Kidney urinary biomarkers in patients with branched-chain amino acid and cobalamin metabolism defects.

There is a clinical need for early detection of chronic kidney disease (CKD) in patients with organic acidurias. We measured kidney markers in a longitudinal study over 5 years in 40 patients with methylmalonic aciduria (Mut0 ), propionic aciduria (PA), cobalamin A (CblA), and cobalamin C (CblC) deficiencies. Neutrophil gelatinase-associated lipocalin (NGAL), calprotectin (CLP), kidney injury molecule-1 (KIM-1), dickkopf-3 (DKK-3), albumin and beta-2-microglobulin (B2MG) in urine, as well as cystatin C (CysC) in serum were quantified. In Mut0 patients, mean concentrations of B2MG, KIM-1, and DKK-3 were elevated compared with healthy controls, all markers indicative of proximal tubule damage. In PA patients, mean B2MG, albumin, and CLP were elevated, indicating signs of proximal tubule and glomerulus damage and inflammation. In CblC patients, mean B2MG, NGAL, and CLP were increased, and considered as markers for proximal and distal tubule damage and inflammation. B2MG, was elevated in all three diseases, and correlated with DKK-3 in Mut0 /CblA and with eGFR(CysC) and KIM-1 in PA patients, respectively. None of the markers were elevated in CblA patients. Significant deterioration of kidney function, as determined by steady increase in CysC concentrations was noted in seven patients within the observation period. None of the investigated biomarker profiles showed a clear increase or added value for early detection. In conclusion, we identified disease-specific biomarker profiles for inflammation, tubular, and proximal damage in the urine of Mut0 , PA, and CblC patients. Whether these biomarkers can be used for early detection of CKD requires further investigation, as significant kidney function deterioration was observed in only a few patients.

Carnosinase-1 Knock-Out Reduces Kidney Fibrosis in Type-1 Diabetic Mice on High Fat Diet.

Carnosine and anserine supplementation markedLy reduce diabetic nephropathy in rodents. The mode of nephroprotective action of both dipeptides in diabetes, via local protection or improved systemic glucose homeostasis, is uncertain. Global carnosinase-1 knockout mice (Cndp1-KO) and wild-type littermates (WT) on a normal diet (ND) and high fat diet (HFD) (n = 10/group), with streptozocin (STZ)-induced type-1 diabetes (n = 21-23/group), were studied for 32 weeks. Independent of diet, Cndp1-KO mice had 2- to 10-fold higher kidney anserine and carnosine concentrations than WT mice, but otherwise a similar kidney metabolome; heart, liver, muscle and serum anserine and carnosine concentrations were not different. Diabetic Cndp1-KO mice did not differ from diabetic WT mice in energy intake, body weight gain, blood glucose, HbA1c, insulin and glucose tolerance with both diets, whereas the diabetes-related increase in kidney advanced glycation end-product and 4-hydroxynonenal concentrations was prevented in the KO mice. Tubular protein accumulation was lower in diabetic ND and HFD Cndp1-KO mice, interstitial inflammation and fibrosis were lower in diabetic HFD Cndp1-KO mice compared to diabetic WT mice. Fatalities occurred later in diabetic ND Cndp1-KO mice versus WT littermates. Independent of systemic glucose homeostasis, increased kidney anserine and carnosine concentrations reduce local glycation and oxidative stress in type-1 diabetic mice, and mitigate interstitial nephropathy in type-1 diabetic mice on HFD.

A Global Cndp1-Knock-Out Selectively Increases Renal Carnosine and Anserine Concentrations in an Age- and Gender-Specific Manner in Mice.

Carnosinase 1 (CN1) is encoded by the Cndp1 gene and degrades carnosine and anserine, two natural histidine-containing dipeptides. In vitro and in vivo studies suggest carnosine- and anserine-mediated protection against long-term sequelae of reactive metabolites accumulating, e.g., in diabetes mellitus. We have characterized the metabolic impact of CN1 in 11- and 55-week-old Cndp1-knockout (Cndp1-KO) mice and litter-matched wildtypes (WT). In Cndp1-KO mice, renal carnosine and anserine concentrations were gender-specifically increased 2- to 9-fold, respectively in the kidney and both most abundant in the renal cortex, but remained unchanged in all other organs and in serum. Renal oxidized/reduced glutathione concentrations, renal morphology and function were unaltered. In Cndp1-KO mice at week 11, renal asparagine, serine and glutamine levels and at week 55, renal arginine concentration were reduced. Renal heat-shock-protein 70 (Hspa1a/b) mRNA declined with age in WT but not in Cndp1-KO mice, transcription factor heat-shock-factor 1 was higher in 55-week-old KO mice. Fasting blood glucose concentrations decreased with age in WT mice, but were unchanged in Cndp1-KO mice. Blood glucose response to intraperitoneal insulin was gender- but not genotype-dependent, the response to intraperitoneal glucose injection was similar in all groups. A global Cndp1-KO selectively, age- and gender-specifically, increases renal carnosine and anserine concentrations, alters renal amino acid- and HSP70 profile and modifies systemic glucose homeostasis. Increase of the natural occurring carnosine and anserine levels in the kidney by modulation of CN1 represents a promising therapeutic approach to mitigate or prevent chronic kidney diseases such as diabetic nephropathy.

Allosteric inhibition of carnosinase (CN1) by inducing a conformational shift.

In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2 ± 0.4, 2.0 ± 0.3, 1.6 ± 0.2 µmol/mg/h/mM; p < .05) lowered dose-dependently recombinant CN1 (rCN1) efficiency (5.2 ± 0.2 µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1C102S) and 229 (Mut2C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.

Relevance of allosteric conformations and homocarnosine concentration on carnosinase activity.

Activity of carnosinase (CN1), the only dipeptidase with substrate specificity for carnosine or homocarnosine, varies greatly between individuals but increases clearly and significantly with age. Surprisingly, the lower CN1 activity in children is not reflected by differences in CN1 protein concentrations. CN1 is present in different allosteric conformations in children and adults since all sera obtained from children but not from adults were positive in ELISA and addition of DTT to the latter sera increased OD450 values. There was no quantitative difference in the amount of monomeric CN1 between children and adults. Further, CN1 activity was dose dependently inhibited by homocarnosine. Addition of 80 microM homocarnosine lowered V (max) for carnosine from 440 to 356 pmol/min/microg and increased K (m) from 175 to 210 microM. The estimated K (i) for homocarnosine was higher (240 microM). Homocarnosine inhibits carnosine degradation and high homocarnosine concentrations in cerebrospinal fluid (CSF) may explain the lower carnosine degradation in CSF compared to serum. Because CN1 is implicated in the susceptibility for diabetic nephropathy (DN), our findings may have clinical implications for the treatment of diabetic patients with a high risk to develop DN. Homocarnosine treatment can be expected to reduce CN1 activity toward carnosine, resulting in higher carnosine levels.