Florian Brinkert

Twenty-one additional cases of familial renal glucosuria: absence of genetic heterogeneity, high prevalence of private mutations and further evidence of volume depletion

INTRODUCTION Familial renal glucosuria (FRG) is a rare renal tubular disorder caused by mutations within the SLC5A2 gene. It is characterized by persistent glucosuria in the absence of hyperglycaemia and any other signs of generalized tubular dysfunction. In small series of patients previously reported, the molecular and phenotypic findings in FRG families, including first hints of extracellular volume depletion and activation of the renin-angiotensin-aldosterone system induced by natriuresis, have been described. We have now extended this analysis to another 21 consecutive cases from 17 pedigrees, including 11 cases with severe glucose excretion. METHODS Mutation analysis was performed by direct sequencing of the genomic coding segments of the SLC5A2 gene. In two cases with severe glucosuria, basal plasma renin activity and serum aldosterone concentrations were determined. RESULTS Within the 17 pedigrees, we have identified a total of 20 different SLC5A2 mutations. Fifteen have not been previously reported. In all glucosuric individuals tested, at least one SLC5A2 mutation could be identified. Heterozygous individuals were found to have only mild glucose excretion whereas homozygous or compound heterozygous patients had severe glucosuria, ranging from 10 to 86.5 g/1.73 m(2)/24 h. In two patients of the latter group, basal plasma renin activity and serum aldosterone concentration were determined and found to be raised to an average of 4.6-fold and 3.1-fold of the upper limit of the normal range, respectively. Discussion. The identification of at least one mutated allele in every affected individual in this cohort of 17 consecutively investigated families strongly suggests that genetic heterogeneity is not prevalent in FRG. Although 5 of the detected alleles have been described previously, 15 are novel, confirming that most mutations in FRG are private. Our finding of an activation of compensatory mechanisms for salt loss may warrant more detailed studies of long-term hormonal and metabolic imbalances in patients with FRG.

Transplantation procedures in children with primary hyperoxaluria type 1: outcome and longitudinal growth.

Background. Cure of the metabolic defect in primary hyperoxaluria type 1 (PH1) is possible with liver transplantation (LTx). Preemptive LTx (PLTx) was promoted to prevent chronic kidney disease due to nephrocalcinosis and urolithiasis. However, timing of this procedure is difficult in view of the heterogeneity of PH1 and effective conservative treatment. Combined liver-kidney transplantation (LKTx) is able to cure metabolic defect and replace renal function at the same time and is effective and indicated for patients with or approaching end-stage renal disease (ESRD). Sometimes a sequential approach for LKTx (first liver, then kidney) has been recommended. Methods. We report on 13 patients with PH1 since 1995 who underwent transplantation procedures in our center for PH1 at a median age of 4.7 (range 1.4–8.9) years. Results. The first two patients, planned for a sequential strategy, died early after LTx because of infectious complications. Four patients underwent PLTx at a median glomerular filtration rate of 65 (range 27–98) mL/min/1.73 m2/day (Hoppe et al., Pediatr Nephrol 1996; 10: 488), and three patients still have sufficient residual renal function after a follow-up of median 11.6 years. Seven patients with ESRD received a combined LKTx, including four with infantile oxalosis, and three weighing less than 10 kg. There was no mortality and catch-up growth was observed in most patients. Conclusion. In summary and conclusion, transplantation procedures are challenging in PH1, but our results including growth data are encouraging. PLTx remains an option despite the difficulties in timing the procedure. LKTx is indicated for patients with ESRD and is possible even in patients with infantile oxalosis and may improve longitudinal growth.

Long-term Clinical Relevance of De Novo Donor-Specific Antibodies After Pediatric Liver Transplantation.

Background Anti-HLA antibodies and especially donor-specific antibodies (DSA) play a significant role in graft survival after solid organ transplantation. Their impact on long-term survival in adult liver transplantation (LT) is controversial, but they may be a risk factor. The effects of DSA after pediatric LT are still unclear. Methods We performed a retrospective evaluation of DSA in sera from 43 children who had received transplants at our tertiary center. Twenty-four patients had good long-term clinical and laboratory graft function (group 1), whereas 19 LT recipients suffered from histologically confirmed and clinically relevant chronic allograft rejection (group 2); 16 of these have already undergone retransplantation due to graft dysfunction. Inclusion criteria were availability of sera before the first LT to identify preformed antibodies in case of DSA positivity after LT and long-term follow-up at our institution. Sera were analyzed for anti-HLA antibodies using Luminex single antigen beads, where a mean fluorescence intensity value of more than 1500 was considered positive. Results The prevalence of DSA was 33% for group 1 and 68% for group 2. Antibodies were predominantly HLA class II. Values of mean fluorescence intensity were comparable in both groups. Only one of the DSA+ ve patients from group 1 exhibited preformed antibodies. In conclusion, pediatric patients with chronic rejection revealed a higher rate of de novo DSA, especially of HLA-class II DSA. Further studies are necessary to confirm these data with a larger pediatric cohort.

Combined liver-kidney transplantation for children with autosomal recessive polycystic kidney disease (ARPKD): indication and outcome

In ARPKD, mutations in the PKHD1 gene lead to remodeling of the kidneys and liver. These may result in progressive liver fibrosis with portal hypertension requiring combined liver and kidney transplantation (CLKT). There is currently no consensus on the indication for CLKT and data on long‐term outcomes are scarce. We analyzed in detail the pretransplant liver symptomatology, laboratory and ultrasound data, histological studies, and genotypes in eight patients undergoing CLKT. The median age was 10.1 years (range 1.7–16) and median follow‐up was 4.6 years (range 1.1–8.9). All patients had clinical signs of portal hypertension and abnormal ultrasound findings. Congenital hepatic fibrosis was present in all pretransplant biopsies (6 out of 8 patients) and in all explanted livers. All patients survived; liver and kidney graft survival was 72% and 88%, respectively. Liver and kidney function were stable in all patients with a median eGFR of 70 ml/min/1.73 m² (range 45–108 ml/min/1.73 m²). Height‐SDS improved significantly after 12, 24, and 36 months (P = 0.016, 0.022 and 0.018 respectively). The indication for CLKT remains challenging and controversial. A favorable outcome for patients with ARPKD can be achieved by using the degree of portal hypertension, longitudinal ultrasound examinations, and preoperative liver histology as parameters for CLKT.

Immediate postoperative intensive care treatment of pediatric combined liver-kidney transplantation: outcome and prognostic factors.

Background. Studies reporting the immediate pediatric intensive care unit (PICU) treatment after combined liver-kidney transplantation (CLKT) are scarce, although this period is pivotal for survival and long-term outcome. Methods. We retrospectively analyzed all pediatric CLKT performed in our center between 1998 and 2010. Results. Sixteen patients underwent 17 CLKT at a median age of 5.3 years (range, 1.3–15.9 years). Median body weight at CLKT was 17.7 kg (range, 9.2–55 kg). Underlying diagnosis was primary hyperoxaluria type 1 in nine patients and autosomal recessive polycystic kidney disease in seven patients. Median time on PICU was 8.5 days (range, 3–68 days); however, patients with primary hyperoxaluria type 1 had a significantly longer stay (P=0.031). Median duration of ventilation was 1 day; however, five patients required ventilation for 25 to 52 days. Continuous veno-venous hemofiltration was applied in nine patients due to delayed kidney graft function, volume overload, or high plasma oxalate. Overall, the survival rate after CLKT was 100% and long-term outcome was very good at a mean follow-up of 3.6 years (range, 0.5–12.2 years). Waiting time, donor age, and donor-to-recipient weight ratio were found to be significant risk factors for an extended PICU stay (P=0.02, 0.0031, and 0.014, respectively). Conclusions. Immediate postoperative course after CLKT may be challenging and complex. However, excellent results can be achieved, even in small children.

Bile salt export pump‐reactive antibodies form a polyclonal, multi‐inhibitory response in antibody‐induced bile salt export pump deficiency

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)

Surgical aspects and outcome of combined liver and kidney transplantation in children

In children with renal insufficiency and accompanying or underlying liver disease, combined liver and kidney transplantations (CLKT) are indicated. However, because of the rare indications, the number of paediatric CLKT is low. Our aim was to analyse CLKT in children with special regard to surgical aspects and outcome. All paediatric CLKT performed at our institution between 1998 and 2009 were retrospectively analysed. Between 1998 and 2009, 15 CLKT were performed in 14 paediatric patients (median age 8 years, range 1–16 years). The indications for CLKT were autosomal recessive polycystic kidney disease (n = 7), primary hyperoxaluria type 1 (n = 7) and retransplantation because of primary liver nonfunction (n = 1). In the postoperative course, six patients showed bleeding complications, thereof three patients needed operative revision for intra‐abdominal bleeding. Eight of 15 patients (53%) needed dialysis. The 1‐ and 5‐year patient survival was 100%; and 1‐ and 5‐year graft survival was 80% for the liver and 93% for the kidney allograft. A number of different complications, especially secondary haemorrhage have to be anticipated after CLKT, requiring a timely and interdisciplinary treatment approach. With this management, our patients showed an excellent graft and patient survival.

Prevalence of polyomavirus viruria (JC virus/BK virus) in children following liver transplantation

Brinkert F, Briem‐Richter A, Ilchmann C, Kemper MJ, Ganschow R. Prevalence of polyomavirus viruria (JC virus/BK virus) in children following liver transplantation.
Pediatr Transplantation 2010: 14: 105–108.

Bile Salt Export Pump‐reactive Antibodies Form a Polyclonal, Multi‐inhibitory Response in Antibody‐induced BSEP Deficiency

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)

High prevalence of renal dysfunction in children after liver transplantation: non-invasive diagnosis using a cystatin C-based equation

BACKGROUND Chronic kidney disease (CKD) has been increasingly shown to be a negative prognostic factor after liver transplantation (Ltx). Creatinine-based glomerular filtration rate (GFR) formulas are notoriously insensitive. In children, non-invasive determination of GFR by measurement of serum cystatin C is feasible and repeatedly correlated to the gold standards of GFR measurements. The aim of our study was to determine GFR using cystatin C (GFR(cys)) in comparison with conventional calculated creatinine clearance (GFR(crea)) in the long-term follow-up after paediatric liver transplantation (pLtx) in a large number of patients. METHODS GFR of 168 children following liver transplantation was determined using cystatin C (GFR(cys)) and the Schwartz formula (GFR(crea)). In order to evaluate risk factors for CKD, a logistic regression analysis was performed. A multivariate model was applied to assess the impact of immunosuppressive treatment. RESULTS The mean follow-up after transplantation was 7.8 (0.44-15.72) years. Due to a high overestimation of GFR as demonstrated in a Bland-Altman plot, only three patients with CKD stages 2-3 were detected with GFR(crea) compared with 34 with GFR(cys) (P < 0.001). Thus, prevalence of CKD with GFR((cys)) < 90 mL/min/1.73 m2; was 30.4%, 7.6% and 27% in patients with 5, 10 and > 10 years of follow-up, respectively. Patients on cyclosporine had a significantly lower GFR than patients on tacrolimus. Logistic regression analysis did not show any significant risk factor for the development of CKD. CONCLUSIONS The cystatin C equation is a non-invasive and sensitive diagnostic tool to detect renal dysfunction in children after Ltx at an early stage. The choice of first-line calcineurin inhibitor has an important impact on the development of CKD.