Predicting and Defining Steroid Resistance in Pediatric Nephrotic Syndrome Using Plasma Metabolomics.

INTRODUCTION: Nephrotic syndrome (NS) is a kidney disease that affects both children and adults. Glucocorticoids have been the primary therapy for >60 years but are ineffective in approximately 20% of children and approximately 50% of adult patients. Unfortunately, patients with steroid-resistant NS (SRNS; vs. steroid-sensitive NS [SSNS]) are at high risk for both glucocorticoid-induced side effects and disease progression. METHODS: We performed proton nuclear magnetic resonance (1H NMR) metabolomic analyses on plasma samples (n = 86) from 45 patients with NS (30 SSNS and 15 SRNS) obtained at initial disease presentation before glucocorticoid initiation and after approximately 7 weeks of glucocorticoid therapy to identify candidate biomarkers able to either predict SRNS before treatment or define critical molecular pathways/targets regulating steroid resistance. RESULTS: Stepwise logistic regression models identified creatinine concentration and glutamine concentration (odds ratio [OR]: 1.01; 95% confidence interval [CI]: 0.99-1.02) as 2 candidate biomarkers predictive of SRNS, and malonate concentration (OR: 0.94; 95% CI: 0.89-1.00) as a third candidate predictive biomarker using a similar model (only in children >3 years). In addition, paired-sample analyses identified several candidate biomarkers with the potential to identify mechanistic molecular pathways/targets that regulate clinical steroid resistance, including lipoproteins, adipate, pyruvate, creatine, glucose, tyrosine, valine, glutamine, and sn-glycero-3-phosphcholine. CONCLUSION: Metabolomic analyses of serial plasma samples from children with SSNS and SRNS identified elevated creatinine and glutamine concentrations, and reduced malonate concentrations, as auspicious candidate biomarkers to predict SRNS at disease onset in pediatric NS, as well as additional candidate biomarkers with the potential to identify mechanistic molecular pathways that may regulate clinical steroid resistance.

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.