of aHUS that did not recover from anuria, despite the administration of eculizumab, with impressive histopathological findings. A 3-month-old girl was admitted because of poor feeding, vomiting, and diarrhoea without haemorrhage. She had anuria and severe hypertension, and laboratory results showed haemolytic anaemia with schizocytes, thrombocytopenia, and renal impairment. Although no mutations in the complement system or diacylglycerol kinase epsilon were detected, she was diagnosed with aHUS owing to the clinical course and by the exclusion of Escherichia coli infection and thrombotic thrombocytopenic purpura. Plasma exchange was performed once at day 2 and eculizumab therapy was started from day 18, with a severe infusion reaction at the first administration. After the initiation of eculizumab, although the serum lactate dehydrogenase level improved gradually, she did not recover from anuria. Pathological findings of the kidney biopsy at day 37 included diffuse arteriolar and arterial luminal stenosis with remarkable thickness and sclerotic changes of the media and intima, which are suggestive of aHUS. In addition, most glomeruli had global sclerosis and were collapsed, and 80% of the tubulointerstitial compartment showed atrophic changes with infiltration of inflammatory cells. The present case is possibly a kidney-specific fulminant type of aHUS. Although showing efficacy against thrombotic microangiopathy, eculizumab did not improve kidney function. The pathological findings reflected the severe and irreversible kidney injury.

Vesicoureteral reflux (VUR) is associated with an increased risk of kidney disorders. It is unclear whether VUR is associated with progression from chronic kidney disease (CKD) to end-stage kidney disease (ESKD) in children with congenital anomalies of the kidney and urinary tract (CAKUT). We conducted a 3-year follow-up survey of a cohort of 447 children with CKD (stage 3–5). Rates of and risk factors for progression to ESKD were determined using the Kaplan–Meier method and Cox regression respectively. Congenital anomaly of the kidney and urinary tract was the primary etiology in 278 out of 447 children; 118 (42.4 %) had a history of VUR at the start of the cohort study. There were significantly more boys than girls with VUR, whereas the proportions were similar in children without VUR. The types of urinary anomalies/complications of the two groups were significantly different. Three-year renal survival rates of the groups were not significantly different, irrespective of CKD stage. Age

Aim C3 glomerulonephritis (C3GN) is a recently described disease that is related to membranoproliferative glomerulonephritis (MPGN). We retrospectively compared the frequencies, clinical characteristics, treatment modalities, and outcomes of C3GN and MPGN in a cohort of Japanese children. Methods Children who were pathologically diagnosed with MPGN (type I or III) in our hospital were divided into two groups based on immunofluorescence imaging of renal biopsies: children with MPGN induced by classical complement pathway activation (classical MPGN) and children with C3GN. Results Of 14 children with MPGN (five boys), four had classical MPGN, eight had C3GN, and two had unclassifiable glomerulonephritis. Four children with classical MPGN and seven with C3GN received methylprednisolone pulse therapy followed by oral prednisolone for 2 years (MPT+PSL therapy). Subsequently, six of seven children with C3GN received combined therapy (prednisolone, azathioprine, and anticoagulants) for 2 years because they responded poorly to MPT+PSL therapy. At the last follow-up visit, two children with classical MPGN and seven with C3GN had not achieved remission. One child with classical MPGN and five with C3GN had hypocomplementaemia at the last follow-up. None of the children had renal impairment. Conclusion More than half of the patients previously diagnosed with MPGN fulfilled the criteria for C3GN in children. C3GN may be more refractory than classical MPGN to immunosuppressant therapy.

The long-term outcome of pediatric IgA nephropathy (IgAN) is unclear. Objective IgAN remission criteria were proposed by the Japanese Society of Nephrology in 2013. Children with newly developed IgAN followed for >5 years were analyzed. They were divided into two groups based on histological findings at initial kidney biopsy: the focal mesangial proliferation group (Focal group) and diffuse mesangial proliferation group (Diffuse group). The primary outcome was the remission rate according to the newly proposed IgAN remission criteria. The patients comprised 53 children (31 boys; mean age at IgAN onset, 10.0 years). The Focal and Diffuse groups comprised 21 and 32 patients, respectively. No significant differences in patient characteristics were found between the groups except for steroid administration. The median follow-up period from onset was 9.9 years. Sixteen patients in the Diffuse group and 10 in the Focal group had not achieved remission at the last observation. Patient conditions 2 years after the initial treatment were almost identical to those at the last observation. Multivariate analysis revealed that proteinuria, particularly

Recently eculizumab, a monoclonal antibody to C5, was found to improve the disease course of atypical haemolytic uraemic syndrome (aHUS) and has been recommended as the first line treatment by an international consensus guideline. However, several practical issues in the use of eculizumab for the acute phase of aHUS have yet to be resolved.Children who received eculizumab with diagnosis of aHUS between March 2010 and December 2015 at Tokyo Metropolitan Children's Medical Center were enrolled. aHUS was diagnosed according to the haemolytic uraemic syndrome (HUS) criteria after excluding Shiga toxin-inducing Escherichia coli (STEC) -associated HUS and thrombocytopaenic purpura. We retrieved and analyzed data from the electronic medical records at our institution.We reviewed four patients with suspected aHUS. Eculizumab was discontinued in one patient in whom STEC-HUS was later diagnosed. Treatment was continued in the remaining three patients without recurrence. Practical issues included difficulty in diagnosing aHUS, particularly in the acute phase, risk of infection by encapsulated organisms, especially Neisseria meningitis, and infusion reaction. In addition to issues relating to the acute phase, discontinuing eculizumab in stable patients in the chronic phase must be considered.Eculizumab, the first line treatment for children with aHUS, is usually effective. However, certain problems associated with its use require caution to be exercised. As clinical information on eculizumab are still very limited, and the rationale for its long-term use has yet to be established, physicians are advised to exercise care when using eculizumab to manage aHUS.

Background Proteinuria is the most important risk factor for IgA nephropathy progression. The purpose of this study is to evaluate the long-term outcome and risk factors for poor prognosis in childhood IgA nephropathy. Methods Patients who were diagnosed with IgA nephropathy between 1972 and 1992 at the Tokyo Metropolitan Kiyose Children’s Hospital were included. We analyzed risk factors for progression to end-stage kidney disease (ESKD) and chronic renal insufficiency (CRI) using Kaplan-Meier method and multivariate analyses of Cox proportional hazard model. Results One hundred patients were included and the median observation period was 11.8 years. Twelve and 17 patients progressed to ESKD and CRI, respectively. The survival probabilities were 90.0% at 10 years and 79.8% at 20 years for ESKD, and 86.1% at 10 years and 72.3% at 20 years for CRI. Notably, patients with heavy proteinuria with hypoalbuminemia during follow-up period showed extremely poor prognosis. In this group, the survival rate at 10 years from ESKD and CRI was 40.6% and 20.8%, respectively. By multivariate analysis, proteinuria at diagnosis and proteinuria during follow-up period were risk factors for ESKD, whereas glomeruli showing mesangial proliferation ≥50% and proteinuria during follow-up period were risk factors for CRI. Patients without heavy proteinuria during follow-up period did not develop CRI and 63% of patients with mild proteinuria during follow-up period showed no proteinuria at the last observation. Conclusions The degree of proteinuria during follow-up period is the strongest risk factor for ESKD and CRI.

The p110 Cut homeobox 1 (CUX1) transcription factor regulates genes involved in DNA replication and chromosome segregation. Using a genome-wide-approach, we now demonstrate that CUX1 also modulates the constitutive expression of DNA damage response genes, including ones encoding ATM and ATR, as well as proteins involved in DNA damage-induced activation of, and signaling through, these kinases. Consistently, RNAi knockdown or genetic inactivation of CUX1 reduced ATM/ATR expression and negatively impacted hallmark protective responses mediated by ATM and ATR following exposure to ionizing radiation (IR) and UV, respectively. Specifically, abrogation of CUX1 strongly reduced ATM autophosphorylation after IR, in turn causing substantial decreases in (i) levels of phospho-Chk2 and p53, (ii) γ-H2AX and Rad51 DNA damage foci and (iii) the efficiency of DNA strand break repair. Similarly remarkable reductions in ATR-dependent responses, including phosphorylation of Chk1 and H2AX, were observed post-UV. Finally, multiple cell cycle checkpoints and clonogenic survival were compromised in CUX1 knockdown cells. Our results indicate that CUX1 regulates a transcriptional program that is necessary to mount an efficient response to mutagenic insult. Thus, CUX1 ensures not only the proper duplication and segregation of the genetic material, but also the preservation of its integrity.

The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).

Sasaki K, Kasahara M, Fukuda A, Shigeta T, Tanaka H, Nakagawa S, Mitsui K, Harada R, Nakagawa A. Living donor liver transplantation for hepatoblastoma with Beckwith–Wiedemann syndrome. Pediatr Transplantation 2010: 14:E89–E92. © 2009 John Wiley & Sons A/S. Abstract: BWS is one of the most well-known somatic overgrowth syndromes, which is characterized by macroglossia, organomegaly, abdominal wall defects, and predisposition to embryonal tumors, such as Wilms’ tumor, hepatoblastoma, and adrenocortical carcinoma. We report a case of BWS in a girl with unresectable hepatoblastoma, who received a planned LVDT following neo-adjuvant chemotherapy. This is the first case report of liver transplantation for patients with BWS. Tumor surveillance after transplantation would be necessary to detect possible recurrence of the original disease and development of other malignancies.

Small GTP-binding proteins of the Ras superfamily regulate a wide array of cellular functions and play critical roles in signal transduction, gene regulation, intracellular trafficking of vesicles, cell proliferation, and survival (37). Proteins in the Rho subfamily (Rho, Rac, and Cdc42) are best characterized for their effects on actin polymerization and cytoskeleton organization (15). Through their effects on the cytoskeleton, Rho GTPases are essential in cell morphology, cell migration and chemotaxis, and establishment of cell polarity (6, 18, 44). They also participate in several aspects of cell cycle progression. During mitosis, Cdc42 regulates spindle microtubule attachment to kinetochores in metaphase, whereas RhoA, which localizes at the cleavage furrow during telophase and ends up at the midbody at the abcission step, is involved in cytokinesis (25, 43, 59). Indeed, interfering with RhoA activity, by treating cells with Clostridium botulinum C3 exoenzyme, or with RhoA expression, by small interfering RNA (siRNA) silencing, prevents contraction of the actomyosin ring and leads to failure of cytokinesis and multinucleation in various cell types (25, 60). During these steps, RhoA activity is regulated by the exchange factor Ect2 and the GTPase activating protein MgcRacGAP (hereafter, RacGAP) which colocalize with a kinesin known as MKLP1, Kif23, or kinesin-like protein 5 (35, 52, 61). MKLP1 associates with RacGAP in a complex known as centralspindlin and is thought to mediate recruitment of RacGAP to the mitotic spindle. Protein interactions between Ect2 and RacGAP lead to conformational changes that activate the exchange activity of Ect2 (20, 26). During mitosis progression, dynamic protein interactions between Ect2, RacGAP, and MKLP1 are regulated by phosphorylation/dephosphorylation events mediated by the Cdk1 and Aurora B kinases (34, 36, 53). The dynamics and functions of this complex are evolutionarily conserved as the Drosophila orthologs (Pebble/RacGAP50/Pavarotti) and the Caenorhabditis elegans orthologs (LET-21/CYK4/ZEN4) also control cytokinesis with similar regulations. Genes whose protein products play specific roles in cell cycling and exert their functions at defined steps of cycle progression are often regulated in a cell cycle-dependent manner (7, 14). These genes include not only the well-characterized mitotic cyclins and their associated kinases or inhibitors but also additional kinases (e.g., Plk-1 and Aurora) and components of the anaphase promoting complex/cyclosome (3, 56, 58). Promoters for cell cycle-regulated genes contain binding sites for specific transcription factors (14). The E2F family contains eight identified members acting as transcriptional activators or repressors (11). E2F1 was initially characterized as an activator of genes expressed at the G1/S transition to regulate cell cycle progression but was also shown to regulate G2/M genes as well as the apoptosis pathway (19, 62). Besides its coactivator DP, E2F may interact with other transcription factors such as B-Myb, itself an E2F target in G1/S (24, 45, 62). CUX1 (Cut homeobox 1) belongs to the family of Cut homeodomain-containing transcription factors that is present in all metazoans. In vertebrates, the protein was first identified as the CCAAT-displacement protein and has been variously called CDP, CUX, or CUTL1 (2, 41, 57). Whereas the CCAAT-displacement activity, which depends on rapid but transient interactions with DNA, appears to be constant throughout the cell cycle, a more stable interaction with DNA arises at the end of the G1 phase as the result of dephosphorylation by Cdc25A and proteolytic processing by a nuclear isoform of cathepsin L (8, 17, 38). The N-terminally truncated isoform thus generated, p110 CUX1, was shown to accelerate entry into S phase (46). The regulatory effect of p110 CUX1 was found to be promoter dependent: it repressed the p21cip and cyclin H genes but activated most genes including those coding for cyclin A2, DNA polymerase (Pol) α, and the Cdc25 proteins (8, 21, 46, 55). Another signature of cell cycle-regulated genes is the frequent occurrence of cell cycle-dependent element/cycle homology region (CDE/CHR) sequences close to the transcription start site. Although factors binding to such elements have not yet been unambiguously characterized, their functionality has clearly been established by mutation or deletion analysis in several cell cycle genes such as cdc25C, Cyclin A, or aurora B (22, 27, 29, 30). We report here that the mitotic complex genes Ect2, RacGAP, and MKLP1 are coordinately induced in S phase in proliferating T lymphocytes as well as in epithelial cells, depending upon activity of the CUX1 and E2F1 transcription factors. We have also identified CHR elements that mediate repression of these promoters in the G1 phase of the cell cycle.

The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase alpha gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase alpha gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).

Histopathological findings can play an important role in the management of atypical haemolytic uraemic syndrome (aHUS). We report a case of aHUS that did not recover from anuria, despite the administration of eculizumab, with impressive histopathological findings. A 3-month-old girl was admitted because of poor feeding, vomiting, and diarrhoea without haemorrhage. She had anuria and severe hypertension, and laboratory results showed haemolytic anaemia with schizocytes, thrombocytopenia, and renal impairment. Although no mutations in the complement system or diacylglycerol kinase epsilon were detected, she was diagnosed with aHUS owing to the clinical course and by the exclusion of Escherichia coli infection and thrombotic thrombocytopenic purpura. Plasma exchange was performed once at day 2 and eculizumab therapy was started from day 18, with a severe infusion reaction at the first administration. After the initiation of eculizumab, although the serum lactate dehydrogenase level improved gradually, she did not recover from anuria. Pathological findings of the kidney biopsy at day 37 included diffuse arteriolar and arterial luminal stenosis with remarkable thickness and sclerotic changes of the media and intima, which are suggestive of aHUS. In addition, most glomeruli had global sclerosis and were collapsed, and 80% of the tubulointerstitial compartment showed atrophic changes with infiltration of inflammatory cells. The present case is possibly a kidney-specific fulminant type of aHUS. Although showing efficacy against thrombotic microangiopathy, eculizumab did not improve kidney function. The pathological findings reflected the severe and irreversible kidney injury.

No large cohort study has yet determined the incidence of acute kidney injury (AKI) in children with heart failure treated with renin-angiotensin system (RAS) inhibitors. We thus retrospectively analyzed the incidence and risk factors for severe AKI (stages 2-3 according to the Kidney Disease Improving Global Outcomes (KDIGO) guidelines) at our institutions from 2008 to 2011. Among 312 children (162 boys; median age, 7.3 months), 59 cases of AKI occurred in 45 children. The incidence of AKI was 14.3 cases per 100 person-years overall (follow-up 413.6 person-years), or 27.3, 16.8, and 4.5 cases per 100 person-years in children aged1, 1-3, and ≥4 years, respectively. Among them, 23 (39.0 %) children had metabolic acidosis and 14 (23.7 %) had hyperkalemia. Younger age, myocardial disease, cyanotic congenital heart disease, use of spironolactone, and cardiac surgery were independent risk factors for AKI. Furthermore, 37.3 % of children suffered dehydration during AKI.AKI incidence is relatively high in children, particularly younger children, with heart failure treated using RAS inhibitors. Careful monitoring of renal function and serum electrolytes is essential. Proper management of fluid balance after infection and cardiac surgery may reduce the risk of AKI. Temporary discontinuation in RAS inhibitors should be considered during dehydration or surgery.• Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are the two main classes of renin-angiotensin system (RAS) inhibitors used to treat hypertension, heart failure, and chronic kidney disease. Acute kidney injury (AKI) and hyperkalemia are potentially life-threatening complications associated with the use of ACEIs and ARBs. Some reports have suggested that dehydration and cardiac surgery are risk factors for AKI in children. However, no large-scale cohort studies have determined the incidence of AKI, its risk factors, and its outcomes in children with heart failure treated with ACEIs and/or ARBs. What is new: • In this retrospective cohort study, we determined the incidence, severity, and risk factors for severe AKI in children with heart failure treated with ACEIs and/or ARBs. The incidence of AKI in these children was relatively high (14.3 episodes per 100 person-years). In addition, younger age, myocardial disease, cyanotic congenital heart disease, concomitant use of spironolactone, and cardiac surgery were risk factors for AKI. Furthermore, 37.3 % of children had dehydration during AKI episodes. • Our results suggested that appropriate fluid balance after infection and cardiac surgery might reduce the risk of AKI and its complications. Temporary discontinuation or reductions in the levels of ACEIs and/or ARBs during dehydration or before surgery may also be warranted in these patients.

C3 glomerulonephritis (C3GN) is a recently described disease that is related to membranoproliferative glomerulonephritis (MPGN). We retrospectively compared the frequencies, clinical characteristics, treatment modalities, and outcomes of C3GN and MPGN in a cohort of Japanese children.Children who were pathologically diagnosed with MPGN (type I or III) in our hospital were divided into two groups based on immunofluorescence imaging of renal biopsies: children with MPGN induced by classical complement pathway activation (classical MPGN) and children with C3GN.Of 14 children with MPGN (five boys), four had classical MPGN, eight had C3GN, and two had unclassifiable glomerulonephritis. Four children with classical MPGN and seven with C3GN received methylprednisolone pulse therapy followed by oral prednisolone for 2 years (MPT+PSL therapy). Subsequently, six of seven children with C3GN received combined therapy (prednisolone, azathioprine, and anticoagulants) for 2 years because they responded poorly to MPT+PSL therapy. At the last follow-up visit, two children with classical MPGN and seven with C3GN had not achieved remission. One child with classical MPGN and five with C3GN had hypocomplementaemia at the last follow-up. None of the children had renal impairment.More than half of the patients previously diagnosed with MPGN fulfilled the criteria for C3GN in children. C3GN may be more refractory than classical MPGN to immunosuppressant therapy.

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

Background Overexpression of the Cut homeobox 1 gene, CUX1, inverselycorrelates with patient survival in breast cancers. Cell-based assays andmolecular studies have revealed that transcriptional regulation byCUX1 involves mostly the proteolytically processed p110isoform. As there is no antibody specific to p110 CUX1 only, an alternatestrategy must be employed to identify its targets. Results We expressed physiological levels of a tagged-p110 CUX1 protein and performedchromatin affinity purification followed by hybridization on ENCODE andpromoter arrays. Targets were validated by chromatin immunoprecipitation andtranscriptional regulation by CUX1 was analyzed in expression profiling andRT-qPCR assays following CUX1 knockdown or p110 CUX1 overexpression.Approximately 47% and 14% of CUX1 binding sites were respectively mappedless than 4 Kbp, or more than 40 Kbp, away from a transcription start site.More genes exhibited changes in expression following CUX1 knockdown thanp110 CUX1 overexpression. CUX1 directly activated or repressed 7.4% and 8.4%of putative targets identified on the ENCODE and promoter arraysrespectively. This proportion increased to 11.2% for targets with 2 bindingsites or more. Transcriptional repression was observed in a slightly higherproportion of target genes. The CUX1 consensus binding motif, ATCRAT, wasfound at 47.2% of the CUX1 binding sites, yet only 8.3% of the CUX1consensus motifs present on the array were bound in vivo. Thepresence of a consensus binding motif did not have an impact on whether atarget gene was repressed or activated. Interestingly, the distance betweena binding site and a transcription start site did not significantly reducedthe ability of CUX1 to regulate a target gene. Moreover, CUX1 not only wasable to regulate the next adjacent gene, but also regulated the gene locatedbeyond this one as well as the gene located further away in the oppositedirection. Conclusion Our results demonstrate that p110 CUX1 can activate or repress transcriptionwhen bound at a distance and can regulate more than one gene on certaingenomic loci.

Chromatin immunoprecipitation (ChIP) has become an essential assay in the field of transcriptional regulation. It is currently the most popular method to monitor the in vivo interaction between a protein and specific genomic sites. The method can also serve to identify novel transcriptional targets when the immunoprecipitated chromatin, sometimes called chipped DNA, is used either as a probe in hybridization experiments with microarrays of genomic DNA (ChIP-chip) or as template in DNA sequencing (ChIP-Seq). ChIP assays rely on the availability of good antibodies that can specifically and efficiently immunoprecipitate the protein under study even after cross-linking. However, good antibodies are not always available. To circumvent this problem, we have developed and validated the method of chromatin affinity purification (ChAP). The subsequent microarray analysis is then referred to as ChAP-chip. In brief, the protein under study is expressed together with two tags in order to allow the purification of chromatin by tandem affinity purification. To ensure that only true targets are identified, it is important to express the recombinant tagged-protein at physiological level. This requirement is not trivial as most expression vectors are designed to express proteins at high levels. We found most convenient to use an inducible retroviral vector in the absence of inducer and transactivator protein. We describe the procedure to generate cells stably expressing recombinant tagged-proteins at physiological level and then to purify the associated chromatin by affinity purification. Targets identified in this manner were validated in independent ChAP assays as well as in ChIP assays using antibodies against the endogenous protein.

Chromatin immunoprecipitation (ChIP) has become an essential assay in the field of transcriptional regulation. It is currently the most popular method to monitor the in vivo interaction between a protein and specific genomic sites. The method can also serve to identify novel transcriptional targets when the immunoprecipitated chromatin, sometimes called chipped DNA, is used either as a probe in hybridization experiments with microarrays of genomic DNA (ChIP-chip) or as template in DNA sequencing (ChIP-Seq). ChIP assays rely on the availability of good antibodies that can specifically and efficiently immunoprecipitate the protein under study even after cross-linking. However, good antibodies are not always available. To circumvent this problem, we have developed and validated the method of chromatin affinity purification (ChAP). The subsequent microarray analysis is then referred to as ChAP-chip. In brief, the protein under study is expressed together with two tags in order to allow the purification of chromatin by tandem affinity purification. To ensure that only true targets are identified, it is important to express the recombinant tagged-protein at physiological level. This requirement is not trivial as most expression vectors are designed to express proteins at high levels. We found most convenient to use an inducible retroviral vector in the absence of inducer and transactivator protein. We describe the procedure to generate cells stably expressing recombinant tagged-proteins at physiological level and then to purify the associated chromatin by affinity purification. Targets identified in this manner were validated in independent ChAP assays as well as in ChIP assays using antibodies against the endogenous protein.

A girl was born with sclerocornea of the right eye, corneal staphyloma of the left eye and lumbar myelomeningocele. The myelomeningocele was repaired soon after birth. The corneal staphyloma was perforated during infancy. She received keratoplasty and achieved light perception. Her right kidney revealed multicystic dysplasia and was non-functioning at birth. She had neurogenic bladder, and her renal function deteriorated gradually. Peters plus syndrome was diagnosed based on anterior ocular segment anomalies, short stature, developmental delay and characteristic face. Anterior ocular segment anomalies are rare findings, but seem to be occasionally associated with spina bifida and renal anomalies. Myelomeningocele and chronic renal failure in patients with Peters plus syndrome have not been reported to our knowledge.

Background Lats1 (large tumor suppressor 1) codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog wts (warts) in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of Lats1 inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation. Results Genome-wide location arrays in cell lines of various cell types revealed that Lats1 was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of Lats1. Expression of Lats1 was reduced in cux1-/- MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of Lats1 was sufficient to confer transcriptional activation by CUX1. Lats1 was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1. Conclusion While inactivation of Lats1/wts in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that Lats1 is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of LATS1 in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be associated with either loss-of-function or overexpression of Lats1.