Roberta Donadelli

Nitric Oxide Synthesis by Cultured Endothelial Cells Is Modulated by Flow Conditions

In the present study, we examined the hypothesis that dynamic characteristics of flow modulate the production of vasoactive mediators, namely nitric oxide (NO) and endothelin-1 (ET-1), by human umbilical vein endothelial cells (HUVECs). Cells were exposed for 6 hours in a cone-and-plate apparatus to different types of flow: steady laminar, with shear stresses of 2, 8, and 12 dyne/cm2, pulsatile laminar, with shear stress from 8.2 to 16.6 dyne/cm2 and a frequency of 2 Hz; periodic laminar, with square wave cycles of 15 minutes and shear stress from 2 to 8 dyne/cm2, and turbulent, with shear stress of 8 dyne/cm2 on average. A second culture dish was kept in a normal incubator as a static control for each experiment. Laminar flow induced synthesis of NO by HUVECs that was dependent on shear-stress magnitude. Laminar shear stress at 8 dyne/cm2 also upregulated the level of NO synthase mRNA. As observed with steady laminar flow, pulsatile flow also induced an increase in NO release by endothelial cells. When HUVECs were subjected to step-change increases of laminar shear, a further increase of NO synthesis was observed, compared with steady laminar shear of the same magnitude. Turbulent flow did not upregulate NO synthase mRNA or increase NO release. Both laminar and turbulent shear stress reduced, although not significantly, ET-1 mRNA and ET-1 production compared with the static condition. These results indicate that local blood flow conditions modulate the production of vasoactive substances by endothelial cells. This may affect vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone.

Relative Role of Genetic Complement Abnormalities in Sporadic and Familial aHUS and Their Impact on Clinical Phenotype

BACKGROUND AND OBJECTIVES Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Most childhood cases are caused by Shiga toxin-producing bacteria. The other form, atypical HUS (aHUS), accounts for 10% of cases and has a poor prognosis. Genetic complement abnormalities have been found in aHUS. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS We screened 273 consecutive patients with aHUS for complement abnormalities and studied their role in predicting clinical phenotype and response to treatment. We compared mutation frequencies and localization and clinical outcome in familial (82) and sporadic (191) cases. RESULTS In >70% of sporadic and familial cases, gene mutations, disease-associated factor H (CFH) polymorphisms, or anti-CFH autoantibodies were found. Either mutations or CFH polymorphisms were also found in the majority of patients with secondary aHUS, suggesting a genetic predisposition. Familial cases showed a higher prevalence of mutations in SCR20 of CFH and more severe disease than sporadic cases. Patients with CFH or THBD (thrombomodulin) mutations had the earliest onset and highest mortality. Membrane-cofactor protein (MCP) mutations were associated with the best prognosis. Plasma therapy induced remission in 55 to 80% of episodes in patients with CFH, C3, or THBD mutations or autoantibodies, whereas patients with CFI (factor I) mutations were poor responders. aHUS recurred frequently after kidney transplantation except for patients with MCP mutations. CONCLUSIONS Results underline the need of genetic screening for all susceptibility factors as part of clinical management of aHUS and for identification of patients who could safely benefit from kidney transplant.

Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion.

We addressed the role of hyperglycemia in leukocyte-endothelium interaction under flow conditions by exposing human umbilical vein endothelial cells for 24 h to normal (5 mM), high concentration of glucose (30 mM), advanced glycosylation end product-albumin (100 microg/ml), or hyperglycemic (174-316 mg/dl) sera from patients with diabetes and abnormal hemoglobin A1c (8.1+/-1.4%). At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dyn/cm2). Rolling and adherent cells were evaluated by digital image processing. Results showed that 30 mM glucose significantly (P < 0. 01) increased the number of adherent leukocytes to endothelial cells in respect to control (5 mM glucose; 151+/-19 versus 33+/-8 cells/mm2). A similar response was induced by endothelial stimulation with IL-1beta, here used as positive control (195+/-20 cells/mm2). The number of rolling cells on endothelial surface was not affected by high glucose level. Stable adhesion of leukocytes to glucose-treated as well as to IL-1beta-stimulated endothelial cells was preceded by short interaction of leukocytes with the endothelial surface. The distance travelled by leukocytes before arrest on 30 mM glucose, or on IL-1beta-treated endothelial cells, was significantly (P < 0.01) higher than that observed for leukocytes adhering on control endothelium (30 mM glucose: 76.7+/-3.5; IL1beta: 69.7+/-4 versus 5 mM glucose: 21.5+/-5 microm). Functional blocking of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells with the corresponding mouse mAb significantly inhibited glucose-induced increase in leukocyte adhesion (67+/-16, 83+/-12, 62+/-8 versus 144+/-21 cells/ mm2). Confocal fluorescence microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Electrophoretic mobility shift assay of nuclear extracts of human umbilical vein endothelial cells (HUVEC) exposed for 1 h to 30 mM glucose revealed an intense NF-kB activation. Treatment of HUVEC exposed to high glucose with the NF-kB inhibitors pyrrolidinedithiocarbamate (100 microM) and tosyl-phe-chloromethylketone (25 microM) significantly reduced (P < 0.05) leukocyte adhesion in respect to HUVEC treated with glucose alone. A significant (P < 0.01) inhibitory effect on glucose-induced leukocyte adhesion was observed after blocking protein kinase C activity with staurosporine (5 nM). When HUVEC were treated with specific antisense oligodesoxynucleotides against PKCalpha and PKCepsilon isoforms before the addition of 30 mM glucose, a significant (P < 0.05) reduction in the adhesion was also seen. Advanced glycosylation end product-albumin significantly increased the number of adhering leukocytes in respect to native albumin used as control (110+/-16 versus 66+/-7, P < 0.01). Sera from diabetic patients significantly (P < 0.01) enhanced leukocyte adhesion as compared with controls, despite normal levels of IL-1beta and TNFalpha in these sera. These data indicate that high glucose concentration and hyperglycemia promote leukocyte adhesion to the endothelium through upregulation of cell surface expression of adhesive proteins, possibly depending on NF-kB activation.

Protein Overload-Induced NF-κB Activation in Proximal Tubular Cells Requires H2O2 through a PKC-Dependent Pathway

Abnormal traffic of proteins through the glomerular capillary has an intrinsic toxicity that results in tubular dysfunction and interstitial inflammation. It has been previously shown that in porcine proximal tubular cells high concentrations of albumin activated NF-kappaB, which is responsible for the enhanced synthesis of the inflammatory chemokine RANTES. This study investigates whether reactive oxygen species (ROS) served as second messengers in protein overload-induced NF-kappaB activation. Human proximal tubular cells (HK-2) were incubated (5 to 60 min) with human albumin and IgG (1 to 30 mg/ml). Both proteins induced a rapid or significant increase in hydrogen peroxide (H(2)O(2)) production at 5 min and persisting at 60 min. This effect was dose-dependent. The contribution of H(2)O(2) in regulating NF-kappaB activation was evaluated by using the antioxidants dimethyl-thiourea and pyrrolidine dithiocarbamate in protein-overloaded HK-2 cells. Both agents, by preventing H(2)O(2) generation, induced human albumin or IgG inhibited NF-kappaB activation. Stimulation of HK-2 with exogenous H(2)O(2) resulted in the activation of a NF-kappaB subunit pattern similar to that obtained after protein challenge. Specific inhibitors of protein kinase C (PKC) activity significantly prevented H(2)O(2) production and consequent NF-kappaB activation, suggesting that ROS generation in HK-2 cells occurs downstream of PKC activation. Either antioxidants or PKC inhibitor almost completely abolished the upregulation of the monocyte chemoattractant protein-1 gene induced by excess albumin, as evaluated by real-time PCR, thus supporting a role for PKC and ROS as critical signals for the expression of NF-kappaB-dependent inflammatory genes. To identify the enzymatic sources responsible for the increased H(2)O(2) production, the effect of dyphenyleneiodonium, an inhibitor of the membrane NADP(H) oxidase, was studied, as was the effect of rotenone, which blocks complex I of the mitochondrial respiratory chain. It was found that both agents significantly reduced the exaggerated H(2)O(2) induced by protein overload. These data indicate that exposure to excess proteins in proximal tubular cells induces the formation of ROS, which are responsible for NF-kappaB activation and consequent induction of NF-kappaB-dependent inflammatory signals.

MYO1E Mutations and Childhood Familial Focal Segmental Glomerulosclerosis

BACKGROUND Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. METHODS We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. RESULTS We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. CONCLUSIONS MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.

Transforming Growth Factor-β1 Is Up-Regulated by Podocytes in Response to Excess Intraglomerular Passage of Proteins : A Central Pathway in Progressive Glomerulosclerosis

Chronic diseases of the kidney have a progressive course toward organ failure. Common pathway mechanisms of progressive injury, irrespectively of the etiology of the underlying diseases, include glomerular capillary hypertension and enhanced passage of plasma proteins across the glomerular capillary barrier because of impaired permselective function. These changes are associated with podocyte injury and glomerular sclerosis. Direct evidence for causal roles is lacking, particularly for the link between intraglomerular protein deposition and sclerosing reaction. Because transforming growth factor-beta1 (TGF-beta1) is the putative central mediator of scarring, we hypothesized that TGF-beta1 can be up-regulated by protein overload of podocytes thereby contributing to sclerosis. In rats with renal mass reduction, protein accumulation in podocytes as a consequence of enhanced transcapillary passage preceded podocyte dedifferentiation and injury, increase in TGF-beta1 expression in podocytes, and TGF-beta1-dependent activation of mesangial cells. Angiotensin-converting enzyme inhibitor prevented both accumulation of plasma proteins and TGF-beta1 overexpression in podocytes and sclerosis. Albumin load on podocytes in vitro caused loss of the synaptopodin differentiation marker and enhanced TGF-beta1 mRNA and protein. Conditioned medium of albumin-stimulated podocytes induced a sclerosing phenotype in mesangial cells, an effect mimicked by TGF-beta1 and blocked by anti-TGF-beta1 antibodies. Thus, the passage of excess plasma proteins across the glomerular capillary wall is the trigger of podocyte dysfunction and of a TGF-beta1-mediated mechanism underlying sclerosis. Agents to reduce TGF-beta1, possibly combined with angiotensin blockade, should have priority in novel approaches to treatment of progressive nephropathies.

Complement Factor H Mutation in Familial Thrombotic Thrombocytopenic Purpura with ADAMTS13 Deficiency and Renal Involvement

Thrombotic thrombocytopenic purpura is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease ADAMTS13, which favors platelet adhesion and aggregation in the microcirculation. The disease manifests mainly with central nervous system symptoms, but cases of renal insufficiency have been reported. Presented are findings of the genetic basis of phenotype heterogeneity in thrombotic thrombocytopenic purpura in two sisters within one family. The patients had ADAMTS13 deficiency as a result of two heterozygous mutations (causing V88M and G1239V changes). In addition, a heterozygous mutation (causing an S890I change) in factor H of complement was found in the patient who developed chronic renal failure but not in her sister, who presented with exclusive neurologic symptoms.

Protein Overload Induces Fractalkine Upregulation in Proximal Tubular Cells through Nuclear Factor κB– and p38 Mitogen-Activated Protein Kinase–Dependent Pathways

Investigated was the effect of high albumin concentrations on proximal tubular cell expression of fractalkine. Human proximal tubular cells (HK-2) were incubated with human serum albumin (HSA), which induced a dose-dependent increase in fractalkine mRNA associated with increased levels of both membrane-bound and soluble forms of the protein. To evaluate the role of nuclear factor kappaB (NF-kappaB) activation in HSA-induced fractalkine mRNA, HK-2 cells were infected with a recombinant adenovirus encoding the natural inhibitor of NF-kappaB, IkBalpha; a 43% reduction of fractalkine mRNA levels resulted. Similarly, when cells were infected with the recombinant adenovirus expressing dominant negative mutant of the IkB kinase 2, a 55% inhibition of fractalkine mRNA was achieved. p38 mitogen-activated protein kinase was activated by HSA and was involved in NF-kappaB-dependent transcription of fractalkine. In kidneys of mice with bovine serum albumin overload proteinuria, fractalkine mRNA levels were 2.3-fold greater than those of controls. Fractalkine expression was also induced in tubular epithelial cells in this model. Anti-CXCR1 antibody treatment limited interstitial accumulation of mononuclear cells. Protein overload is a promoter of fractalkine gene induction mediated by NF-kappaB and p38 activation in proximal tubular cells. Fractalkine might contribute to direct mononuclear cells into peritubular interstitium and enhance their adhesion property, which in turn would favor inflammation and disease progression.

The renoprotective properties of angiotensin-converting enzyme inhibitors in a chronic model of membranous nephropathy are solely due to the inhibition of angiotensin II: evidence based on comparative studies with a receptor antagonist.

In several models of renal disease progression, angiotensin-converting enzyme (ACE) inhibitors reduced proteinuria and limited glomerulosclerosis, which suggested that reduction of renal angiotensin II (Ang II) activity is crucial for the preservation of glomerular structure and function. However, it cannot be ruled out that other hormonal systems, including inhibition of the bradykinin breakdown, also play a role. We compared the effects of chronic treatment with the ACE inhibitor lisinopril with those of a specific Ang II receptor antagonist, L-158,809, on proteinuria and renal injury in passive Heymann nephritis (PHN), a model of immune renal disease that closely resembles human membranous nephropathy, with long-lasting proteinuria followed by tubulointerstitial damage and glomerulosclerosis. Passive Heymann nephritis was induced with 0.5 mL/100 g of rabbit anti-Fx1A antibody in 24 male Sprague-Dawley rats. The animals were divided into three groups of eight rats each, and were given the following in the drinking water on a daily basis: lisinopril (40 mg/L), L-158,809 (50 mg/L), or no therapy. Treatment started at day 7 (proteinuria was already present) and lasted 12 months. Eight normal rats were used as controls. Untreated PHN rats developed hypertension, while rats with PHN given lisinopril or L-158,809 all had systolic blood pressure values even lower than those of normal rats. Urinary protein excretion progressively increased with time in untreated PHN rats, who developed tubulointerstitial damage and glomerulosclerosis. Both lisinopril and L-158,809 exhibited a potent antiproteinuric effect and preserved glomerular and tubular structural integrity at a similar extent. Renal gene expression of transforming growth factor-beta and extracellular matrix proteins was also effectively reduced by the two treatments. These results indicate that ACE inhibitors and Ang II receptor antagonists are equally effective in preventing renal injury in PHN and suggest that the renoprotective effects of ACE inhibitors in this model are solely due to inhibition of Ang II.

The acute effect of FK506 and cyclosporine on endothelial cell function and renal vascular resistance.

We have previously documented that cyclosporine exerts a direct cytotoxic effect on endothelial cells and causes an increase in renal vascular resistance (RVR) in the rat. In the present study we investigated whether FK506, a novel immunosuppressive agent thought to be less nephrotoxic than CsA, impairs endothelial cell function in vitro and affects RVR in vivo. In vitro eicosanoid release and endothelin release were measured in bovine aortic endothelial cells in culture exposed for 1, 6, and 24 hr to increasing concentrations of FK506 (1 nM to 10 μM) or CsA (0.5, 10 μM). No significant changes in TxB2 and 6-keto-PGF1α (the stable breakdown products of TxA2 and PGI2, respectively) and endothelin release were found after 1 and 6 hr of incubation with all the concentrations of FK506 and CsA considered. FK506 did not affect endothelin release even after 24 hr of incubation. In contrast, cell exposure to CsA was associated with a dose-dependent increase in TxB2, 6-keto-PGF1α, and endothelin release that reached statistical significance after incubation with 10 μM CsA. FK506 did not induce cell detachment or lysis at any concentration and time considered, while 10 μM CsA induced a significant reduction in cell count accompanied by cell lysis. In vivo studies showed that a single i.v. injection of FK506 to rats within a broad range of doses (28 ng/kg to 2.8 μg/kg) did not modify RVR. This was true even for a dose as high as 20 mg/kg, while 20 mg/kg CsA caused a dramatic increase in RVR. We conclude that FK506, unlike CsA, does not induce endothelial cell injury in vitro. Whether this explains the differences in renovascular resistance observed in vivo after acute injection of FK506 and CsA is an attractive possibility that needs to be further explored.