Hyperfiltration can be detected by transcutaneous assessment of glomerular filtration rate in diabetic obese mice.

We addressed if hyperfiltration can be assessed transcutaneously in male diabetic obese mice (BTBRob/ob) at 12 and 24 wk and how this relates to glomerular parameters indicative for hyperfiltration. Transcutaneous assessment of FITC-Sinistrin clearance [transcutaneous assessment of glomerular filtration rate (tGFR)] was compared against classical plasma clearance. Kidney from SV620C-01-PEI perfused mice were harvested at 24 wk and processed for tissue clearing and classical histology. Perfusion patterns of glomerular capillaries, glomerular size, and vasodilation of the afferent arterioles were assessed. Although at 12 wk FITC-Sinistrin half-life (t1/2) for both tGFR and plasma clearance suggested hyperfiltration, this was not significant anymore at 24 wk. In kidneys of diabetic mice the diameter of the afferent arteriole was significantly larger and positively correlated with glomerular size. Glomerular perfusion pattern in these mice was heterogeneous ranging from non- to well-perfused glomeruli. Nonperfused glomerular areas displayed a strong periodic acid-Schiff's (PAS) positive staining. Collectively our data demonstrate that tGFR is a valid method to detect hyperfiltration. Hyperfiltration occurs early in BTBRob/ob mice and disappears with disease progression as a consequence of a reduced filtration surface. It remains to be assessed if tGFR is also a valid method in diabetic mice with severely compromised renal function.NEW & NOTEWORTHY tGFR measurement is a relatively new method to assess kidney function in conscious rodents, which can be repeated multiple times in the same animal to track the course of the disease and/or the effect of potential treatments. Since the literature was inconclusive on the suitability of this technique in obese mice, we validated it for the first time against classical plasma clearance in the commonly used BTBRob/ob mouse model.

Lipopolysaccharide Tolerance in Human Primary Monocytes and Polarized Macrophages.

Innate immune memory allows macrophages to adequately respond to pathogens to which they have been pre-exposed. To what extent different pattern recognition receptors, cytokines and resolution signals influence innate immune memory needs further elucidation. The present study assessed whether lipopolysaccharide (LPS) tolerance in monocytes and macrophages is affected by these factors. Human CD14+ cells were isolated from peripheral blood, stimulated by LPS and re-stimulated after 3 days of resting. Hereafter, immune-responsive gene 1 (IRG-1), heme oxygenase 1 (HO-1), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expression were assessed. Our study revealed the following findings: (1) While pre-stimulation with the Toll-like receptor 4 ligand LPS inhibits the induction of IRG-1, TNF-α and IL-6 expression, pre-stimulation with TLR 1/2 ligands only affects cytokine production but not IRG-1 expression upon subsequent TLR4 engagement. (2) Prior TNF-α stimulation does not affect LPS tolerance but rather increases LPS-mediated cytokine expression. (3) Dimethyl itaconate (DMI) inhibits the expression of IRG-1 in a dose-dependent manner but does not affect TNF-α or IL-6 expression. (4) Docosahexaenoic acid (DHA) partly inhibits IRG-1 expression in monocytes but not in M(IFNγ) and M(IL-4) polarized macrophages. LPS tolerance is not affected in these cells by DHA. The data presented in this study partly corroborate and extend previous findings on innate immune memory and warrant further studies on LPS tolerance to gain a better understanding of innate immune memory at the molecular level.

Adipose-Derived Mesenchymal Stem Cells Protect Endothelial Cells from Hypoxic Injury by Suppressing Terminal UPR In Vivo and In Vitro.

Adipose-derived stem cells (ASCs) have been used as a therapeutic intervention for peripheral artery disease (PAD) in clinical trials. To further explore the therapeutic mechanism of these mesenchymal multipotent stromal/stem cells in PAD, this study was designed to test the effect of xenogeneic ASCs extracted from human adipose tissue on hypoxic endothelial cells (ECs) and terminal unfolded protein response (UPR) in vitro and in an atherosclerosis-prone apolipoprotein E-deficient mice (ApoE-/- mice) hindlimb ischemia model in vivo. ASCs were added to Cobalt (II) chloride-treated ECs; then, metabolic activity, cell migration, and tube formation were evaluated. Fluorescence-based sensors were used to assess dynamic changes in Ca2+ levels in the cytosolic- and endoplasmic reticulum (ER) as well as changes in reactive oxygen species. Western blotting was used to observe the UPR pathway. To simulate an acute-on-chronic model of PAD, ApoE-/- mice were subjected to a double ligation of the femoral artery (DLFA). An assessment of functional recovery after DFLA was conducted, as well as histology of gastrocnemius. Hypoxia caused ER stress in ECs, but ASCs reduced it, thereby promoting cell survival. Treatment with ASCs ameliorated the effects of ischemia on muscle tissue in the ApoE-/- mice hindlimb ischemia model. Animals showed less muscle necrosis, less inflammation, and lower levels of muscle enzymes after ASC injection. In vitro and in vivo results revealed that all ER stress sensors (BIP, ATF6, CHOP, and XBP1) were activated. We also observed that the expression of these proteins was reduced in the ASCs treatment group. ASCs effectively alleviated endothelial dysfunction under hypoxic conditions by strengthening ATF6 and initiating a transcriptional program to restore ER homeostasis. In general, our data suggest that ASCs may be a meaningful treatment option for patients with PAD who do not have traditional revascularization options.

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.

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 Ki of 11 nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30 mg/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-naïve 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.

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 < 0.0001; median CNU 0.1 vs 0.2 vs 1.5 mg/24 h, p < 0.0001; prevalence of CNU 61 vs. 81 vs. 97% p < 0.05 in normo- (n = 241), micro- (n = 80) and macroalbuminuria (n = 40), respectively). Patients with estimated glomerular filtration rate (eGFR) < 30 ml/min/1.73 m2 displayed higher median CNU excretion rates in comparison to patients with preserved eGFR (> 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.

Carnosinase concentration, activity, and CNDP1 genotype in patients with type 2 diabetes with and without nephropathy.

This study assessed if serum carnosinase (CNDP1) activity and concentration in patients with type 2 diabetes mellitus (T2D) with diabetic nephropathy (DN) differs from those without nephropathy. In a cross-sectional design 127 patients with T2D with DN ((CTG)5 homozygous patients n = 45) and 145 patients with T2D without nephropathy ((CTG)5 homozygous patients n = 47) were recruited. Univariate and multivariate regression analyses were performed to predict factors relevant for serum CNDP1 concentration. CNDP1 (CTG)5 homozygous patients with T2D with DN had significantly lower CNDP1 concentrations (30.4 ± 18.3 vs 51.2 ± 17.6 µg/ml, p < 0.05) and activity (1.25 ± 0.5 vs 2.53 ± 1.1 µmol/ml/h, p < 0.05) than those without nephropathy. This applied for patients with DN on the whole, irrespective of (CTG)5 homozygosity. In the multivariate regression analyses, lower serum CNDP1 concentrations correlated with impaired renal function and to a lesser extend with the CNDP1 genotype (95% CI of regression coefficients: eGFR: 0.10-1.94 (p = 0.001); genotype: - 0.05 to 5.79 (p = 0.055)). Our study demonstrates that serum CNDP1 concentrations associate with CNDP1 genotype and renal function in patients with T2D. Our data warrant further studies using large cohorts to confirm these findings and to delineate the correlation between low serum CNDP1 concentrations and renal function deterioration in patients with T2D.

The Cholinergic Anti-Inflammatory Pathway as a Conceptual Framework to Treat Inflammation-Mediated Renal Injury.

BACKGROUND: The cholinergic anti-inflammatory pathway, positioned at the interface of the nervous and immune systems, is the efferent limb of the "inflammatory reflex" which mainly signals through the vagus nerve. As such, the brain can modulate peripheral inflammatory responses by the activation of vagal efferent fibers. Importantly, immune cells in the spleen express most cholinergic system components such as acetylcholine (ACh), choline acetyltransferase, acetylcholinesterase, and both muscarinic and nicotinic ACh receptors, making communication between both systems possible. In general, this communication down-regulates the inflammation, achieved through different mechanisms and depending on the cells involved. SUMMARY: With the awareness that the cholinergic anti-inflammatory pathway serves to prevent or limit inflammation in peripheral organs, vagus nerve stimulation has become a promising strategy in the treatment of several inflammatory conditions. Both pharmacological and non-pharmacological methods have been used in many studies to limit organ injury as a consequence of inflammation. Key Messages: In this review, we will highlight our current knowledge of the cholinergic anti-inflammatory pathway, with emphasis on its potential clinical use in the treatment of inflammation-triggered kidney injury.

Carnosine Catalyzes the Formation of the Oligo/Polymeric Products of Methylglyoxal.

BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS: MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.

Donor Dopamine Does Not Affect Liver Graft Survival: Evidence of Safety From a Randomized Controlled Trial.

Treatment of donation after brain death (DBD) donors with low-dose dopamine improves the outcomes after kidney and heart transplantation. This study investigates the course of liver allografts from multiorgan donors enrolled in the randomized dopamine trial between 2004 and 2007 (clinicaltrials.gov identifier: NCT00115115). There were 264 hemodynamically stable DBDs who were randomly assigned to receive low-dose dopamine. Dopamine was infused at 4 µg/kg/minute for a median duration of 6.0 hours (interquartile range, 4.4-7.5 hours). We assessed the outcomes of 212 liver transplantations (LTs) performed at 32 European centers. Donors and recipients of both groups were very similar in baseline characteristics. Pretransplant laboratory Model for End-Stage Liver Disease score was not different in recipients of a dopamine-treated versus untreated graft (18 ± 8 versus 20 ± 8; P = 0.12). Mean cold ischemia time was 10.6 ± 2.9 versus 10.1 ± 2.8 hours (P = 0.24). No differences occurred in biopsy-proven rejection episodes (14.4% versus 15.7%; P = 0.85), requirement of hemofiltration (27.9% versus 31.5%; P = 0.65), the need for early retransplantation (5.8% versus 6.5%; P > 0.99), the incidence of primary nonfunction (7.7% versus 8.3%; P > 0.99), and in-hospital mortality (15.4% versus 14.8%; P > 0.99). Graft survival was 71.2% versus 73.2% and 59.6% versus 62.0% at 2 and 3 years (log-rank P = 0.71). Patient survival was 76.0% versus 78.7% and 65.4% versus 69.4% at 1 and 3 years (log-rank P = 0.50). In conclusion, donor pretreatment with dopamine has no short-term or longterm effects on outcome after LT. Therefore, low-dose dopamine pretreatment can safely be implemented as the standard of care in hemodynamically stable DBDs.

Dopamine in transplantation: Written off or comeback with novel indication?

Renal-dose dopamine has fallen out of favor in the intensive care unit (ICU) during past years due to its ineffectiveness to prevent impending or to ameliorate overt renal failure in the critically ill. By contrast, growing evidence indicates that low-dose dopamine administered to the stable organ donor after brain death confirmation improves the clinical course of transplanted organs after kidney and heart transplantation. Ensuring a thorough monitoring for potential circulatory side effects, employment of dopamine at a dose of 4 µg/kg/min is safe in the deceased donor. Among recipients, the advantageous effect is easy to achieve, inexpensive, and devoid of adverse side effects. The mode of action relies on dopamine's propensity to mitigate injury in various cell systems from isolated transplantable organs under cold storage conditions. The present review article summarizes the clinical evidence of dopamine donor pretreatment in solid organ transplantation and focuses on the underlying molecular mechanisms of cellular protection. Introducing the routine use of low-dose dopamine for the management of the brain-dead donor in the ICU before procurement provides an evidence-based strategy to improve graft outcome after kidney transplantation without conferring harm to non-renal grafts, namely to livers and hearts, in cases of multi-organ donation.

The adenosinergic system: a potential player in the pathogenesis of ANCA-associated vasculitis?

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a potentially lethal autoimmune disease whose pathology comprises disturbed T cell differentiation and functionality accompanied by dysfunctional autoreactive immunoglobulin development, culminating in destructive innate immune response as well. Purines, adenine nucleotides and adenosine in particular, have been elucidated as potent extracellular mediators for fine adjustment of these pivotal processes establishing human immunity. Therefore, the extracellular purinergic microenvironment is under control of ectonucleotidases CD39 and CD73 degrading pro-inflammatory adenosine triphosphate (ATP) to anti-inflammatory adenosine as well as adenosine deaminase bound to CD26 deactivating adenosine. Accordingly, the ATP P2X7 receptor was elicited to be responsible for promotion of inflammation, while predominantly the adenosine A2A receptor demonstrated the opposite. Recent reports pointed at the adenosinergic system to be crucially involved in AAV pathogenesis. Here, experimental evidence on ecto-enzymes controlling extracellular adenine nucleotide concentrations and purinergic signaling in the immune system with respect to its contribution to the AAV pathomechanism is reviewed besides unsolved problems being identified that require further investigation in order to develop new treatment strategies for AAV.

N-octanoyl dopamine treatment exerts renoprotective properties in acute kidney injury but not in renal allograft recipients.

BACKGROUND: N-octanoyl dopamine (NOD) treatment improves renal function when applied to brain dead donors and in the setting of warm ischaemia-induced acute kidney injury (AKI). Because it also activates transient receptor potential vanilloid type 1 (TRPV1) channels, we first assessed if NOD conveys its renoprotective properties in warm ischaemia-induced AKI via TRPV1 and secondly, if renal transplant recipients also benefit from NOD treatment. METHODS: We induced warm renal ischaemia in Lewis, wild-type (WT) and TRPV1(-/-) Sprague-Dawley (sd) rats by clamping the left renal artery for 45 min. Transplantations were performed in allogeneic and syngeneic donor-recipient combinations (Fisher to Lewis and Lewis to Lewis) with a cold ischaemia time of 20 h. Treatment was instituted directly after restoration of organ perfusion. Renal function, histology and perfusion were assessed by serum creatinine, microscopy and magnetic resonance imaging (MRI) using arterial spin labelling (ASL). RESULTS: NOD treatment significantly improved renal function in Lewis rats after warm ischaemia-induced AKI. It was, however, not effective after prolonged cold ischaemia. The renoprotective properties of NOD were only observed in Lewis or WT, but not in TRPV1(-/-) sd rats. Renal inflammation was significantly abrogated by NOD. MRI-ASL showed a significantly lower cortical perfusion in ischaemic when compared with non-ischaemic kidneys. No overall differences were observed in renal perfusion between NOD- and NaCl-treated rats. CONCLUSIONS: NOD treatment reduces renal injury in warm ischaemia, but is not effective in renal transplant in our experimental animal models. The salutary effect of NOD appears to be TPRV1-dependent, not involving large changes in renal perfusion.

Optimized donor management and organ preservation before kidney transplantation.

Kidney transplantation is a major medical improvement for patients with end-stage renal disease, but organ shortage limits its widespread use. As a consequence, the proportion of grafts procured from extended criteria donors (ECD) has increased considerably, but this comes along with increased rates of delayed graft function (DGF) and a higher incidence of immune-mediated rejection that limits organ and patient survival. Furthermore, most grafts are derived from brain dead organ donors, but the unphysiological state of brain death is associated with significant metabolic, hemodynamic, and pro-inflammatory changes, which further compromise patient and graft survival. Thus, donor interventions to preserve graft quality are fundamental to improve long-term transplantation outcome, but interventions must not harm other potentially transplantable grafts. Several donor pretreatment strategies have provided encouraging results in animal models, but evidence from human studies is sparse, as most clinical evidence is derived from single-center or nonrandomized trials. Furthermore, ethical matters have to be considered especially concerning consent from donors, donor families, and transplant recipients to research in the field of donor treatment. This review provides an overview of clinically proven and promising preclinical strategies of donor treatment to optimize long-term results after kidney transplantation.

Dopamine and lipophilic derivates protect cardiomyocytes against cold preservation injury.

Donor heart allografts are extremely susceptible to prolonged static cold storage. Because donor treatment with low-dose dopamine improves clinical outcome after heart transplantation, we tested the hypothesis that dopamine and its lipophilic derivate, N-octanoyl dopamine (NOD), protect cardiomyocytes from cold storage injury. Neonatal rat cardiomyocytes were treated with dopamine or NOD or left untreated and subsequently subjected to static cold storage (8-12 hours). Dopamine- and NOD-treated cardiomyocytes displayed a better viability compared with untreated cells after hypothermia. In untreated cardiomyocytes, cell damage was reflected by lactate dehydrogenase (LDH) release and a decrease in intracellular ATP. NOD was approximately 20-fold more potent than dopamine. Similarly to cardiomyocytes in vitro, rat hearts perfused with NOD before explantation showed significantly lower LDH release after static cold storage. ATP regeneration and spontaneous contractions after cold storage and rewarming only occurred in treated cardiomyocytes. Hypothermia severely attenuated isoprenaline-induced cAMP formation in control but not in dopamine- or NOD-treated cells. Esterified derivates of NOD with redox potential and lipophilic side chains reduced cell damage during cold storage similarly to NOD. In contrast to dopamine, neither NOD nor its derivates induced a significant ß-adrenoceptor-mediated elevation of cellular cAMP levels. The ß1-adrenoceptor antagonist atenolol and D1/D2 receptor antagonist fluphenazine had no impact on the protective effect of NOD or dopamine. We conclude that dopamine as well as NOD treatment mitigates cold preservation injury to cardiomyocytes. The beneficial effects are independent of ß-adrenoceptor or dopaminergic receptor stimulation but correlate with redox potential and lipophilic properties.

Vagal stimulation in brain dead donor rats decreases chronic allograft nephropathy in recipients.

BACKGROUND: Although it has been shown that a vagus nerve stimulation of brain dead (BD) donors leads to an improvement of renal function in recipients in an acute allograft rejection model, its influence on chronic allograft nephropathy is still unknown. In the present study, we assessed the influence of donor vagus nerve stimulation on survival, renal function and histology in a chronic allograft model. METHODS: Brain death was induced in Fisher rats, and electro-stimulation of the vagus nerve was applied in one group (BD + vagus) during the whole course of BD (6 h). Unstimulated BD Fisher donor rats served as controls. Allogeneic Lewis rats were used as recipients and no immunosuppressive medication was administered. Blood and urine samples were collected every second week. Banff classification was assessed from harvested allografts. RESULTS: Vagal stimulation of BD donors resulted in an improved survival of recipients. Long-term renal function was significantly better in these recipients as reflected by improved creatinine clearance. Banff classification revealed significantly reduced vasculopathy and less tubulopathy in the BD + vagus group. CONCLUSIONS: In conclusion, our data demonstrate a long-lasting beneficial effect of vagus nerve stimulation in BD donors on the renal transplantation outcome. Hence, activation of the cholinergic anti-inflammatory pathway in BD donors may represent a novel therapeutic modality to reduce chronic allograft nephropathy without any side effects for the recipient.

Serum carnosinase 1, an early indicator for incident microalbuminuria in type 1 diabetes.

Aims: Carnosinase (CN1) polymorphisms have been linked to diabetic kidney disease (DKD), as CN1 degrades dipeptides which scavenge oxidative metabolites and prevent the formation of advanced glycation end-products. In this work, we studied the association between serum CN1, the systemic redox status and long-term renal outcome in type 1 diabetes. Methods: Serum CN1 was measured in a prospective type 1 diabetes cohort (n = 218) with a 16-year follow-up. A total of 218 patients treated at the Diabetes Outpatient Clinic of the Weezenlanden Hospital (nowadays Isala Hospital, Zwolle, The Netherlands) were included in this analysis. We assessed whether serum CN1 was associated with renal function and development of DKD as well as other diabetic complications. Results: At baseline, age, systemic redox status and N-terminal pro brain-natriuretic peptide (NT-proBNP) were associated with serum CN1 concentration (p < 0.05). During follow-up, CN1 concentration in the middle tertile was associated with less incident microalbuminuria (odds ratio = 0.194, 95% C.I.: 0.049-0.772, p = 0.02) after adjustment for age, systemic redox status, NT-proBNP and sex. Discussion: Serum CN1 could predict incident microalbuminuria and may be used as a novel parameter to identify patients at risk for DKD.

Gene regulation for inflammation and inflammation resolution differs between umbilical arterial and venous endothelial cells.

Systemic inflammation affects the whole vasculature, yet whether arterial and venous endothelial cells differ in their abilities to mediate inflammation and to return to homeostasis after an inflammatory stimulus has not been addressed thoroughly. We assessed gene-expression profiles in isolated endothelial cells from human umbilical arteries (HUAEC) or veins (HUVEC) under basal conditions, after TNF-α stimulation and various time points after TNF-α removal to allow reinstatement of homeostasis. TNF-α regulates the expression of different sets of transcripts that are significantly changed only in HUAEC, only in HUVEC or changed in both. We identified three types of gene regulation, i.e. genes that were significantly regulated after 24 h of TNF-α stimulation but no longer when TNF-α was removed (homeostatic regulation), genes that maintained significantly regulated after TNF-α removal (not homeostatic regulation) and genes that were only significantly regulated when TNF-α was removed (post-regulation). HUAEC and HUVEC quantitatively differed in these types of gene regulation, with relatively more genes being post-regulated in HUAEC. In conclusion our data demonstrate that HUAEC and HUVEC respond intrinsically different to an inflammatory insult. Whether this holds true for all endothelial cells and its relevance for inflammatory insults in different organs during systemic inflammation warrants further studies.

Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations.

Serum carnosinase (CN-1) measurements are at present mainly performed by assessing enzyme activity. This method is time-consuming, not well suited for large series of samples and can be discordant to measurements of CN-1 protein concentrations. To overcome these limitations, we developed sandwich ELISA assays using different anti-CN-1 antibodies, i.e., ATLAS (polyclonal IgG) and RYSK173 (monoclonal IgG1). With the ATLAS-based assay, similar amounts of CN-1 were detected in serum and both EDTA and heparin plasma. The RYSKS173-based assay detected CN-1 in serum in all individuals at significantly lower concentrations compared to the ATLAS-based assay (range: 0.1-1.8 vs. 1-50 µg/ml, RYSK- vs. ATLAS-based, P<0.01). CN-1 detection with the RYSK-based assay was increased in EDTA plasma, albeit at significantly lower concentrations compared to ATLAS. In heparin plasma, CN-1 was also poorly detected with the RYSK-based assay. Addition of DTT to serum increased the detection of CN-1 in the RYSK-based assay almost to the levels found in the ATLAS-based assay. Both ELISA assays were highly reproducible (R: 0.99, P<0.01 and R: 0.93, P<0.01, for the RYSK- and ATLAS-based assays, respectively). Results of the ATLAS-based assay showed a positive correlation with CN-1 activity (R: 0.62, P<0.01), while this was not the case for the RYSK-based assay. However, there was a negative correlation between CN-1 activity and the proportion of CN-1 detected in the RYSK-based assay, i.e., CN-1 detected with the RYSK-based assay/CN-1 detected with the ATLAS-based assay × 100% (Spearman-Rang correlation coefficient: -0.6, P<0.01), suggesting that the RYSK-based assay most likely detects a CN-1 conformation with low CN-1 activity. RYSK173 and ATLAS antibodies reacted similarly in Western blot, irrespective of PNGase treatment. Binding of RYSK173 in serum was not due to differential N-glycosylation as demonstrated by mutant CN-1 cDNA constructs. In conclusion, our study demonstrates a good correlation between enzyme activity and CN-1 protein concentration in ELISA and suggests the presence of different CN-1 conformations in serum. The relevance of these different conformations is still elusive and needs to be addressed in further studies.

Association Between Serum Carnosinase Concentration and Activity and Renal Function Impairment in a Type-2 Diabetes Cohort.

Introduction: Genetic studies have identified associations of carnosinase 1 (CN1) polymorphisms with diabetic kidney disease (DKD). However, CN1 levels and activities have not been assessed as diagnostic or prognostic markers of DKD in cohorts of patients with type 2 diabetes (T2D). Methods: We established high-throughput, automated CN1 activity and concentration assays using robotic systems. Using these methods, we determined baseline serum CN1 levels and activity in a T2D cohort with 970 patients with no or only mild renal impairment. The patients were followed for a mean of 1.2 years. Baseline serum CN1 concentration and activity were assessed as predictors of renal function impairment and incident albuminuria during follow up. Results: CN1 concentration was significantly associated with age, gender and estimated glomerular filtration rate (eGFR) at baseline. CN1 activity was significantly associated with glycated hemoglobin A1c (HbA1c) and eGFR. Serum CN1 at baseline was associated with eGFR decline and predicted renal function impairment and incident albuminuria during the follow-up. Discussion: Baseline serum CN1 levels were associated with presence and progression of renal function decline in a cohort of T2D patients. Confirmation in larger cohorts with longer follow-up observation periods will be required to fully establish CN1 as a biomarker of DKD.