Francesco Addabbo

Kidney-derived mesenchymal stem cells contribute to vasculogenesis, angiogenesis and endothelial repair

We isolated a clonal cell line (4E) from kidneys of mice expressing green fluorescent protein controlled by the endothelial-specific Tie2 promoter. When grown in a three-dimensional matrigel matrix they formed a fluorescent capillary network. In vivo angiogenesis assays using growth factor-depleted matrigel implanted plugs promoted a moderate angiogenesis of host endothelial cells. Using vascular endothelial growth factor (VEGF)-A and fibroblast growth factor-2 in the plugs containing 4E-cells resulted in a robust vasculogenesis. Transplantation of 4E cells into mice with acute renal ischemia showed selective engraftment in the ischemic kidney which promoted tubular regeneration by increasing epithelial proliferation and inhibiting apoptosis. This resulted in an accelerated functional recovery 3 days after ischemia. These mice showed a 5-fold increase in tissue VEGF expression compared to controls, but no difference in plasma VEGF level corresponding with better preservation of peritubular capillaries, perhaps due to a local paracrine effect following systemic 4E infusion. One month after ischemia, 9% of engrafted 4E cells expressed green fluorescent protein in the peritubular region while half of them expressed alpha-smooth muscle actin. Our study shows that kidney mesenchymal stem cells are capable of differentiation toward endothelial and smooth muscle cell lineages in vitro and in vivo, support new blood vessel formation in favorable conditions and promote functional recovery of an ischemic kidney.

Mitochondria and Reactive Oxygen Species

Fascination by the mitochondria, “the colonial posterity of migrant prokaryocytes, probably primitive bacteria that swam into ancestral precursors of our eukaryotic cells and stayed there,”1 stems from the above-mentioned nebulous endosymbiotic theory of their origin, as well as from the growing realization of a very special role that they play in the pathogenesis of diverse diseases. These organelles generate energy primarily in the form of the electrochemical proton gradient (ΔμH+), which fuels ATP production, ion transport, and metabolism.2 Generation of this universal energy currency, ΔμH+, occurs through the series of oxidative reactions conducted by the respiratory chain complexes at the ion-impermeable, almost cholesterol-free inner membrane. Reduced nicotinamide adenine dinucleotide represents the entry point to the complex I (reduced nicotinamide adenine dinucleotide:ubiquinone reductase), whereas the reduced ubiquinol enters the respiratory chain in the complex III (ubiquinol:cytochrome c [cyt-c] reductase) to reduce cyt-c, the electron carrier to the complex IV, cyt-c oxidase. Each of these steps generates ΔμH+ by electrogenic pumping of protons from the mitochondrial matrix to the intermembrane space and is coupled to electron flow, thus generating the electric membrane potential of −180 to −220 mV and a pH gradient of 0.4 to 0.6 U across the inner mitochondrial membrane resulting in the negatively charged matrix side of the membrane and alkaline matrix. Ultimately, accumulated ΔμH+ is converted into the influx of protons into the matrix driving ATP synthesis or protein transport. In addition, these end points are necessary for the execution of 2 major enzymatic metabolic pathways within the mitochondrial matrix: the tricarboxylic acid (TCA) oxidation cycle and the fatty acid β-oxidation pathway. This intricate system fueling cellular functions is as elegant as it is vulnerable: practically every component of the system, from the electron transport chain complexes to the permeability properties of the membranes, is a …

Vascular actions of insulin with implications for endothelial dysfunction

Hemodynamic actions of insulin depend largely on the hormones ability to stimulate synthesis and release of endothelial mediators, whose balanced activity ensures dynamic control of vascular function. Nitric oxide (NO), endothelin-1 (ET-1), and reactive oxygen species (ROS) are important examples of endothelial mediators with opposing properties on vascular tone, hemostatic processes, and vascular permeability. Reduced NO bioavailability, resulting from either insufficient production or increased degradation of NO, characterizes endothelial dysfunction. In turn, endothelial dysfunction predicts vascular complications of metabolic and hemodynamic disorders. In the cardiovascular system, insulin stimulates the production and release of NO, ET-1, and ROS via activation of distinct intracellular signaling pathways. Under insulin-resistant conditions, increased insulin concentrations and/or impaired insulin-signaling pathways in the vasculature may contribute to imbalance in secretion of endothelial mediators that promote pathogenesis of vascular abnormalities. This short review describes signaling pathways involved in insulin-stimulated release of NO, ROS, and ET-1 and suggests possible molecular mechanisms by which abnormal insulin signaling may contribute to endothelial dysfunction.

Heme Oxygenase-Mediated Increases in Adiponectin Decrease Fat Content and Inflammatory Cytokines Tumor Necrosis Factor-α and Interleukin-6 in Zucker Rats and Reduce Adipogenesis in Human Mesenchymal Stem Cells

Adiponectin, an abundant adipocyte-derived plasma protein that modulates vascular function in type 2 diabetes, has been shown to provide cytoprotection to both pancreatic and vascular systems in diabetes. Therefore, we examined whether up-regulation of heme oxygenase (HO)-1 ameliorates the levels of inflammatory cytokines and influences serum adiponectin in Zucker fat (ZF) rats. ZF rats displayed a decrease in both HO activity and HO-1 and HO-2 protein levels and an increase in tumor necrosis factor (TNF)-α and interleukin (IL)-6 compared with Zucker lean (ZL) rats. Treatment of ZF animals with 2 mg/kg cobalt protoporphyrin IX (CoPP) increased protein levels of HO-1 and HO activity, but HO-2 was unaffected. The increase in HO-1 was associated with a decrease in superoxide levels (p < 0.05) and an increase in plasma adiponectin (p < 0.005), compared with untreated ZF rats. CoPP treatment decreased visceral and s.c. fat content, and it reduced weight gain (p < 0.01). In addition, the inflammatory cytokines TNF-α and IL-6 were decreased (p < 0.04 and p < 0.008, respectively). Treatment of human bone marrow-derived adipocytes cultured with CoPP resulted in an increase in HO-1 and a decrease in superoxide levels. Up-regulation of HO-1 caused adipose remodeling, smaller adipocytes, and increased adiponectin secretion in the culture medium of human bone marrow-derived adipocytes. In summary, this study demonstrates that the antiobesity effect of HO-1 induction results in an increase in adiponectin secretion, in vivo and in vitro, a decrease in TNF-α and IL-6, and a reduction in weight gain. These findings highlight the pivotal role and symbiotic relationship of HO-1 and adiponectin in the modulation of the metabolic syndrome phenotype.

Heme oxygenase-mediated increases in adiponectin decrease fat content and inflammatory cytokines, TNF and IL-6, in Zucker rats and reduce adipogenesis in human mesenchymal stem cells

Adiponectin, an abundant adipocyte-derived plasma protein that modulates vascular function in type 2 diabetes, has been shown to provide cytoprotection to both pancreatic and vascular systems in diabetes. Therefore, we examined whether up-regulation of heme oxygenase (HO)-1 ameliorates the levels of inflammatory cytokines and influences serum adiponectin in Zucker fat (ZF) rats. ZF rats displayed a decrease in both HO activity and HO-1 and HO-2 protein levels and an increase in tumor necrosis factor (TNF)-α and interleukin (IL)-6 compared with Zucker lean (ZL) rats. Treatment of ZF animals with 2 mg/kg cobalt protoporphyrin IX (CoPP) increased protein levels of HO-1 and HO activity, but HO-2 was unaffected. The increase in HO-1 was associated with a decrease in superoxide levels (p < 0.05) and an increase in plasma adiponectin (p < 0.005), compared with untreated ZF rats. CoPP treatment decreased visceral and s.c. fat content, and it reduced weight gain (p < 0.01). In addition, the inflammatory cytokines TNF-α and IL-6 were decreased (p < 0.04 and p < 0.008, respectively). Treatment of human bone marrow-derived adipocytes cultured with CoPP resulted in an increase in HO-1 and a decrease in superoxide levels. Up-regulation of HO-1 caused adipose remodeling, smaller adipocytes, and increased adiponectin secretion in the culture medium of human bone marrow-derived adipocytes. In summary, this study demonstrates that the antiobesity effect of HO-1 induction results in an increase in adiponectin secretion, in vivo and in vitro, a decrease in TNF-α and IL-6, and a reduction in weight gain. These findings highlight the pivotal role and symbiotic relationship of HO-1 and adiponectin in the modulation of the metabolic syndrome phenotype.

Adriamycin Nephropathy : A Failure of Endothelial Progenitor Cell-Induced Repair

Adriamycin-associated nephropathy (AAN) remains poorly understood. We hypothesized that adriamycin affects endothelial progenitor cells (EPCs), leading to impaired regeneration. We analyzed renal hematopoietic stem cells (HSCs) and EPCs in mice with AAN and examined the potential contribution of adoptive transfer of intact EPCs to the repair processes. FACS analyses revealed that populations of HSCs and EPCs were scarcely represented in control kidneys and did not change numerically in kidneys obtained from mice with AAN. The observed defect in engraftment was attributable to the decreased viability and increased senescence of EPCs. Adoptive transfer of intact EPCs improved proteinuria and renal function, with a threefold decrease in mortality. Infusion of EPCs to adriamycin-treated mice reduced plasma levels of interleukin-1alpha and -beta and granulocyte-colony stimulating factor as well as increased the level of vascular endothelial growth factor with concomitant improvement of vascular density and reduction of apoptosis. An additional mechanism of tissue repair is proposed based on tunneling nanotube formation between EPCs and endothelial cells exposed to adriamycin, leading to the multiple rounds of exchange between EPCs and mature cells. In conclusion, AAN is associated with development of EPC incompetence; adoptive transfer of intact EPCs blunts morphological and functional manifestations of AAN; and the proposed mechanisms of repair by EPCs include direct incorporation into blood vessels, paracrine signaling, and tunneling nanotube renewal of mitochondrial pool in endothelial cells.

Diagnostic Potential of Urine Proteome: A Broken Mirror of Renal Diseases

This brief overview of studies into the urine proteome illustrates its potential value for diagnostic, prognostic, and pathophysiologic discovery. Hypothesis-targeted investigations of individual proteins as well as proteome-wide searches for urinary biomarkers of various diseases and their progression are reviewed. The majority of urine proteins appear as cleavage products that are found not only as free solutes but also in secreted membrane vesicles called exosomes. Described are several recent examples of important diagnostic findings using urine proteomics along with the idea that signature profiles of injury to individual nephron segments can be measured by this technology. Shared are some thoughts on the most challenging step: Integration of seemingly unrelated findings of various protein fragments into a rational pathogenetic pathway(s). The future chance that the centuries-old technique of uroscopy will reveal its secrets using modern proteomic approaches makes gradual improvement.

Association of anemia and erythropoiesis stimulating agents with inflammatory biomarkers in chronic kidney disease

Inflammatory cytokines are important predictors of cardiovascular mortality especially in patients with chronic kidney disease. Here we explored the relationship of anemia and epoetin treatment to inflammatory cytokine levels in patients with chronic kidney disease. One hundred non-dialysis patients with chronic kidney disease over 18 years of age were evenly split into anemic and non-anemic cohorts. Of the 50 anemic patients, 23 were receiving erythropoiesis stimulating agents treatments. Levels of tumor necrosis factor (TNF)-alpha were found to be significantly higher and serum albumin was significantly lower with trends towards higher interleukin (IL)-6 and IL-8 in anemic compared to non-anemic patients. Further analysis by multiple logistic regression found that anemic patients treated with erythropoiesis stimulating agents had significantly higher odds for the upper two quartiles for IL-6, IL-8 and TNF-alpha compared to non-anemic patients. Our study found that the anemia of chronic kidney disease was associated with up regulation of TNF-alpha, and possibly IL-6 and IL-8 along with increased levels of these proinflammatory cytokines in patients treated with epoetin.

Selective Decrease in Urinary Aquaporin 2 and Increase in Prostaglandin E2 Excretion Is Associated with Postobstructive Polyuria in Human Congenital Hydronephrosis

This study was undertaken to determine the role of aquaporin 2 (AQP2) in the impaired urinary concentrating capacity observed in patients who underwent pyeloplasty because of congenital unilateral hydronephrosis as a result of pyeloureteral junction disease. Twelve children (mean age, 8 +/- 2 mo) were examined in the study. From day 1 to day 5 after surgery, the urine was collected separately from pyelostomy draining only from the postobstructed kidney and from the bladder catheter draining mostly from the contralateral kidney used as internal control. After pyeloplasty, the postobstructed kidney was characterized by a reduced urinary excretion of AQP2 (approximately 54%) associated with polyuria that persisted from day 1 to day 5 (433 +/- 58 versus 310 +/- 74 ml/24 h at day 1; 326 +/- 44 versus 227 +/- 26 ml/24 h at day 5). In parallel, urine osmolality from the postobstructed kidney was significantly reduced compared with the contralateral kidney (111 +/- 12 versus 206 +/- 49 at day 1; 136 +/- 24 versus 235 +/- 65 mOsm/kg at day 5). Creatinine clearance from the postobstructed kidney was not significantly different compared with the contralateral kidney throughout the 4 d after surgery. However, on day 5, creatinine clearance from the postobstructed kidney became significantly lower. Prostaglandin E2 in the urine from postobstructed kidneys was found to be twofold higher than in the contralateral samples (26.0 +/- 6.7 versus 13.5 +/- 2.5 at day 5). It is concluded that (1) the selective downregulation of AQP2 in postobstructed kidney may account for the higher excretion of hypotonic urine, and (2) the local increase in prostaglandin E2 synthesis in postobstructed kidney may be involved in AQP2 downregulation and in maintaining a GFR similar to that of the contralateral kidney.

Characterization of two novel missense mutations in the AQP2 gene causing nephrogenic diabetes insipidus

Here, we report the aquaporin 2 (AQP2) mutational analysis of a patient with nephrogenic diabetes insipidus heterozygote due to two novel missense mutations. Direct sequencing of DNA in the male patient revealed that he was compound heterozygote for two mutations in the AQP2 gene: a thymine-to-adenine transversion at position 450 (c.450T>A) in exon 2 and a guanine-to-thymine at nucleotide position 643 (c.643G>T) in exon 4. The double heterozygous 450T>A and 643G>T transversion causes the amino acid substitution D150E and G215C. Direct sequencing of exons 2 and 4 of the AQP2 gene from each of the parents revealed that the c.450T>A mutation was inherited from the father while the c.643G>T mutation was inherited from the mother. Analysis of AQP2 excretion demonstrated that no AQP2 was detectable in the urine of the proband, whereas normal AQP2 levels were measured in both parents. When expressed in renal cells, both proteins were retarded in the endoplasmic reticulum and no redistribution was observed after forskolin stimulation. Of note, homology modeling revealed that the two mutations involve two highly conserved residues providing important clues about the role of the wt residues in AQP2 stability and function.