Urinary liver-type fatty acid-binding protein is independently associated with graft failure in outpatient kidney transplant recipients.

Urinary liver-type fatty acid-binding protein (uL-FABP) is a biomarker of kidney hypoxia and ischemia, and thus offers a novel approach to identify early kidney insults associated with increased risk of graft failure in outpatient kidney transplant recipients (KTR). We investigated whether uL-FABP is associated with graft failure and whether it improves risk prediction. We studied a cohort of 638 outpatient KTR with a functional graft ≥1-year. During a median follow-up of 5.3 years, 80 KTR developed graft failure. uL-FABP (median 2.11, interquartile range 0.93-7.37 µg/24"/>h) was prospectively associated with the risk of graft failure (hazard ratio 1.75; 95% confidence interval 1.27-2.41 per 1-SD increment; P = .001), independent of potential confounders including estimated glomerular filtration rate and proteinuria. uL-FABP showed excellent discrimination ability for graft failure (c-statistic of 0.83) and its addition to a prediction model composed by established clinical predictors of graft failure significantly improved the c-statistic to 0.89 (P for F-test <.001). These results were robust to several sensitivity analyses. Further validation studies are warranted to evaluate the potential use of a risk-prediction model including uL-FABP to improve identification of outpatient KTR at high risk of graft failure in clinical care.

No renal phenotype in human phospholipid transfer protein transgenic apolipoprotein E deficient mice despite severe aortic atherosclerosis.

BACKGROUND: Phospholipid transfer protein (PLTP) is an emerging cardiometabolic risk factor. Plasma PLTP is elevated in humans with end-stage kidney disease and glomerular proteinuria, but the contribution of systemic PLTP elevation to the development of renal damage is unknown. We tested whether human PLTP expression in ApoE deficient mice (an atherosclerosis-prone model) results in renal insufficiency, albuminuria, or glomerulosclerosis. METHODS: Serum creatinine, albuminuria, as well as kidney and aortic arch histology were determined in 6 male huPLTPtgApoE-/- mice and 8 similarly aged male wild type mice fed a regular chow diet. RESULTS: huPLTPtgApoE-/- mice (2- to 3-fold elevated PLTP activity) showed marked aortic atherosclerosis. However, serum creatinine (p = 0.11) and albuminuria (p = 0.87) were not increased, whereas renal arteriolar atherosclerosis and glomerulosclerosis were not evident in this PLTP transgenic model. CONCLUSIONS: High systemic PLTP expression does not contribute significantly to a renal phenotype despite being implicated in systemic atherosclerosis.

Vascular endothelial growth factor C levels are modulated by dietary salt intake in proteinuric chronic kidney disease patients and in healthy subjects.

BACKGROUND: Recent experimental findings demonstrate vascular endothelial growth factor C (VEGF-C)-mediated water-free storage of salt in the interstitium, which prevents a salt-sensitive blood pressure state. It is unknown whether this mechanism plays a role in salt homeostasis and regulation of blood pressure in humans as well. Therefore, we investigated circulating VEGF-C levels and blood pressure during different well-controlled salt intake in chronic kidney disease (CKD) patients and in healthy subjects. METHODS: In two crossover studies, non-diabetic proteinuric CKD patients (n = 32) and healthy subjects (n = 31) were treated with consecutively a low-sodium diet (LS, aim 50 mmol Na(+)/day) and a high-sodium diet (HS, aim 200 mmol Na(+)/day) in random order, during two 6-week (CKD patients) and two 1-week periods (healthy subjects). RESULTS: We found that VEGF-C levels are higher during HS than during LS in CKD patients (P = 0.034) with a trend towards higher VEGF-C in healthy subjects as well (P = 0.070). In CKD patients, HS was associated with higher NT-proBNP levels (P = 0.005) and body weight (P = 0.013), consistent with extracellular volume (ECV) expansion and with higher blood pressure (P < 0.001), indicating salt sensitivity. In healthy subjects, blood pressure was not affected by dietary salt (P = 0.14), despite a rise in ECV (P = 0.023). DISCUSSION: Our findings support a role for VEGF-C-mediated salt homeostasis in humans. Considering the salt sensitivity of blood pressure, this buffering mechanism appears to be insufficient in proteinuric CKD patients. Future studies are needed to provide causality and to substantiate the clinical and therapeutic relevance of this VEGF-C regulatory mechanism in humans.

Celecoxib treatment reduces peritoneal fibrosis and angiogenesis and prevents ultrafiltration failure in experimental peritoneal dialysis.

BACKGROUND: Daily peritoneal exposure to peritoneal dialysis fluid (PDF) induces severe morphological alterations including fibrosis and angiogenesis that lead to a loss of peritoneal ultrafiltration (UF) capacity. Since cyclooxygenase (COX)-2 is involved in fibrosis and angiogenesis, we investigated the in vivo effects of a selective COX-2 inhibitor (celecoxib) in a rat-PD model. METHODS: Sixteen rats daily received 10 ml of conventional PDF for 4-5 weeks intraperitoneally. Half of them (n = 8) daily received celecoxib (20 mg/kg BW) via oral gavage, and the other half (n = 8) received vehicle via oral gavage. The study also included two control groups (no PDF instillations), each consisting of n = 8 animals that daily received celecoxib or vehicle, respectively, via oral gavage. Functional, morphological and cellular parameters were analysed. RESULTS: PDF exposure induced an inflammatory condition evidenced by the increased leucocyte number and synthesis of MCP-1, VEGF and hyaluronic acid. After PDF exposure, the omentum showed intense angiogenesis and milky spots formation. Parietal peritoneum showed increased angiogenesis, lymphangiogenesis, submesothelial matrix thickness and enhanced expression of mesothelial aquaporin1 (Aqp1). Concomitant PDF and celecoxib exposure drastically reduced PGE2 levels, angiogenesis, lymphangiogenesis, fibrosis and milky spot formation in studied tissues, but did not modify mesothelial Aqp1 expression nor the tissue expression of VEGF and inflammatory markers. PDF exposure induced severe UF failure that celecoxib treatment completely prevented. CONCLUSIONS: Altogether, celecoxib treatment improves UF capacity and reduces morphological alterations in our rat PD model.

Correction: Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173.

[This corrects the article DOI: 10.1371/journal.pone.0146831.].

Hyperglycemia Does Not Affect Iron Mediated Toxicity of Cultured Endothelial and Renal Tubular Epithelial Cells: Influence of L-Carnosine.

Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3 challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity.

Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173.

BACKGROUND AND AIMS: The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn2+ and Cu2+ concentration during hemodialysis. METHODS AND RESULTS: Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn2+ or Cu2+ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn2+ and Cu2+ concentrations after dialysis. CONCLUSIONS: Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme.

Oxytocin Neurotransmission in the A1-area of the Brainstem Induces Hormonal Vasopressin Release in Rats.

To investigate the role of the oxytocin innervation of the caudal ventrolateral medulla, immunocytochemical techniques were used to demonstrate the presence of oxytocin fibres and terminals in close apposition to noradrenergic neurons of the A1-area. Subsequently, in freely moving animals fitted with an indwelling jugular venous catheter and a bilaterally implanted chronic cannula in the A1-area, it was examined whether infusions of oxytocin in this area were able to influence hormonal vasopressin release. It appeared that nanomolar (50-500 nM) concentrations of oxytocin induce a fourfold rise in plasma vasopressin values. The specificity of this effect was established with control infusions of Ringer, vasopressin, and the addition of an antagonist to oxytocin. It was not possible to demonstrate a major role for oxytocin in the A1-area in the release of hormonal vasopressin occurring during haemorrhage. These data permit us to conclude that oxytocin acts on presumably noradrenergic neurons of the A1-area leading to the release of vasopressin into the peripheral circulation. The circumstances under which oxytocin is released in this area remain to be established.

Identification of novel genes associated with renal tertiary lymphoid organ formation in aging mice.

A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, 12 and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10(-137)) in male mice; Ctnnbip1 (P = 6.42×10(-267)) and Tnfrsf8 (P = 5.42×10(-245)) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the Wnt-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the Wnt-signaling pathway that may be causally linked to tertiary lymphoid organ formation.

Lectin complement pathway gene profile of the donor and recipient does not influence graft outcome after kidney transplantation.

In kidney transplantation, complement activation was found to be induced by donor brain death, renal ischemia-reperfusion injury and allograft rejection. There are three known pathways of complement activation: the classical, lectin and the alternative pathway. The lectin complement pathway can be activated upon pattern recognition by mannan binding lectin (MBL) or ficolins (FCN). Single nucleotide polymorphisms (SNPs) in the genes encoding the lectin pathway proteins determine their functional activity and serum levels. The aim of this study was to investigate the role of the lectin gene profile of the donor and recipient on post-transplant outcome. A total of 12 functional SNPs in the MBL2, FCN2 and MBL-associated serine proteases 2 (MASP2) genes of 1271 donor-recipient pairs were determined. Lectin genotypic variants were analyzed for association with primary non-function (PNF), delayed graft function (DGF), biopsy proven acute rejection, death-censored graft survival and patient survival. Multivariate analyses found no association of donor and recipient MBL2 and MASP2 genotype with allograft outcome. Analysis of separate functional SNPs and haplotypes in the FCN2 gene of the donor and recipient did not reveal an association with transplant outcome. Also, the joint effect of the MBL2 and FCN2 genotype was not associated with allograft outcome.This study shows that the genetic profile of the lectin pathway of complement activation of the donor and recipient is not associated with allograft outcome after kidney transplantation.

Vitamin D in chronic kidney disease: new potential for intervention.

Prevention of progressive renal function loss and its complications remains the main challenge in clinical nephrology. Although current therapeutic strategies aiming at reduction of blood pressure and proteinuria often slow down deterioration of renal function, still many patients progress to end-stage renal disease. The development of novel pharmacological approaches for treatment of chronic kidney disease (CKD) is therefore instrumental. Here we review the renoprotective potential of vitamin D and its analogues. In CKD patients, vitamin D deficiency is common and progression of CKD is associated with low (active) vitamin D levels. Moreover, in animal models of CKD, treatment with vitamin D (analogues) alone or in combination with renin-angiotensin-aldosterone system (RAAS) blockade reduces proteinuria, glomerulosclerosis and tubulointerstitial fibrosis. Potential underlying mechanisms include suppression of the RAAS, modulation of immune cell function and direct protective effects on renal cells such as podocytes. Whether vitamin D analogues could further optimize existing therapies in human renal disease is currently under investigation.