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Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro.

ABSTRACT: In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11-->Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 degrees C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 degrees C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.

SUBMITTER: Lumb MJ

PROVIDER: S-EPMC1223567 | biostudies-other | 2003 Aug

REPOSITORIES: biostudies-other

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Project description:Individuals with autosomal dominant polycystic kidney disease have a higher incidence of stone formation than the general population. However, there are no cystic animal models known to develop stones. Cystic mice compound heterozygous for hypomorphic Pkd1V and Pkd1RC alleles develop cystic kidneys within a few weeks of birth but live beyond 20 wk of age, allowing for the study of cystic comorbidities including stone formation. Cystic Pkd1V/RC mice were euthanized at 3, 13, or 26 wk of age, and their kidneys were analyzed by microcomputed tomography (µCT) for stone formation. Mice had occasional mineral aggregates that could be detected by µCT analysis at 3 wk of age. At 13 or 26 wk of age, numerous white masses were visible beneath the kidney surface. µCT analysis confirmed the masses to be large mineral stone deposits throughout the renal cortex, with mineral content increasing with age. Staining of histological sections with alizarin red and von Kossa suggested that the stone deposits were composed primarily of calcium and phosphate. Microdissection confirmed stones localized within cyst lumens. Analysis of individual stones by µCT and infrared spectroscopy confirmed apatite mineral composition. Urinalysis revealed elevated levels of phosphate and citrate at 3 wk of age and lower pH and elevated levels of calcium and citrate at 13 wk of age, suggesting altered phosphate and calcium homeostasis as a potential cause of mineralization and renal stone formation. This is the first animal model exhibiting overt kidney stone formation in the context of cystic kidney disease.NEW & NOTEWORTHY Compound heterozygous Pkd1V/RC mice were found to form calcium phosphate-containing stones within cysts of the renal cortex by 13 wk of age. This is the first polycystic kidney disease animal model exhibiting spontaneous stone formation. A growing body of evidence suggests a link between renal stone formation and cystic kidney disease. This mouse model may be useful for studying the interplay between stone and cyst formation and the functional role of polycystins in mineral homeostasis.

Project description:BackgroundKidney stones or nephrolithiasis is a chronic metabolic disease characterized by renal colic and hematuria. Currently, a pathogenetic mechanism resulting in kidney stone formation remains elusive. We performed a multi-omic study investigating urinary microbial compositions and metabolic alterations during nephrolithiasis.MethodUrine samples from healthy and individuals with nephrolithiasis were collected for 16S rRNA gene sequencing and liquid chromatography-mass spectroscopy. Microbiome and metabolome profiles were analyzed individually and combined to construct interactome networks by bioinformatic analysis.ResultsDistinct urinary microbiome profiles were determined in nephrolithiasis patients compared with controls. Thirty-nine differentially abundant taxa between controls and nephrolithiasis patients were identified, and Streptococcus showed the most significant enrichment in nephrolithiasis patients. We also observed significantly different microbial compositions between female and male nephrolithiasis patients. The metabolomic analysis identified 112 metabolites that were differentially expressed. Two significantly enriched metabolic pathways, including biosynthesis of unsaturated fatty acids and tryptophan metabolism, were also identified in nephrolithiasis patients. Four potentially diagnostic metabolites were also identified, including trans-3-hydroxycotinine, pyroglutamic acid, O-desmethylnaproxen, and FAHFA (16:0/18:2), and could function as biomarkers for the early diagnosis of nephrolithiasis. We also identified three metabolites that contributed to kidney stone size. Finally, our integrative analysis of the urinary tract microbiome and metabolome identified distinctly different network characteristics between the two groups.ConclusionsOur study has characterized important profiles and correlations among urinary tract microbiomes and metabolomes in nephrolithiasis patients for the first time. These results shed new light on the pathogenesis of nephrolithiasis and could provide early clinical biomarkers for diagnosing the disease.

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Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro.

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