Implications of the carnosine-carnosinase system in diabetic nephropathy

In humans, both carnosine-degrading and synthesizing enzymes, i.e. carnosinase (CN1) and Carnosine synthase (CARNS) are expressed in the kidney. While polymorphisms that are associated with the susceptibility to develop diabetic nephropathy (DN) have been described for CN1, no such polymorphisms for CARNS have been reported. Therefore, the studies described herein particularly focus on the role of CN1 in the progression of DN and try to elucidate if carnosine has vasoprotective properties and if this is possibly mediated via improvement of the intracellular redox milieu in methylglyoxal (MGO)-challenged cultured endothelial cells. This thesis partly makes use of clinical cohorts to understand the role of urinary CN1 in the progression of DN and describes in vitro studies to assess the relation between MGO, redox homeostasis and carnosine in the context of vascular damage. Two main hypotheses were put forward: 1) in patients with DN the extent of urinary CN1 is increased due to impairment of the glomerular filtration barrier and leakage from the serum. Because high renal CN1 concentrations may lead to depletion of renal carnosine stores, an association between the extent of urinary CN1 and renal function deterioration is expected. 2) MGO affects the redox status in cultured endothelial cells thereby causing cellular structural damage. Carnosine can ameliorate this damage by virtue of its anti-oxidant properties. The main findings of this study are as follows: 1) CN1 can be reliably detected in spot and in 24-hr urine samples by ELISA. 2) In T2DM patients the prevalence and extent of urinary CN1 increased in parallel with albuminuria (median urinary CN1 0.1 vs 0.2 vs 1.5 mg/24-hr, p<0.0001; prevalence: 61 vs. 81 vs. 97% p<0.05 in normo-, micro- and macroalbuminuria, respectively). Patients with poor eGFR displayed higher median urinary CN1 concentration rates in comparison to patients with preserved eGFR. Multivariate linear regression analysis revealed that albuminuria and eGFR are the main independent predictors of urinary CN1 (R2=0.37, p<0.0001). 3) The correlations between urinary CN1 and albuminuria were also found in spot urine samples of chronic kidney disease patients irrespective of the baseline disease (non-diabetic vs diabetic). 4) MGO affects cell viability in a dose and cell density dependent manner. Based on the low cell number of TUNEL positive cells, MGO-induced cell death is unlikely mediated through apoptosis mechanisms, albeit that apoptosis might contribute to the overall cell death. Carnosine counteracts MGO-mediated toxicity in short and long term MGO exposure conditions. Carnosine but not MGO affects the intracellular redox milieu confirming the anti-oxidant properties of carnosine

Methylglyoxal induces p53 activation and inhibits mTORC1 in human umbilical vein endothelial cells

Methylglyoxal (MGO), a precursor of advanced glycation end products (AGEs), is regarded as a pivotal mediator of vascular damage in patients with diabetes. We have previously reported that MGO induces transcriptional changes compatible with p53 activation in cultured human endothelial cells. To further substantiate this finding and to explore the underlying mechanisms and possible consequences of p53 activation, we aimed (1) to provide direct evidence for p53 activation in MGO-treated human umbilical vein endothelial cells (HUVECs), (2) to assess putative mechanisms by which this occurs, (3) to analyze down-stream effects on mTOR and autophagy pathways, and (4) to assess the potential benefit of carnosine herein. Exposure of HUVECs to 800 mu M of MGO for 5 h induced p53 phosphorylation. This was paralleled by an increase in TUNEL and gamma-H2AX positive cells, indicative for DNA damage. Compatible with p53 activation, MGO treatment resulted in cell cycle arrest, inhibition of mTORC1 and induction of autophagy. Carnosine co-treatment did not counteract MGO-driven effects. In conclusion, our results demonstrate that MGO elicits DNA damage and p53 activation in HUVECs, resulting in modulation of downstream pathways, e.g. mTORC1

Human carnosinase 1 overexpression aggravates diabetes and renal impairment in BTBR(Ob/Ob)mice

Objective: To assess the influence of serum carnosinase (CN1) on the course of diabetic kidney disease (DKD). Methods: hCN1 transgenic (TG) mice were generated in a BTBROb/Ob genetic background to allow the spontaneous development of DKD in the presence of serum carnosinase. The influence of serum CN1 expression on obesity, hyperglycemia, and renal impairment was assessed. We also studied if aggravation of renal impairment in hCN1 TG BTBROb/Ob mice leads to changes in the renal transcriptome as compared with wild-type BTBROb/Ob mice. Results: hCN1 was detected in the serum and urine of mice from two different hCN1 TG lines. The transgene was expressed in the liver but not in the kidney. High CN1 expression was associated with low plasma and renal carnosine concentrations, even after oral carnosine supplementation. Obese hCN1 transgenic BTBROb/Ob mice displayed significantly higher levels of glycated hemoglobin, glycosuria, proteinuria, and increased albumin-creatinine ratios (1104 ± 696 vs 492.1 ± 282.2 μg/mg) accompanied by an increased glomerular tuft area and renal corpuscle size. Gene-expression profiling of renal tissue disclosed hierarchical clustering between BTBROb/Wt, BTBROb/Ob, and hCN1 BTBROb/Ob mice. Along with aggravation of the DKD phenotype, 26 altered genes have been found in obese hCN1 transgenic mice; among them claudin-1, thrombospondin-1, nephronectin, and peroxisome proliferator–activated receptor-alpha have been reported to play essential roles in DKD. Conclusions: Our data support a role for serum carnosinase 1 in the progression of DKD. Whether this is mainly attributed to the changes in renal carnosine concentrations warrants further studies. Key messages: Increased carnosinase 1 (CN1) is associated with diabetic kidney disease (DKD).BTBROb/Ob mice with human CN1 develop a more aggravated DKD phenotype.Microarray revealed alterations by CN1 which are not altered by hyperglycemia.These genes have been described to play essential roles in DKD.Inhibiting CN1 could be beneficial in DKD

Serum Carnosinase-1 and Albuminuria Rather than the CNDP1 Genotype Correlate with Urinary Carnosinase-1 in Diabetic and Nondiabetic Patients with Chronic Kidney Disease

Background. Carnosinase-1 (CN-1) can be detected in 24 h urine of healthy individuals and patients with type 2 diabetes (T2DM). We aimed to assess whether urinary CN-1 is also reliably measured in spot urine and investigated its association with renal function and the albumin/creatinine ratio (ACR). We also assessed associations between the CNDP1 (CTG)(n) genotype and CN-1 concentrations in serum and urine. Methods. Patients with T2DM (n=85) and nondiabetic patients with chronic kidney disease (CKD) (n=26) stratified by albuminuria (ACR 300 mg/g) recruited from the nephrology clinic and healthy subjects (n=24) were studied. Results. Urinary CN-1 was more frequently detected and displayed higher concentrations in patients with ACR>300 mg/g as compared to those with AC

Influence of carnosine and carnosinase-1 on diabetes-induced afferent arteriole vasodilation:implications for glomerular hemodynamics

Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Two cohorts of mice including appropriate controls were studied: i.e., diabetic mice that received oral carnosine supplementation (cohort 1) and human (h)CN1 transgenic (TG) diabetic mice (cohort 2). The lumen area ratio (LAR) of the afferent arterioles and glomerular parameters were measured by conventional histology. Three-dimensional analysis using a tissue clearing strategy was also used. In both cohorts, LAR was significantly larger in diabetic BTBRob/ob versus nondiabetic BTBRwt/ob mice (0.41?? 0.05 vs. 0.26 ?? 0.07, P < 0.0001 and 0.42 ?? 0.06 vs. 0.29 ?? 0.04, P < 0.0001) and associated with glomerular size (cohort 1: r = 0.55, P = 0.001 and cohort 2: r = 0.89, P < 0.0001). LAR was partially normalized by oral carnosine supplementation (0.34 ?? 0.05 vs. 0.41?? 0.05, P = 0.004) but did not differ between hCN1 TG and wild-type BTBRob/ob mice. In hCN1 TG mice, serum CN1 concentrations correlated with LAR (r = 0.90, P = 0.006). Diabetic mice displayed decreased nephrin expression and increased glomerular hypertrophy. This was not significantly different in hCN1 TG BTBRob/ob mice (P = 0.06 and P = 0.08, respectively). In conclusion, carnosine and CN1 may affect intraglomerular pressure in an opposing manner through the regulation of afferent arteriolar tone. This study corroborates previous findings on the role of carnosine in the progression of DKD. NEW & NOTEWORTHY Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Our results provide evidence that carnosine feeding and CN1 overexpression likely affect intraglomerular pressure through vasoregulation of the afferent arteriole

Detection of carnosinase-1 in urine of healthy individuals and patients with type 2 diabetes: Correlation with albuminuria and renal function

Low serum carnosinase (CN-1) concentrations are associated with low risk for development of diabetic nephropathy (DN) in patients with type 2 diabetes (T2D). Although CN-1 is expressed in the kidney, urinary CN-1 (CNU) excretion and its pathological relevance in patients with T2D have not been investigated to date. The present study therefore assessed the extent of CNU excretion in healthy subjects (n = 243) and in patients with T2D (n = 361) enrolled in the DIAbetes and LifEstyle Cohort Twente-1 (DIALECT-1) in relation to functional renal parameters. CNU was detected in a high proportion of healthy individuals, 180 (74%); median CNU excretion was 0.25 mg/24 h [(IQR 0–0.65 mg/24 h]. In patients with T2D the prevalence and extent of CNU increased in parallel with albuminuria (r = 0.59, p  90 ml/min/1.73 m2) (1.36 vs 0.13 mg/24 h, p < 0.05). Backward stepwise multivariate linear regression analysis revealed albuminuria, eGFR and glycosuria to be independent factors of CNU excretion rates, all together explaining 37% of variation of CNU excretion rates (R2 = 0.37, p < 0.0001). These results show for the first time that CN-1 can be detected in urine and warrants prospective studies to assess the relevance of CNU for renal function deterioration in diabetes patients

Identification and characterisation of carnostatine (SAN9812), a potent and selective carnosinase (CN1) inhibitor with in vivo activity

Carnosinase 1 (CN1) has been postulated to be a susceptibility factor for developing diabetic nephropathy (DN). Although its major substrate, carnosine, is beneficial in rodent models of DN, translation of these findings to humans has been hampered by high CN1 activity in human serum resulting in rapid degradation of carnosine. To overcome this hurdle, we screened a protease-directed small-molecule library for inhibitors of human recombinant CN1. We identified SAN9812 as a potent and highly selective inhibitor of CN1 activity with a K-i of 11nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30mg/kg SAN9812 led to a sustained reduction in circulating CN1 activity in human CN1 transgenic (TG) mice. Simultaneous administration of carnosine and SAN9812 increased carnosine levels in plasma and kidney by up to 100-fold compared to treatment-naive CN1-overexpressing mice. To our knowledge, this is the first study reporting on a potent and selective CN1 inhibitor with in vivo activity. SAN9812, also called carnostatine, may be used to increase renal carnosine concentration as a potential therapeutic modality for renal diseases linked to glycoxidative conditions