LC-MS-based metabolomics analysis to identify meprin-ß-associated changes in kidney tissue from mice with STZ-induced type 1 diabetes and diabetic kidney injury.

Meprin metalloproteases have been implicated in the pathophysiology of diabetic kidney disease (DKD). Single-nucleotide polymorphisms in the meprin-ß gene have been associated with DKD in Pima Indians, a Native American ethnic group with an extremely high prevalence of DKD. In African American men with diabetes, urinary meprin excretion positively correlated with the severity of kidney injury. In mice, meprin activity decreased at the onset of diabetic kidney injury. Several studies have identified meprin targets in the kidney. However, it is not known how proteolytic processing of the targets by meprins impacts the metabolite milieu in kidneys. In the present study, global metabolomics analysis identified differentiating metabolites in kidney tissues from wild-type and meprin-ß knockout mice with streptozotocin (STZ)-induced type 1 diabetes. Kidney tissues were harvested at 8 wk post-STZ and analyzed by hydrophilic interaction liquid chromatography ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Principal component analysis identified >200 peaks associated with diabetes. Meprin expression-associated metabolites with strong variable importance of projection scores were indoxyl sulfate, N-γ-l-glutamyl-l-aspartic acid, N-methyl-4-pyridone-3-carboxamide, inosine, and cis-5-decenedioic acid. N-methyl-4-pyridone-3-carboxamide has been previously implicated in kidney injury, and its isomers, 4-PY and 2-PY, are markers of peroxisome proliferation and inflammation that correlate with creatinine clearance and glucose tolerance. Meprin deficiency-associated differentiating metabolites with high variable importance of projection scores were cortisol, hydroxymethoxyphenylcarboxylic acid-O-sulfate, and isovaleryalanine. The data suggest that meprin-ß activity enhances diabetic kidney injury in part by altering the metabolite balance in kidneys, favoring high levels of uremic toxins such as indoxyl sulfate and N-methyl-pyridone-carboxamide.

Meprin ß metalloproteases associated with differential metabolite profiles in the plasma and urine of mice with type 1 diabetes and diabetic nephropathy.

BACKGROUND: Meprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules and small intestines. Meprins are also expressed in podocytes and leukocytes (monocytes and macrophages). Meprins are implicated in the pathophysiology of diabetic nephropathy (DN) but underlying mechanisms are not fully understood. Single nucleotide polymophisms (SNPs) in the meprin ß gene were associated with DKD in human subjects. Furthermore, meprin α and ß double deficiency resulted in more severe kidney injury and higher mortality rates in mice with Streptozotocin (STZ)-induced type 1 diabetes. Identification of meprin substrates has provided insights on how meprins could modulate kidney injury. Meprin targets in the kidney include extracellular matrix (ECM) proteins, modulators of inflammation, and proteins involved in the protein kinase A (PKA) and PKC signaling pathways. The current study used a global metabolomics approach to determine how meprin ß expression impacts the metabolite milieu in diabetes and DKD. METHODS: Low dose STZ was used to induce type 1 diabetes in 8-week old wild-type (WT) and meprin ß knockout (ßKO) mice. Blood and urine samples were obtained at 4 and 8 weeks post-STZ injection. Assays for albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule - 1 (KIM-1), and cystatin C were used for biochemical assessment of kidney injury. Data for biomarkers of kidney injury utilized two-way ANOVA. Metabolomics data analysis utilized UPLC-QTOF MS and multivariate statistics. RESULTS: The number of metabolites with diabetes-associated changes in levels were significantly higher in the WT mice when compared to meprin ßKO counterparts. Annotated meprin ß expression-associated metabolites with strong variable importance in projection (VIP) scores play roles in lipid metabolism (LysoPC(16:1(9Z)), taurocholic acid), amino acid metabolism (indoxyl sulfate, hippuric acid), and neurotransmitter/stress hormone synthesis (cortisol, 3-methoxy-4-hydroxyphenylethylene glycolsulfate, homovanillic acid sulfate). Metabolites that associated with meprin ß deficiency include; 3,5-dihydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavone 3-glucuronide, pantothenic acid, and indoxyl glucuronide (all decreased in plasma). CONCLUSION: Taken together, the annotated metabolites suggest that meprin ß impacts complications of diabetes such as DKD by altering distinct metabolite profiles.

A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae.

BACKGROUND: The second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence. RESULTS: Complementary in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization. CONCLUSIONS: These results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of V. cholerae.