Paul Goodyer

Renal and Retinal Effects of Enalapril and Losartan in Type 1 Diabetes

BACKGROUND Nephropathy and retinopathy remain important complications of type 1 diabetes. It is unclear whether their progression is slowed by early administration of drugs that block the renin-angiotensin system. METHODS We conducted a multicenter, controlled trial involving 285 normotensive patients with type 1 diabetes and normoalbuminuria and who were randomly assigned to receive losartan (100 mg daily), enalapril (20 mg daily), or placebo and followed for 5 years. The primary end point was a change in the fraction of glomerular volume occupied by mesangium in kidney-biopsy specimens. The retinopathy end point was a progression on a retinopathy severity scale of two steps or more. Intention-to-treat analysis was performed with the use of linear regression and logistic-regression models. RESULTS A total of 90% and 82% of patients had complete renal-biopsy and retinopathy data, respectively. Change in mesangial fractional volume per glomerulus over the 5-year period did not differ significantly between the placebo group (0.016 units) and the enalapril group (0.005, P=0.38) or the losartan group (0.026, P=0.26), nor were there significant treatment benefits for other biopsy-assessed renal structural variables. The 5-year cumulative incidence of microalbuminuria was 6% in the placebo group; the incidence was higher with losartan (17%, P=0.01 by the log-rank test) but not with enalapril (4%, P=0.96 by the log-rank test). As compared with placebo, the odds of retinopathy progression by two steps or more was reduced by 65% with enalapril (odds ratio, 0.35; 95% confidence interval [CI], 0.14 to 0.85) and by 70% with losartan (odds ratio, 0.30; 95% CI, 0.12 to 0.73), independently of changes in blood pressure. There were three biopsy-related serious adverse events that completely resolved. Chronic cough occurred in 12 patients receiving enalapril, 6 receiving losartan, and 4 receiving placebo. CONCLUSIONS Early blockade of the renin-angiotensin system in patients with type 1 diabetes did not slow nephropathy progression but slowed the progression of retinopathy. (ClinicalTrials.gov number, NCT00143949.)

Paired-Box genes are frequently expressed in cancer and often required for cancer cell survival.

The paired-box (PAX) genes encode a family of nine well-characterized paired-box transcription factors, with important roles in development and disease. Although PAX genes are primarily expressed in the embryo, constitutive expression promotes tissue hyperplasia. Rare tumor-specific mutations of PAX genes implicate an oncogenic role, and persistent PAX expression characterizes several tumors. Yet, a cancer-wide analysis of PAX gene expression to investigate a general role for PAX genes has not been performed. We analysed the pattern and requirement for PAX gene expression in a panel of common cancer cell lines. Very frequent PAX gene expression was identified in tumor cell lines, including lymphoma, breast, ovarian, lung, and colon cancer. In addition, the PAX2 gene was frequently expressed in a panel of 406 common primary tumor tissues. Apoptosis was rapidly induced in ovarian and bladder cancer cell lines following RNA interference to silence PAX2 expression, despite concomitant TP53 and/or HRAS mutations. These data suggest that PAX genes are frequently expressed in cancer, and that endogenous PAX gene expression is required for the growth and survival of cancer cells.

The Wilms’ Tumor Suppressor Gene (wt1) Product Regulates Dax-1 Gene Expression during Gonadal Differentiation

ABSTRACT Gonadal differentiation is dependent upon a molecular cascade responsible for ovarian or testicular development from the bipotential gonadal ridge. Genetic analysis has implicated a number of gene products essential for this process, which include Sry, WT1, SF-1, and DAX-1. We have sought to better define the role of WT1 in this process by identifying downstream targets of WT1 during normal gonadal development. We have noticed that in the developing murine gonadal ridge, wt1 expression precedes expression of Dax-1, a nuclear receptor gene. We document here that the spatial distribution profiles of both proteins in the developing gonad overlap. We also demonstrate that WT1 can activate the Dax-1 promoter. Footprinting analysis, transient transfections, promoter mutagenesis, and mobility shift assays suggest that WT1 regulates Dax-1via GC-rich binding sites found upstream of the Dax-1 TATA box. We show that two WT1-interacting proteins, the product of a Denys-Drash syndrome allele of wt1 and prostate apoptosis response-4 protein, inhibit WT1-mediated transactivation ofDax-1. In addition, we demonstrate that WT1 can activate the endogenous Dax-1 promoter. Our results indicate that the WT1–DAX-1 pathway is an early event in the process of mammalian sex determination.

A Common RET Variant Is Associated with Reduced Newborn Kidney Size and Function

Congenital nephron number varies five-fold among normal humans, and individuals at the lower end of this range may have an increased lifetime risk for essential hypertension or renal insufficiency; however, the mechanisms that determine nephron number are unknown. This study tested the hypothesis that common hypomorphic variants of the RET gene, which encodes a tyrosine kinase receptor critical for renal branching morphogenesis, might account for subtle renal hypoplasia in some normal newborns. A common single-nucleotide polymorphism (rs1800860 G/A) was identified within an exonic splicing enhancer in exon 7. The adenosine variant at mRNA position 1476 reduced affinity for spliceosome proteins, enhanced the likelihood of aberrant mRNA splicing, and diminished the level of functional transcript in human cells. In vivo, normal white newborns with an rs1800860(1476A) allele had kidney volumes 10% smaller and cord blood cystatin C levels 9% higher than those with the rs1800860(1476G) allele. These findings suggest that the RET(1476A) allele, in combination with other common polymorphic developmental genes, may account for subtle renal hypoplasia in a significant proportion of the white population. Whether this gene variant affects clinical outcomes requires further study.

PAX2 Suppresses Apoptosis in Renal Collecting Duct Cells

PAX2 is a transcription factor belonging to the evolutionarily conserved paired box family and is required during development of the central nervous system and genitourinary axis. Mutations in the PAX2 gene cause a rare autosomal dominant renal-coloboma syndrome, characterized by optic nerve colobomas and renal hypoplasia. Recent analysis of a spontaneous PAX2 mutant mouse model (1Neu) revealed that the major cause of renal hypoplasia is reduced branching of the ureteric bud (UB) and fewer nephrons. We have observed that this abnormality is associated with a striking increase in the number of UB cells undergoing programmed cell death during nephrogenesis. To ascertain whether apoptosis is directly linked to the level of PAX2 expression, we have studied the role of PAX2 in cultured renal cells. We show that mIMCD-3 cells, a murine collecting duct cell line with high endogenous PAX2 expression, undergo apoptosis when transfected with anti-sense PAX2. In contrast, HEK293 cells expressing exogenous PAX2 are protected against apoptotic death induced by caspase-2. PAX2 has no effect on proliferation of embryonic kidney or in cultured kidney cells. Our observations imply a direct role for PAX2 in survival of ureteric bud cells.

A Common Variant of the PAX2 Gene Is Associated with Reduced Newborn Kidney Size

Congenital nephron number ranges widely in the human population. Suboptimal nephron number may be associated with increased risk for essential hypertension and susceptibility to renal injury, but the factors that set nephron number during kidney development are unknown. In renal-coloboma syndrome, renal hypoplasia and reduced nephron number are due to heterozygous mutations of the PAX2 gene. This study tested for an association between a common haplotype of the PAX2 gene and subtle renal hypoplasia in normal newborns. A PAX2 haplotype was identified to occur in 18.5% of the newborn cohort, which was significantly associated with a 10% reduction in newborn kidney volume adjusted for body surface area. This haplotype was also associated with reduced allele-specific PAX2 mRNA level in a human renal cell carcinoma cell line. Subtle renal hypoplasia in normal newborns may be partially due to a common variant of the PAX2 gene that reduces mRNA expression during kidney development.

Effect of nitisinone (NTBC) treatment on the clinical course of hepatorenal tyrosinemia in Québec.

BACKGROUND Hepatorenal tyrosinemia (HT1, fumarylacetoacetate hydrolase deficiency, MIM 276700) can cause severe hepatic, renal and peripheral nerve damage. In Québec, HT1 is frequent and neonatal HT1 screening is practiced. Nitisinone (NTBC, Orfadin ®) inhibits tyrosine degradation prior to the formation of toxic metabolites like succinylacetone and has been offered to HT1 patients in Québec since 1994. METHODS We recorded the clinical course of 78 Québec HT1 patients born between 1984 and 2004. There were three groups: those who never received nitisinone (28 patients), those who were first treated after 1 month of age (26 patients) and those treated before 1 month (24 patients). Retrospective chart review was performed for events before 1994, when nitisinone treatment began, and prospective data collection thereafter. FINDINGS No hospitalizations for acute complications of HT1 occurred during 5731 months of nitisinone treatment, versus 184 during 1312 months without treatment (p<0.001). Liver transplantation was performed in 20 non-nitisinone-treated patients (71%) at a median age of 26 months, versus 7 late-treated patients (26%, p<0.001), and no early-treated patient (p<0.001). No early-treated patient has developed detectable liver disease after more than 5 years. Ten deaths occurred in non-nitisinone treated patients versus two in treated patients (p<0.01). Both of the latter deaths were from complications of transplantation unrelated to HT1. One probable nitisinone-related event occurred, transient corneal crystals with photophobia. INTERPRETATION Nitisinone treatment abolishes the acute complications of HT1. Some patients with established liver disease before nitisinone treatment eventually require hepatic transplantation. Patients who receive nitisinone treatment before 1 month had no detectable liver disease after more than 5 years.

Hypercalcemia of the newborn: etiology, evaluation, and management

Abstract Hypercalcemia in infants is uncommon but has potentially serious sequelae. This review examines four cases of neonatal hypercalcemia, emphasizing appropriate investigations and treatment of acute and chronic hypercalcemia. The paper provides additional information as to the mechanisms of calcium dysregulation in idiopathic infantile hypercalcemia, Williams syndrome, vitamin D intoxication, and parathyroid and parathyroid-related protein disturbances.

Suppression of Ureteric Bud Apoptosis Rescues Nephron Endowment and Adult Renal Function in Pax2 Mutant Mice

The molecular mechanisms that set congenital nephron number are unknown. However, humans with modest suboptimal nephron number may be at increased risk for essential hypertension, and those with more severe nephron deficits at birth may develop progressive renal insufficiency. A model of branching morphogenesis during fetal kidney development in which the extent of ureteric bud arborization is dependent on suppression of programmed cell death has been proposed. This study shows that the increased apoptosis and reduced ureteric bud branching of heterozygous Pax2 mutant mice is associated with 40% decrease in nephron number at birth. This leads to postnatal glomerular hypertrophy and long-term renal insufficiency in the absence of glomerulosclerosis. To determine whether restoration of antiapoptotic factors alone is sufficient to rescue the nephron deficit in these mice, a BCL2 transgene that is under the control of the PAX2 promoter was targeted to the ureteric bud. The transgene suppressed programmed cell death in the ureteric bud lineage, increased nephron number to 90% of that of wild-type littermates at birth, and normalized renal function at 1 yr. These observations lend strong support to the hypothesis that factors that control ureteric bud apoptosis are powerful determinants of congenital nephron endowment.

Responsiveness of Hypercalciuria to Thiazide in Dent’s Disease

Hypercalciuria is the major risk factor promoting stone formation in Dents disease, also known as X-linked recessive nephrolithiasis, but the effects of diuretics on calcium excretion and other stone risk factors in this disease are unknown. This study examined urine composition in eight male patients with Dents disease, ages 6 to 49 yr, all of whom were hypercalciuric and had inactivating mutations of CLCN5. Eight males, ages 7 to 34 yr, with idiopathic hypercalciuria (IH) served as controls. Patients were instructed to maintain a consistent intake of sodium, potassium, calcium, and protein. Two consecutive 24-h urine collections were obtained after a baseline period and after 2 wk of chlorthalidone (25 mg), amiloride (5 mg), and the two diuretics in combination, with a week off drug separating the treatment periods in a randomized crossover design. Doses were reduced by half in boys under age 12 yr. Chlorthalidone alone (P < 0.002) and the combination of chlorthalidone and amiloride (P < 0.003) reduced calcium excretion significantly in either patient group. With chlorthalidone, calcium excretion fell to normal (<4.0 mg/kg per d) in all but one patient in each group. Amiloride alone had no significant effect on urinary calcium excretion, in either patient group. In patients with Dents disease during chlorthalidone therapy, the supersaturation ratios for calcium oxalate and calcium phosphate fell by 25% and 35%, respectively. Mean citrate excretion was reduced by chlorthalidone (P <.04) and by chlorthalidone in combination with amiloride (P <.02). There were no significant differences in the responses to these diuretics between the patient groups in any of the urinary parameters. The intact hypocalciuric response to a thiazide diuretic indicates that inactivation of the ClC-5 chloride channel does not impair calcium transport in the distal convoluted tubule and indicates that thiazides should be useful in reducing the risk of kidney stone recurrence in patients with Dents disease.