Structure-activity relationships and pharmacokinetic evaluation of L-cystine diamides as L-cystine crystallization inhibitors for cystinuria.

Cystinuria is a rare genetic disorder characterized by defective l-cystine reabsorption from the renal proximal tubule, resulting in abnormally high concentrations of L-cystine and subsequent l-cystine crystallization and stone formation in urine. l-Cystine diamides have shown great promise as inhibitors of l-cystine crystallization. The free α-amino groups in l-cystine diamides have previously been shown to be necessary for l-cystine crystallization inhibitory activity.

8-l-Cystinyl Bis(1,8-diazaspiro[4.5]decane) as an Orally Bioavailable l-Cystine Crystallization Inhibitor for Cystinuria.

Cystinuria, a rare genetic disorder, is characterized by defective l-cystine reabsorption from the renal proximal tubule, resulting in abnormally high concentrations of l-cystine and subsequent l-cystine crystallization in urine and stone formation in the urinary tract. Inhibition of l-cystine crystallization by l-cystine diamides such as LH708 () represents a promising new approach to prevent stone formation in patients with cystinuria. While shows promising efficacy and a good safety profile in a -knockout mouse model of cystinuria, further structural modification of led to the discovery of 8-l-cystinyl bis(1,8-diazaspiro[4.

Development of convenient crystallization inhibition assays for structure-activity relationship studies in the discovery of crystallization inhibitors.

Kidney stone diseases are increasing globally in prevalence and recurrence rates, indicating an urgent medical need for developing new therapies that can prevent stone formation. One approach we have been working on is to develop small molecule inhibitors that can interfere with the crystallization process of the chemical substances that form the stones. For these drug discovery efforts, it is critical to have available easily accessible assay methods to evaluate the potential inhibitors and rank them for structure-activity relationship studies.

Disrupting Crystal Growth through Molecular Recognition: Designer Therapies for Kidney Stone Prevention.

Aberrant crystallization within the human body can lead to several disease states or adverse outcomes, yet much remains to be understood about the critical stages leading to these events, which can include crystal nucleation and growth, crystal aggregation, and the adhesion of crystals to cells. Kidney stones, which are aggregates of single crystals with physiological origins, are particularly illustrative of pathological crystallization, with 10% of the U.S.

The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac.

The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood.

Metabolic consequences of cystinuria.

Background: Cystinuria is an inherited disorder of renal amino acid transport that causes recurrent nephrolithiasis and significant morbidity in humans. It has an incidence of 1 in 7000 worldwide making it one of the most common genetic disorders in man. We phenotypically characterized a mouse model of cystinuria type A resultant from knockout of Slc3a1.

Cystinuria: genetic aspects, mouse models, and a new approach to therapy.

Cystinuria, a genetic disorder of cystine transport, is characterized by excessive excretion of cystine in the urine and recurrent cystine stones in the kidneys and, to a lesser extent, in the bladder. Males generally are more severely affected than females. The disorder may lead to chronic kidney disease in many patients.

Role of Molecular Recognition in l-Cystine Crystal Growth Inhibition.

l-Cystine kidney stones-aggregates of single crystals of the hexagonal form of l-cystine-afflict more than 20 000 individuals in the United States alone. Current therapies are often ineffective and produce adverse side effects. Recognizing that the growth of l-cystine crystals is a critical step in stone pathogenesis, real-time in situ atomic force microscopy of growth on the (0001) face of l-cystine crystals and measurements of crystal growth anisotropy were performed in the presence of prospective inhibitors drawn from a 31-member library.

α-Lipoic acid treatment prevents cystine urolithiasis in a mouse model of cystinuria.

Cystinuria is an incompletely dominant disorder characterized by defective urinary cystine reabsorption that results in the formation of cystine-based urinary stones. Current treatment options are limited in their effectiveness at preventing stone recurrence and are often poorly tolerated. We report that the nutritional supplement α-lipoic acid inhibits cystine stone formation in the Slc3a1 mouse model of cystinuria by increasing the solubility of urinary cystine.

l-Cystine Diamides as l-Cystine Crystallization Inhibitors for Cystinuria.

l-Cystine bismorpholide (1a) and l-cystine bis(N'-methylpiperazide) (1b) were seven and twenty-four times more effective than l-cystine dimethyl ester (CDME) in increasing the metastable supersaturation range of l-cystine, respectively, effectively inhibiting l-cystine crystallization. This behavior can be attributed to inhibition of crystal growth at microscopic length scale, as revealed by atomic force microscopy. Both 1a and 1b are more stable than CDME, and 1b was effective in vivo in a knockout mouse model of cystinuria.

Cystine growth inhibition through molecular mimicry: a new paradigm for the prevention of crystal diseases.

Cystinuria is a genetic disease marked by recurrent kidney stone formation, usually at a young age. It frequently leads to chronic kidney disease. Treatment options for cystinuria have been limited despite comprehensive understanding of its genetic pathophysiology.

Novel cystine ester mimics for the treatment of cystinuria-induced urolithiasis in a knockout mouse model.

Objective: To assess the effectiveness of l-cystine dimethyl ester (CDME), an inhibitor of cystine crystal growth, for the treatment of cystine urolithiasis in an Slc3a1 knockout mouse model of cystinuria.

Materials And Methods: CDME (200 μg per mouse) or water was delivered by gavage daily for 4 weeks. Higher doses by gavage or in the water supply were administered to assess organ toxicity.

Certification in molecular pathology in the United States: an update from the Association for Molecular Pathology Training and Education Committee.

The past 25 years have witnessed the field of molecular pathology evolving from an imprecisely defined discipline to a firmly established medical subspecialty that plays an essential role in patient care. During this time, the training, certification, and licensure requirements for directing and performing testing in a molecular pathology or molecular diagnostics laboratory have become better defined. The purpose of this document is to describe the various board certifications available to individuals seeking certification in molecular diagnostics at the level of laboratory director, supervisor, or technologist.

2,8-dihydroxyadenine nephrolithiasis induces developmental stage-specific alterations in gene expression in mouse kidney.

Objectives: To identify factors that may be crucial for the initiation and progression of stone-induced injury in the developing mouse kidney by a prospective observational study using microarray analysis. Kidney stone diseases are common in premature infants, but the underlying molecular and cellular mechanisms are not fully defined.

Methods: Mice with adenine phosphoribosyltransferase deficiency develop 2,8-dihydroxyadenine (DHA) nephrolithiasis.

Bladder outlet obstruction in male cystinuria mice.

Background: Cystinuria is the most common inherited cause of urinary tract stones in children. It can lead to obstructive uropathy, which is a major cause of renal failure. Genetic studies have identified two genes, SLC3A1 and SLC7A9, to be directly involved in cystine stone formation.

Preparing DNA from Mammalian sources for genotyping.

INTRODUCTIONThe availability of high-quality DNA from a large number of individuals is a prerequisite for the success of genetic variation studies. This requirement has spurred major technological advances in DNA extraction methodologies. Twenty years ago, large-scale manual extractions took >3 d to complete.

Preparing DNA from saliva for genotyping.

INTRODUCTIONSaliva may be a viable alternative to blood as a source for DNA, and has the advantage that it is collected noninvasively. This protocol describes the Oragene procedure for DNA extraction from 2-mL saliva samples. It yields high-quality DNA suitable for research and diagnostic applications.

Preparing DNA from blood for genotyping.

INTRODUCTIONThis protocol describes the extraction of genomic DNA from whole blood samples (fresh or frozen) and buffy coats. It provides methods for (1) large-scale extraction of DNA using either in-house or commercial (PUREGENE) reagents; (2) mid-scale extraction of DNA using the QIAamp DNA Blood Midi Kit (for 0.5-mL samples); and (3) small-scale extraction of DNA using the QIAamp DNA Blood Mini Kits (for 200-μL samples).

Preparing DNA from cell pellets for genotyping.

INTRODUCTIONThis protocol describes large-scale DNA extraction from cell pellets using either in-house or commercial (PUREGENE) reagents. The cell pellets are prepared from human lymphoblast cell lines (LCLs) that have been established using the Epstein-Barr virus (EBV).

Expression profiling of crystal-induced injury in human kidney epithelial cells.

Background: Deposition of crystals within tubular lumens is a feature of many kidney stone diseases, including crystals of calcium oxalate monohydrate (COM) in primary hyperoxaluria and of 2,8-dihydroxyadenine (DHA) in adenine phosphoribosyltransferase deficiency. Crystals are injurious to renal epithelial cells, but the molecular bases of cell injury have not been well characterized.

Methods: We used a cDNA microarray to identify the time-dependent changes in gene expression associated with the interaction of COM or DHA crystals with primary cultures of normal human kidney cortical epithelial cells.

Aprt/Opn double knockout mice: osteopontin is a modifier of kidney stone disease severity.

Background: Osteopontin (OPN) is reported to have two distinct functions in kidney disease: Promotion of inflammation at sites of tissue injury, and inhibition of calcium oxalate monohydrate stone formation. However, many of the studies supporting these functions were carried out in animal models of acute renal injury or in cultured cells; thus, the role of OPN in chronic renal disease is not well defined. We examined the role of OPN in adenine phosphoribosyltransferase (Aprt) knockout mice, in which inflammation and formation of 2,8-dihydroxyadenine (DHA) kidney stones are prominent features, by generating Aprt/Opn double knockout mice.