Biallelic DICER1 mutations occur in Wilms tumours.

DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two-hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single-base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two 'hits' in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours.

Effects of PAX2 expression in a human fetal kidney (HEK293) cell line.

PAX2, a member of the "paired-box" family of homeotic genes, is a nuclear transcription factor expressed in the early stages of nephrogenesis by induced blastemal cells as they progress from mesenchymal condensates to the "S-shaped" stage and also by the ureteric bud. Spontaneous mutations in one copy of PAX2 in humans causes a syndrome of proteinuric renal failure and coloboma of the eye (P. Sanyanusin et al., Nat. Genet. 9 (1995) 358-363); transgenic mice with disruption of the PAX2 gene are anephric (M. Torres et al., Development 121 (1995) 4057-4067. Although PAX2 is clearly critical for normal kidney development, its direct effects on kidney cell phenotype are unknown. To address this issue, we developed stable transfectants of the HEK293 human fetal kidney epithelial cell line expressing human PAX2 protein under tetracycline-regulatable promoter. In these cells, PAX2 had no effect on the proliferative rate, but increased the expression of the Wilms' tumor gene (2-fold) and E-cadherin (7-fold). PAX2 had a strong inhibitory effect on vimentin; vimentin/GAPDH mRNA ratio was suppressed to 8% of control whereas cytokeratin-18/GAPDH mRNA ratio was unchanged. During nephrogenesis, loss of vimentin and onset of low-level WT1 and E-cadherin expression occur in mesenchymal condensates. Our observations suggest that these events may be, in part, regulated by PAX2.

A gamma-aminobutyric acid-specific transport mechanism in mammalian kidney.

We describe high-affinity, sodium-dependent transport of gamma-aminobutyric acid in slices exposing basal lateral membranes and brush-border membrane vesicles prepared from rat renal cortex. In the presence of aminooxyacetic acid, to block gamma-aminobutyric acid oxidation, uptake into the intracellular space of slices was saturable (apparent Kt, 26 +/- 4 microM, mean and S.E.) and concentrative (steady-state distribution ratio at 50 microM gamma-aminobutyric acid, 47.7 +/- 2.4, mean and S.E.). Brush-border membrane vesicles accumulated gamma-aminobutyric acid in the presence of an inward-directed sodium chloride gradient, (apparent Kt, 30-36 microM) with the peak of 'overshoot' at 10 min. Uptake by vesicles responded to manipulation of the transmembrane potential gradient with valinomycin or impermeant anion. beta-Alanine inhibited gamma-aminobutyric acid transport by slices and brush-border membrane vesicles; inhibitors of neuronal-type gamma-aminobutyric acid transport (e.g., nipecotic and diaminobutyric acids) did not. An 'ABC test' indicated that gamma-aminobutyric acid and beta-alanine do not share a single carrier in either the brush-border or basal-lateral membrane of renal cortex. Influx of gamma-aminobutyric acid into brush-border membrane vesicles, at transequilibrium NaCl, was stimulated by trans-gamma-aminobutyric acid but not by trans-taurine. Ion gradient-driven gamma-aminobutyric acid co-transport was unaffected in freeze-thawed brush-border membrane vesicles; this treatment abolished beta-alanine and taurine co-transport. We conclude that rat kidney membranes (brush-border and basal-lateral) possess a gamma-aminobutyric acid-preferring, high-affinity transport mechanism.

Properties of gamma-aminobutyric acid synthesis by rat renal cortex.

Substantial synthesis of gamma-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3 +/- 2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal gamma-aminobutyric acid content (39.5 +/- 5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which gamma-aminobutyric acid formation from [2,3-3H]glutamate and CO2 release from [1(-14)C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (Ki = 5X10(-5) M); (2) renal glutamate decarboxylase is less sensitive (Ki = 3-5X10(-5) M) to inhibition by aminooxyacetic acid than is the brain enzyme (Ki = 1X10(-6) M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5'-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal gamma-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.

The relationship of 4-aminobutyric acid metabolism to ammoniagenesis in renal cortex.

Mitochondrial 4-aminobutyrate aminotransferase in rat kidney can utilize pyruvate as the acceptor for the amino group of 4-aminobutyrate. Renal 4-aminobutyrate aminotransferase activity at saturating equimolar concentration of 4-aminobutyrate and 5 mM pyruvate is 42.8 +/- 2.5 mumol/g protein per h (mean +/- S.E.M.) or 70% of 4-aminobutyrate aminotransferase activity with equimolar alpha-ketoglutarate. 4-Aminobutyrate aminotransferase in brain does not transaminate with pyruvate. Since pyruvate is an important mitochondrial metabolite in kidney, net disposal of glutamate via the 4-aminobutyrate pathway is possible. The renal 4-aminobutyrate pathway in the rat has other distinctive features when compared with the pathway in rat brain. Most inhibitors of rat neuronal glutamate decarboxylase were ineffective against the renal form of the enzyme, but 20 mM semicarbazide inhibited the latter form by 80% (P < 0.001) in vitro and reduced renal 4-aminobutyrate content by 75% (P < 0.001) in vivo. In the presence of 20 mM semicarbazide, ammoniagenesis by rat renal cortex slices incubated in 1 mM glutamine was inhibited 26% (P < 0.01). Semicarbazide was proportionately less effective (15% inhibition) when ammonia-genesis was stimulated (+243%) in slices prepared from chronically acidotic animals, and was no deterrant to ammoniagenesis when non-acidotic slices were incubated in supraphysiologic concentrations of 10 mM glutamine. We conclude that whereas integrity of the renal 4-aminobutyrate pathway may contribute to glutamate disposal and thus ammoniagenesis under physiologic conditions, the pathway is a passive participant in the overall process of ammoniagenesis.

Renal chloride channel, CLCN5, mutations in Dent's disease.

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. Patients with Dent's disease may also suffer from rickets and other features of the renal Fanconi Syndrome. Patients may have mutations in the X-linked renal chloride channel gene, CLCN5, which encodes a 746-amino-acid protein with 12-13 transmembrane domains. We have investigated the 11 coding exons of CLCN5 for mutations in eight unrelated patients with Dent's disease. Leukocyte DNA was used for the polymerase chain reaction amplification of CLCN5 and the products analyzed for single-stranded conformational polymorphisms (SSCPs). Abnormal SSCPs were sequenced and revealed eight mutations. These consisted of three nonsense mutations (Arg34Stop, Arg648Stop, Arg704Stop), four deletions involving codons 40, 86, 157, and 241, and one acceptor splice consensus sequence mutation tgcag --> tgaag. The mutations were confirmed either by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis. In addition, an analysis of 110 alleles from 74 unrelated normal individuals demonstrated that the DNA sequence changes were not common polymorphisms. All of the mutations predict truncated chloride channels that are likely to result in a functional loss. Thus, our findings expand the spectrum of CLCN5 mutations associated with Dent's disease and the results will help to elucidate further the functional domains of this novel chloride channel.

A perimortem protocol for suspected genetic disease.

A considerable portion of pediatric deaths represent disease with risk of recurrence in subsequent family members. Procedures to obtain samples of body fluids and tissues suitable for diagnosis of mendelian and chromosomal disorders are described. These procedures, the "perimortem protocol," are used in studying children who died of suspected but undiagnosed genetic disease.

Expression of transforming growth factor-alpha and epidermal growth factor receptor in human fetal kidneys.

Beginning at the fifth week of fetal life, successive generations of individual nephrons are induced by contact between metanephric mesenchyme and ureteric bud. Following phenotypic transformation, cells of each primitive renal vesicle undergo a phase of rapid cell division. In order to identify genes which might regulate nephron development in man, we screened adult and fetal kidney RNA for expression of a panel of growth-related genes. Among the genes which were expressed at higher levels in fetal kidney was the epidermal growth factor (EGF) receptor. There is controversy as to the most likely physiologic EGF receptor ligand in fetal kidney; we were able to identify a transcript for transforming growth factor-alpha (TGF-alpha) but not EGF on Northern blots of fetal kidney RNA. Since the abundance of TGF-alpha mRNA is low, we confirmed its presence by polymerase chain reaction amplification. Using specific radioimmunoassays, we also provide direct evidence for TGF-alpha but not EGF peptide in extracts of fetal kidney and mid-gestational amniotic fluid. We suggest that TGF-alpha/EGF receptor interactions may serve an important function in development of human fetal kidney.

Inherited lactic acidosis: correction of the defect in cultured fibroblasts.

We report a case of familial lactic acidosis, lethal in the newborn period. Studies in intact fibroblasts identified a defect in the oxidative pathway of pyruvate metabolism. Although assay of pyruvate dehydrogenase on cell sonicates was not appreciably reduced, flux through the enzyme and other mitochondrial multienzyme dehydrogenases was severely impaired in intact cells. Deficient lactate conversion to carbon dioxide could be repaired by the addition to the incubation medium of electron acceptors such as methylene blue (25 micrograms/ml) or dichlorophenolindophenol (25 micrograms/ml).

Expression of the epidermal growth factor receptor in fetal kidney.

Formation of the human kidney begins at the 6th week of fetal life when the first generations of nephrons are generated from foci of metanephric mesenchyme through contact with the branches of the ureteric bud. This process requires a proliferative burst which must be tightly regulated by local signals. In this report, we review the evidence that the epidermal growth factor receptor molecule is an important arbiter of these events.

Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.

The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.

Expression of growth-related genes in human fetal kidney.

By the end of gestation, nephron formation in the human kidney is complete. Following local induction of metanephric mesenchyma, committed cells of each primitive renal vesicle must undergo a phase of rapid cell division. In order to identify genes which might regulate these events, we examined the expression profile of 22 proto-oncogenes in fetal versus adult human kidney. Among those expressed at especially high levels in the fetal tissue was the gene for epidermal growth factor receptor (EGFR). We were able to detect mRNA (by polymerase chain reaction [PCR] amplification) and peptide (by specific radioimmunoassay) for transforming growth factor-alpha (TGF-alpha) in fetal kidney, whereas epidermal growth factor (EGF) peptide was undetectable in midgestation kidney and amniotic fluid. TGF-alpha/EGFR interactions may direct renal cell proliferation in fetal life.

Turner's syndrome, 46X, del (X) (p 11), persistent complement activation and membranoproliferative glomerulonephritis.

An adolescent girl with short stature and learning disability was found to have an unusual variant of Turner's syndrome, 46X, del (X) (p 11) and an abnormal urinary sediment. Further studies demonstrated persistent depression of C3 and histologic evidence of membranoproliferative glomerulonephritis (MPGN). The occurrence of MPGN in this case may have been a manifestation of the known tendency for Turner patients to develop immunologic disease.

Epidermal growth factor receptor activation in developing rat kidney.

Epidermal growth factor (EGF) binding increases in late-gestational rat kidney and then falls toward basal adult levels postnatally during the 1st wk. We report that the increase in EGF binding is accompanied by an increase in EGF receptor (EGFR) protein and activation of EGFR tyrosine kinase. Multiple proteins were endogenously tyrosine phosphorylated in kidney membranes from fetal rats, and the phosphorylation pattern was similar in rats ranging from 16 to 21 days of gestation. Tyrosine phosphorylation was, however, almost undetectable in 12-wk adult rat kidneys (controls). Among the phosphoproteins in fetal kidney, a prominent 170-kDa protein was identified as EGFR. Endogenous tyrosine phosphorylation of EGFR (reflecting receptor activation) was 30-fold higher in fetal kidney membranes than in adult (3- to 7-fold higher when adjusted for differences in EGF binding or EGFR protein content). The EGFR substrate, phospholipase C-gamma 1, was tyrosine phosphorylated in fetal kidneys but not adult, and a greater proportion was membrane-associated in fetal kidneys, consistent with activation of phospholipase C-gamma 1. Thus EGFR tyrosine kinase activity is increased in late-gestational rat kidney. Induction and activation of EGFR may mediate perinatal renal cell growth and development.

Analysis of the Heymann nephritogenic glycoprotein in rat, mouse, and human kidney.

Passive Heymann nephritis is induced in rats by intravenous administration of antiserum raised against antigens of the renal proximal tubule. Evidence by Kerjaschki and Farquhar indicates that the critical nephritogenic is a high molecular weight glycoprotein (HMWgp) of rat renal brush border membrane. Their immunocytochemical studies also localize the nephritogenic antigen to the glomerular epithelial cell surface and may explain in situ formation of immune complexes at this locus in Heymann nephritis. We have confirmed the observations of Kerjaschki and Farquhar by demonstrating the HMWgp in extracts of rat brush border membrane and isolated glomeruli on sodium dodecyl sulfate-polyacrylamide (SDS-PA) (5%) gels. An antiserum raised to purified rat HMWgp identifies the antigen from rat or mouse kidney on Western blots. However, unlike rodent kidney, we were unable to detect a comparable HMWgp in extracts of human kidney on SDS-PA gels and found no cross-reactive material on Western blots of human brush border membrane proteins. Our observations suggest that human kidney lacks the nephritogenic antigen critical to initiation of Heymann nephritis in rodents.

Prospective analysis and classification of patients with cystinuria identified in a newborn screening program.

Patients who inherit mutant cystinuria genes excrete high concentrations of cystine, ornithine, arginine, and lysine in the urine. At least three variants of cystinuria can be distinguished in heterozygotes. To determine whether certain combinations of mutant genes are more disadvantageous than others, we analyzed amino acid excretion in families of 17 probands with cystinuria identified by the Quebec neonatal screening program. Parents of the probands were classified into the three known phenotypes by calculating the sum of cystine, ornithine, arginine, and lysine excretion. Although parents of type I/I homozygotes excreted amounts of cystine in the normal range, their offspring excreted significantly greater amounts of urinary cystine than did children who have type I/III genetic compounds. This observation suggests that types I and III cystinuria mutations might involve two distinct genetic loci. Children with type I/I homozygous cystinuria often excrete cystine at levels greater than the theoretic solubility limit and may be at greatest risk for nephrolithiasis. We outline an approach to monitoring children with cystinuria who come to medical attention before formation of cystine stones.

Rabbit pancreatic acini express CFTR as a cAMP-activated chloride efflux pathway.

Cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-activated chloride transport in epithelial cells. Isolated rabbit pancreatic acini possess a cAMP-activated chloride efflux mechanism distinct from zymogen granule secretion. To determine whether CFTR is expressed in acini, we used polymerase chain reaction (PCR) to amplify a 480-base pair (bp) sequence from reverse-transcribed rabbit acinar RNA. The PCR product was consistent with a 480-bp band amplified in T84 cells, and its sequence was > 90% homologous to human CFTR. CFTR antibody M3A7 recognized a 180- and a 160-kDa protein from acinar membranes consistent with bands seen in Chinese hamster ovary (CHO) cells transfected with CFTR. To determine if CFTR was responsible for the cAMP-activated chloride efflux previously demonstrated in pancreatic acini, we incubated acinar cells for 20 h with 1.75 microM CFTR antisense or sense oligodeoxynucleotide. Chloride efflux, in response to 8-bromoadenosine 3',5'-cyclic monophosphate and phorbol ester but not to calcium ionophore, was selectively inhibited by CFTR antisense oligodeoxynucleotide. Antisense oligodeoxynucleotide did not inhibit acinar amylase secretion. These findings indicate that isolated pancreatic acini can be used for future studies of CFTR expression and function.

Rapid loss of renal parenchyma after acute obstruction.

Urinary tract obstruction (UTO) is a frequent cause of renal failure in the pediatric population. We report a patient with type I/I cystinuria, followed prospectively from birth with yearly ultrasonography, who developed acute UTO due to a cystine stone at 10 years of age. In animal models of UTO, acute obstruction produces rapid loss of renal parenchyma secondary to apoptosis of tubular cells. Since we had prospectively obtained serial ultrasonographic measurements of renal growth, we were able to document sudden decrease in kidney size and function following UTO, suggesting that programmed cell death may similarly have caused the rapid irreversible loss of renal parenchyma in our patient. Despite surgical relief of the obstruction, kidney size decreased for at least 3-4 months. We speculate that anti-apoptotic drugs might be considered as a therapeutic strategy to protect ongoing renal parenchyma loss in UTO.

Excretion of epidermal growth factor-like material in acute Henoch-Schönlein purpura nephritis.

In Henoch-Schönlein purpura nephritis (HSPN), glomeruli may develop cellular "crescents" composed of infiltrating monocytes and proliferating renal epithelia. In this study, we demonstrate that peripheral human monocytes can release an epidermal growth factor (EGF)-like substance detectable by a radioreceptor assay, which recognizes both EGF and transforming growth factor-alpha (TGF-alpha), but not with a radioimmunoassay, which recognizes only EGF. Furthermore, we report that urine from pediatric patients during the acute phase of HSPN contains a similar EGF-like species in addition to the endogenous EGF which is normally present. The EGF-like material was not present in urine from nine healthy children or from six children with acute post-streptococcal glomerulonephritis. The extent of crescent formation in our patients is uncertain, since renal biopsy was performed in only one case. However, we speculate that the urinary material resembling TGF-alpha which appears during the acute phase of HSPN may derive from monocytes infiltrating the kidney.

Antenatal betamethasone and renal ammoniagenesis in the newborn.

As part of a double-blind clinical trial of antenatal betamethasone, we studied the effects of this drug on urinary ammonia excretion in 28 premature infants. Betamethasone was administered before 34 weeks of gestation according to dosage schedules which have been shown to alter the incidence of respiratory distress syndrome. Although glucocorticoids affect renal ammoniagenesis in adults, the antenatal betamethasone trial did not augment ammonia excretion measured during the first day of postnatal life. We speculate that precocious maturation of renal ammoniagenesis cannot be triggered by glucocorticoids during the gestational period studied.