Pharmacokinetics, Pharmacodynamics, and Safety of Lisinopril in Pediatric Kidney Transplant Patients: Implications for Starting Dose Selection.

Hypertension in pediatric kidney transplant recipients contributes to long-term graft loss, yet treatment options--including angiotensin-converting enzyme inhibitors--are poorly characterized in this vulnerable population. We conducted a multicenter, open-label pharmacokinetic (PK) study of daily oral lisinopril in 22 children (ages 7-17 years) with stable kidney transplant function. Standard noncompartmental PK analyses were performed at steady state. Effects on blood pressure were examined in lisinopril-naïve patients (n = 13). Oral clearance declined in proportion to underlying kidney function; however, in patients with low estimated glomerular filtration rate (30-59 ml/min per 1.73m(2)), exposure (standardized to 0.1 mg/kg/day dose) was within the range reported previously in children without a kidney transplant. In lisinopril-naïve patients, 85% and 77% had a ≥ 6 mmHg reduction in systolic and diastolic blood pressure, respectively. Lisinopril was well tolerated. Our study provides initial insight on lisinopril use in children with a kidney transplant, including starting dose considerations.

3-5 year longitudinal follow-up of pediatric patients after acute renal failure.

Few data exist regarding the long-term sequelae of acute renal failure (ARF), and these studies are limited to a few renal conditions. We aim to assess the 3-5-year survival and incidence of renal injury in children who previously developed ARF of varying causes. We queried parents, physicians, and hospital/state vital statistics records to find patient survival in 174 children who previously had ARF and survived to hospital discharge. We assessed the following in 29 children for residual renal injury: (a) microalbuminuria, (b) glomerular filtration rate (GFR) by Schwartz formula, (c) hypertension, and (d) hematuria. The 3-5-year survival of children with ARF who survived to hospital discharge was 139/174 (79.9%). Most deaths (24/35 (68.5%)) occurred within 12 months after initial hospitalization. Combining those who died during initial hospitalization and in subsequent 3-5 years, the overall survival rate was 139/245 (56.8%). In all, 16 children progressed to end-stage renal disease; thus, renal survival was 127/173 (91%). Those with primary renal/urologic conditions had lower renal survival than others (24/35 (68.6%) vs 134/139 (96.4%); P<0.0001). Among the 29 patients assessed for long-term sequelae at 3-5 years, 17/29 (59%) subjects had at least one sign of renal injury; microalbuminuria (n=9), hyperfiltration (n=9), decreased GFR (n=4), and hypertension (n=6). A pediatric nephrologist was involved in care of only 6/17 (35%) with chronic renal injury. Patients have high risks of ongoing residual renal injury and death after ARF; therefore, periodic evaluation after the initial insult is necessary.

Mutagenesis by metal-induced oxygen radicals.

To assess the contribution of reactive oxygen species (ROS) to metal-induced mutagenesis, we have determined the spectrum of mutations in the lacZ alpha gene after exposure of M13mp2 DNA to Fe2+, Cu2+, and Ni2+. With iron and copper ions, mutations are clustered and are predominantly single-base substitutions. Fe, Cu, and phorbol ester-stimulated neutrophils also produced tandem double CC-->TT mutations. This mutation may provide a marker for the role of oxidative damage in carcinogenesis. Mutagenesis by Ni2+ required the complexing of the metal to a tripeptide and the addition of H2O2. To assess the contribution of ROS in mammalian cells, we determined the spectrum of mutations produced when purified DNA polymerases-alpha and -beta synthesized DNA using a template that had been damaged by ROS. The mutation spectra produced by the two polymerases indicates that these enzymes substitute different nucleotides opposite the same lesions.

Reverse chemical mutagenesis: identification of the mutagenic lesions resulting from reactive oxygen species-mediated damage to DNA.

An understanding of the contribution of reactive oxygen species to mutagenesis has been hampered by the vast number of different chemical modifications they cause in DNA. Even though many of these DNA alterations have been catalogued, the identification of specific lesions that cause mutations has depended on testing one modification at a time. In this study we present another approach to identify key mutagenic lesions from a pool of oxidatively modified nucleotides. dCTP was treated with an oxygen radical-generating system containing FeSO4, H2O2, and ascorbic acid. The modification products were separated by reverse-phase and anion-exchange HPLC and then incorporated by human immunodeficiency virus reverse transcriptase into a DNA that contains a target gene for scoring for mutations. One of the mutagenic species isolated was identified as 5-hydroxy-2'-deoxycytidine. It is incorporated efficiently into DNA and causes C-->T transitions in Escherichia coli at a frequency of 2.5%, which is more mutagenic than any previously identified oxidative DNA lesion.

Reactive oxygen species in tumorigenesis.

In this review we will summarize recent data on reactive oxygen species-induced mutagenesis and consider its relationship to tumorigenesis in humans. With the use of a single-stranded DNA template it has been possible to correlate oxygen radical-induced chemical alterations at specific nucleotides with the types of mutations that occur when these altered bases are copied by DNA polymerases. This has allowed us to identify the types of mutations that occur secondary to a variety of oxidative stresses and study several of the mechanisms by which they arise. The most frequent mutations that result from reactive oxygen species-induced damage to DNA in bacteria are C to T transitions. These mutations, however, are not pathoneumonic for mutagenesis by oxygen-free radicals since they result from DNA damage caused by other genotoxic agents as well as by DNA polymerase errors. One type of mutation, a tandem CC to TT double substitution, has been shown to be induced by reactive oxygen species generated by a variety of systems and may be diagnostic for such damage. In studies with mammalian DNA polymerases, DNA damaged by reactive oxygen species yields mutations different from those observed in Escherichia coli. This diversity of mutagenic changes in these in vitro studies highlights the role of DNA replicating enzymes in specifying the types of mutations produced by reactive oxygen species. In conclusion, we will consider the role of reactive oxygen species in the pathogenesis of three common tumors, carcinoma of the liver, lung, and prostate with consideration on the possible use of antioxidant preventive therapy to slow tumorigenesis sufficiently to prevent clinical presentation of these cancers during the life span of a patient.

Oxygen radical induced mutagenesis is DNA polymerase specific.

Oxygen free radicals are produced in large amounts by normal cellular processes. Damage to DNA by these reactive species has been implicated in mutagenesis and may be important in the etiology of a variety of human diseases. In this study we investigate the types of mutations produced in vitro as a result of DNA damage by oxygen free radicals. We used a lacZ alpha forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase alpha or beta, transfected into E. coli, and screened for mutations by reduced alpha-complementation of beta-galactosidase activity. By determining the effects of damaged templates on the fidelity of individual DNA polymerases involved in replication and repair, we address the role of specific DNA polymerases in mutagenesis induced by reactive oxygen species. Aerobic incubation of DNA with 100 microM CuCl, 10 microM H2O2 and 100 microM ascorbic acid results in a 3.3-fold and a 3.6-fold elevation in mutation frequency for polymerases alpha and beta, respectively. The specificity and location of the induced mutations, however, are entirely different. For polymerase alpha, A to C, and C to A transversions and deletions of C are each elevated more than 10-fold over their frequencies on undamaged template. For polymerase beta, A to T, C to T, C to A, G to C, and G to T substitutions, and deletions of G are elevated by damage. The frequency of mutants containing two or more closely spaced substitutions is also markedly increased by template damage although the types of mutations and their positions are again specific to each DNA polymerase. We conclude that, for oxidative lesions, the frequency and the types of mutations are determined in part by the DNA polymerase that encounters the site of damage.

Mechanisms of mutation by oxidative DNA damage: reduced fidelity of mammalian DNA polymerase beta.

Reactive oxygen species, produced in cells by a variety of mechanisms, damage DNA and cause mutations. To characterize the types of mutations produced in mammalian cells, we copied DNA damaged by reactive oxygen species with mammalian DNA polymerase beta. Double-stranded circular M13mp2 DNA containing a 361-nucleotide single-stranded gap within the lacZ gene was damaged by aerobic incubation with Fe2+ and H2O2. The gap then was filled by purified recombinant rat DNA polymerase beta, and the DNA was transfected into Escherichia coli. Mutations within the nonessential lacZ gene for beta-galactosidase were identified by reduced alpha-complementation. In this system, oxidative damage increased the mutation frequency within the target region by an average of 4.3-fold. At certain sites, the base substitution rate is nearly 300 times greater than would be expected to result from a random distribution of damage. The oxidatively induced mutations fall into two categories: those apparently caused by direct miscoding of modified DNA and those associated with enhanced misincorporation at prexisting polymerase-specific hot spots. The latter group may be due to a conformational change in the DNA caused by oxidative modification and could be indicative of a novel mutagenic mechanism.

The C proteins of heterogeneous nuclear ribonucleoprotein complexes interact with RNA sequences downstream of polyadenylation cleavage sites.

The heterogeneous nuclear ribonucleoprotein C1 and C2 proteins were preferentially cross-linked by treatment with UV light in nuclear extracts to RNAs containing six different polyadenylation signals. The domain required for the interaction was located downstream of the poly(A) cleavage site, since deletion of this segment from several polyadenylation substrate RNAs greatly reduced cross-linking efficiency. In addition, RNAs containing only downstream sequences were efficiently cross-linked to C proteins, while fully processed, polyadenylated RNAs were not. Analysis of mutated variants of the simian virus 40 late polyadenylation signal showed that uridylate-rich sequences located in the region between 30 and 55 nucleotides downstream of the cleavage site were required for efficient cross-linking of C proteins. This downstream domain of the simian virus 40 late poly(A) addition signal has been shown to influence the efficiency of the polyadenylation reaction. However, there was not a strict correlation between cross-linking of C proteins and the efficiency of polyadenylation.