Search

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli. [ABSTRACT FROM AUTHOR]

The endothelial glycocalyx is a gel-like layer which covers the luminal side of blood vessels. The glomerular endothelial cell (GEnC) glycocalyx is composed of proteoglycan core proteins, glycosaminoglycan (GAG) chains, and sialoglycoproteins and has been shown to contribute to the selective sieving action of the glomerular capillary wall. Damage to the systemic endothelial glycocalyx has recently been associated with the onset of albuminuria in diabetics. In this study, we analyze the effects of high glucose on the biochemical structure of the GEnC glycocalyx and quantify functional changes in its protein-restrictive action. We used conditionally immortalized human GEnC. Proteoglycans were analyzed by Western blotting and indirect immunofluorescence. Biosynthesis of GAG was analyzed by radiolabeling and quantified by anion exchange chromatography. FITC-albumin was used to analyze macromolecular passage across GEnC monolayers using an established in vitro model. We observed a marked reduction in the biosynthesis of GAG by the GEnC under high-glucose conditions. Further analysis confirmed specific reduction in heparan sulfate GAG. Expression of proteoglycan core proteins remained unchanged. There was also a significant increase in the passage of albumin across GEnC monolayers under high-glucose conditions without affecting interendothelial junctions. These results reproduce changes in GEnC barrier properties caused by enzymatic removal of heparan sulfate from the GEnC glycocalyx. They provide direct evidence of high glucose-induced alterations in the GEnC glycocalyx and demonstrate changes to its function as a protein-restrictive layer, thus implicating glycocalyx damage in the pathogenesis of proteinuria in diabetes. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF)-A is an autocrine survival factor for podocytes, which express two VEGF receptors, VEGF-R1 and VEGF-R3. As VEGF-A is not a known ligand for VEGF-R3, the aim of this investigation was to examine whether VEGF-C, a known ligand for VEGF-R3, served a function in podocyte biology and whether this was VEGF-R3 dependent. VEGF-C protein expression was localized to podocytes in contrast to VEGF-D, which was expressed in parietal epithelial cells. Intracellular calcium ([Ca2+]i) experiments demonstrated that VEGF-C induced a 0.74 ± 0.09-fold reduction in [Ca2+]i compared with baseline in human conditionally immortalized podocytes (hCTPs; P < 0.05, one sample f-test, n = 8). Cytotoxicity experiments revealed that in hCIPs VEGF-C reduced cytotoxicity to 81.4 ± 1.9% of serum-starved conditions (P < 0.001, paired t-test, n = 16), similar to VEGF-A (82.8 ± 4.5% of serum-starved conditions, P < 0.05, paired t-test). MAZ51 (a VEGF-R3 kinase inhibitor) inhibited the VEGF-C-induced reduction in cytotoxicity (106.2 ± 2.1% of serum-starved conditions), whereas MAZ51 by itself had no cytotoxic effects on hClPs. VEGF-C was also shown to induce a 0.5 ± 0.13-fold reduction in levels of MAPK phosphorylation compared with VEGF-A and VEGF-A-Mab treatment (P < 0.05, ANOVA, n = 4), yet had no effect on Akt phosphorylation. Surprisingly, immunoprecipitation studies detected no VEGF-C-induced autophosphorylation of VEGF-R3 in hCIPs but did so in HMVECs. Moreover, SU-5416, a tyrosine kinase inhibitor, blocked the VEGF-C-induced reduction in cytotoxicity (106 ± 2.8% of serum-starved conditions) at concentrations specific for VEGF-R1. Together, these results suggest for the first time that VEGF-C acts in an autocrine manner in cultured podocytes to promote survival, although the receptor or receptor complex activated has yet to be elucidated. [ABSTRACT FROM AUTHOR]

Hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations are the most common cause of maturity-onset diabetes of the young. HNF-1alpha homozygous knockout mice exhibit a renal Fanconi syndrome with glucosuria and generalized aminoaciduria in addition to diabetes. We investigated glucosuria and aminoaciduria in patients with HNF-1alpha mutations. Sixteen amino acids were measured in urine samples from patients with HNF-1alpha mutations, age-matched nondiabetic control subjects, and age-matched type 1 diabetic patients, type 2 diabetic patients, and patients with diabetes and chronic renal failure. The HNF-1alpha patients had glucosuria at lower glycemic control (as shown by HbA1c) than type 1 and type 2 diabetic patients, consistent with a lower renal glucose threshold. The HNF-1alpha patients had a generalized aminoaciduria with elevated levels of 14 of 16 amino acids and an increased mean Z score for all amino acids compared with control subjects (0.66 vs. 0.00; P < 0.0005). Generalized aminoaciduria was also present in type 1 diabetic (Z score, 0.80; P < 0.0001), type 2 diabetic (Z score, 0.71; P < 0.0002), and chronic renal failure (Z score, 0.65; P < 0.01) patients. Aminoaciduria was not associated with microalbuminuria or proteinuria but was associated with glucosuria (1.00 glucosuria vs. 0.19 no glucosuria; P = 0.002). In type 1 diabetic patients, urine samples taken on the same day showed significantly more aminoaciduria when glucosuria was present compared with when it was absent (P < 0.01). In conclusion, HNF-1alpha mutation carriers have a mutation-specific defect of proximal tubular glucose transport, resulting in increased glucosuria. In contrast, the generalized aminoaciduria seen in patients with HNF-1alpha mutations is a general feature of patients with diabetes and glucosuria. Glucose may depolarize and dissipate the electrical gradient of the sodium-dependent amino acid transporters in the proximal renal tubule, causing a reduction in amino acid resorption. [ABSTRACT FROM AUTHOR]