Increased renal calcium and magnesium transporter abundance in streptozotocin-induced diabetes mellitus.

Diabetes is associated with renal calcium and magnesium wasting, but the molecular mechanisms of these defects are unknown. We measured renal calcium and magnesium handling and investigated the effects of diabetes on calcium and magnesium transporters in the thick ascending limb and distal convoluted tubule in streptozotocin (STZ)-induced diabetic rats. Rats were killed 2 weeks after inducing diabetes, gene expression of calcium and magnesium transporters in the kidney was determined by real-time polymerase chain reaction, and the abundance of protein was assessed by immunoblotting. Our results showed that diabetic rats had significant increase in the fractional excretion for calcium and magnesium (both P < 0.01), but not for sodium. Reverse transcriptase-polymerase chain reaction revealed significant increases in messenger RNA abundance of transient potential receptor (TRP) V5 (223 +/- 10%), TRPV6 (177 +/- 9%), calbindin-D28k (231 +/- 8%), and TRPM6 (165 +/- 8%) in diabetic rats. Sodium chloride cotransporter was also increased (207 +/- 10%). No change was found in paracellin-1 (cortex: 108 +/- 8%; medulla: 110 +/- 10%). Immunofluorescent studies of renal sections showed significant increase in calbindin-D28k (238 +/- 10%) and TRPV5 (211 +/- 10%), but no changes in paracellin-1 in Western blotting (cortex: 110 +/- 7%; medulla: 99 +/- 7%). Insulin administration completely corrected the hyperglycemia-associated hypercalciuria and hypermagnesiuria, and reversed the increase of calcium and magnesium transporter abundance. In conclusion, our results demonstrated increased renal calcium and magnesium transporter abundance in STZ-induced diabetic rats, which may represent a compensatory adaptation for the increased load of calcium and magnesium to the distal tubule.

Analysis of splice-site mutations of the alpha-galactosidase A gene in Fabry disease.

Fabry disease is an X-linked disease caused by a defective lysosomal enzyme, alpha-galactosidase A, and characterized by skin lesions and multiorgan involvement, including kidney, heart, and the central nervous system. Currently more than 200 genotypes have been identified, including several aberrant splicing. However, most of the mutation analyses were performed using genomic sequencing only, and therefore some of the splicing mutations were misclassified as missense mutations. In order to predict the splicing event caused by each mutation, we conducted a literature search for all published mutations located near the splice sites, including exonic point mutations, and performed a splice-site score (SSS) analysis. The literature search identified 13 donor-site mutations, including four exonic mutations (S65T, D183S, K213N, and M267I), located at the end of exons 1, 3, 4, and 5, respectively, six acceptor-site mutations, and one new exon creation. All mutated splice sites, except for the one associated with the new exon creation, had a lower SSS than their respective natural sites. Cryptic or newly created sites were identified with SSS from 0.09 to 1.0. The predictions, based on SSS analysis, are in agreement with all six mutations with known cDNA sequence from the literature, including five mutations with exon skipping and one mutation with creation of a new acceptor site. For the S65T genotype, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis using RNA isolated from the whole-blood sample. We verified that a weak cryptic site (SSS = 0.09) 14 nucleotides downstream was activated and resulted in an insertion of 14 bp and a frameshift stop at codon 106. This change is more consistent with the clinical presentation of the patient, the classical Fabry disease, than the amino acid substitution (S65T), which does not affect the enzyme function. In conclusion, the SSS analysis is very useful for predicting splicing events and genotype/phenotype correlation in Fabry disease. As different mechanisms may be involved in pre-mRNA splicing, it is important to obtain cDNA sequencing for molecular diagnosis.

Two novel mutations in the alpha-galactosidase A gene in Chinese patients with Fabry disease.

Fabry disease is an X-linked disorder caused by a deficiency of the lysosomal alpha-galactosidase A [EC 3.2.1.22]. The molecular diagnosis of Fabry disease is important for genotype/phenotype correlation, pre-natal or early diagnosis, and detection of carrier status. Although more than 200 genotypes of the alpha-galactosidase A gene have been identified, mutation data on the Chinese population is sparse. We recently identified two unrelated Chinese families with Fabry disease. Mutation analysis was performed by polymerase chain reaction (PCR) sequencing of the seven exons and adjacent introns of the alpha-galactosidase A gene. Two novel mutations were identified: in family I, a C-to-A transversion resulted in an early termination at amino acid 222 (Y222X), while in family II, an A-to-G transition resulted in a substitution of alanine for threonine at amino acid 410 (T410A). Carrier status was identified in all four females in the two families. The genotype Y222X is associated with classic Fabry disease, with unexpectedly rapid deterioration of visual acuity, while T410A is associated with a milder Fabry disease, with ventricular hypertrophy and neuropathic pain.