Functions of PAX2 and PAX8 genes during kidney development

The 9 PAX genes constitute a family of developmental transcriptional regulators characterized by a highly conserved "paired-box" domain. Mutations in 6 of the 9 PAX genes result in autosomal dominant congenital malformations in mice and in humans. PAX2 transcripts are detected in the CNS, throughout the genitourinary tract, eye and ear. In humans, mutations of PAX2 cause Renal-Coloboma Syndrome (RCS), a constellation of renal anomalies, eye defects and, less frequently, hearing loss and mild CNS manifestations. Homozygous PAX2 mutations in mice cause complete renal agenesis and perinatal death. PAX8 transcripts are found in the CNS, kidney and thyroid glands. PAX8 mutations cause congenital thyroid hypoplasia. No renal defects have been detected either in human or murine PAX8 mutants.In spite of considerable information about PAX2 and PAX8 genes, their precise functions in development are poorly understood. This project was aimed at elucidating the functions of PAX2 and PAX8 in nephrogenesis and is subdivided into two parts: mutational screening of the PAX8 gene (Chapter 4) and delineation of the role of PAX2 in developing kidney (Chapters 5--7). The latter evolved into the major portion of this work.Familial juvenile nephronophthisis (NPH1) is a rare disease characterized by multiple small cysts at the cortico-medullary junction and end-stage renal failure in pediatric populations. Because of its proximity to the disease locus, we considered PAX8 a candidate gene for NPH1. During analysis of PAX8 exons from patients, we identified a rare non-conservative polymorphic amino acid change, but found no causative mutations. Subsequently, other groups isolated the novel NPH1 gene. Following reports that PAX8 knockout mice have no obvious renal anomaly, we considered the possibility that PAX8 mutations might, nevertheless, affect proximal tubule function. In patients with thyroid hypoplasia and proven PAX8 mutations we found no aminoaciduria, indicating that haploinsufficiency for PAX8 does not alter tubular transport function.In order to define PAX2 function in the two primordial cell lineages of developing kidney (induced metanephric mesenchyme and ureteric epithelium), we used both a cell culture approach and an in vivo PAX2 mutant mouse model (1Neu). We demonstrated that PAX2 plays a dual role. In mesenchymally-derived HEK293 cells expressing regulatable exogenous PAX2, we showed that PAX2 is responsible for expression of genes involved in differentiation of mesenchyme. Contrary to one published hypothesis, we found that PAX2 does not affect cell division. In ureteric epithelium, however, PAX2 plays a different role, serving as a survival factor, critical for sustaining the ureteric bud stem cell population. Attenuation of PAX2 dosage (1Neu mouse mutants or collecting duct cells transfected with an antisense PAX2 vector) results in increased apoptosis. We demonstrate that the primary renal defect in RCS is reduced nephron number associated with excessive apoptosis and simplified branching of the ureteric bud. We hypothesize that arborization of the uretric bud requires accumulation of sufficient stem cell mass to allow the next round of branching---possibly by lifting the branch point beyond a putative local inhibitory field.In summary, we establish that inactivation of PAX8 gene expression does not disturb normal kidney development and function. Conversely, PAX2 plays a crucial dual role in the two primordial kidney cell lineages: being a differentiating factor in mesenchyme and a survival factor in ureteric epithelium

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

AbstractPAX2, 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

Recurrent Focal Segmental Glomerulosclerosis: A Discrete Clinical Entity

Focal segmental glomerulosclerosis refers to a set of particular histopathologic lesions in which steroid-resistant podocyte injury leads to patchy adhesions between the glomerular tuft and Bowman's capsule, followed by progressive glomerulosclerosis and proteinuric renal failure. Because of the nonspecific nature of this lesion, it has been difficult to classify the various forms of primary nephrotic syndrome in children. However, with the recognition of hereditary FSGS caused by mutations podocyte slit diaphragm genes, it is increasingly clear that the steroid-resistant form of FSGS that recurs in the renal allografts (R-FSGS) constitutes a distinct clinical entity. Capitalizing on recent studies in which patients have been screened for slit diaphragm gene mutations, this review focuses on the natural history and pathogenesis of R-FSGS

Endolymphatic Sac Enlargement in a Girl with a Novel Mutation for Distal Renal Tubular Acidosis and Severe Deafness

Hereditary distal renal tubular acidosis (dRTA) is caused by mutations of genes encoding subunits of the H+-ATPase (ATP6V0A4 and ATP6V1B1) expressed in α-intercalated cells of the distal renal tubule and in the cochlea. We report on a 2-year-old girl with distal RTA and profound speech delay which was initially misdiagnosed as autism. Genetic analysis showed compound heterozygous mutations with one known and one novel mutation of the ATP6V1B1 gene; cerebral magnetic resonance imaging (MRI) revealed bilateral enlargement of the endolymphatic sacs of the inner ear. With improved cooperation, audiometric testing showed that hearing loss was most profound on the right, where endolymphatic sac enlargement was greatest, demonstrating a clear link between the degree of deafness and the degree of inner ear abnormality. This case indicates the value of MRI for diagnosis of inner ear involvement in very young children with distal RTA. Although citrate therapy quickly corrects the acidosis and restores growth, early diagnosis of deafness is crucial so that hearing aids can be used to assist acquisition of speech and to provide enough auditory nerve stimulation to assure the affected infants remain candidates for cochlear implantation