George Dunea

Publications of George Dunea

• Stem cells from foreign body granulation tissue accelerate recovery from acute kidney injury.

  Authors: Jilpa Patel, Nishit Pancholi, Krishnamurthy P Gudehithlu, Perianna Sethupathi, Peter D Hart, George Dunea, Jose A L Arruda, Ashok K Singh

  Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 10/2011;

  BACKGROUND: In previous studies, we obtained mesenchymal stem cells called granulation tissue stem cells (GTSC) from a regenerating granulation tissue created by placing a foreign body in theBACKGROUND: In previous studies, we obtained mesenchymal stem cells called granulation tissue stem cells (GTSC) from a regenerating granulation tissue created by placing a foreign body in the subcutaneous tissue of rats. Here, we used GTSC to ameliorate ischemia/reperfusion-induced acute kidney injury (AKI) in rats.METHODS: In two groups of Fischer rats, we induced ischemia/reperfusion injury. Group 1 (treated rats) received one intravenous injection of GTSC 3 h after injury; Group 2 (control rats) received vehicle. Both groups were subsequently studied by renal function tests, kidney histology and immunohistochemistry.RESULTS: At 24 and 48 h after injury, plasma creatinine and blood urea nitrogen were significantly lower in the treated rats as compared to control rats. The levels remained low and declined to near baseline levels by Day 4 in the treated group. At the cortico-medullary region, the treated rats showed significantly higher renal tubular cell proliferation and less tubular cell apoptosis. Histological analysis of the kidney for tubular dilatation, necrosis, congestion and casts was not significantly different in the two groups. To understand the mechanism of the GTSC effect, messenger RNA levels of several growth and immune modulatory factors were quantified in cultured GTSC and compared with those in cultured glomerular epithelial cell (GEC; a non-stem cell line). GTSC had 2- to 8-fold higher expression of FGF2, HGF, IGF-1, vascular endothelial growth factor (growth factors) and IL-4, IL-6 (anti-inflammatory factors) than GEC.CONCLUSIONS: Administration of GTSC accelerates recovery in rats with ischemia/reperfusion-induced AKI. This effect may be mediated by the paracrine action of growth and immune-suppressive factors secreted by these cells.

• Culture of omentum-induced regenerating liver yielded hepatocyte-committed stem cells.

  Authors: Nishit Pancholi, Jilpa Patel, Krishnamurthy P Gudehithlu, Mark A Kraus, George Dunea, Jose A L Arruda, Ashok K Singh

  Translational research : the journal of laboratory and clinical medicine. 12/2010; 156(6):358-68.

  Earlier we showed that when omentum, activated by inert particles, is allowed to fuse to a wedge cut in the liver, it induces stem cell proliferation in the liver resulting in massive liverEarlier we showed that when omentum, activated by inert particles, is allowed to fuse to a wedge cut in the liver, it induces stem cell proliferation in the liver resulting in massive liver regeneration. Here, we attempt to culture stem cells from the omentum-induced regenerating liver tissue. Cells from regenerating liver tissue were harvested and cultured. Cultured cells were characterized by immune staining, fluorescence activated cell sorting analysis, growth factor assay, in vitro differentiation, and their ability to engraft to injured sites in vivo. Culture yielded cells with a mesenchymal stem cell phenotype that could be maintained in culture indefinitely. These cells, called regenerating liver stem cells, expressed both adult and embryonic stem cell markers, secreted high levels of vascular endothelial growth factor, and expressed albumin. When grown on matrigel in the presence of hepatocyte growth factor, these cells differentiated into hepatocyte-like cells in culture, but they did not differentiate to adipogenic and osteogenic lineages when grown in specific differentiation medium. The differentiated cells expressed α-fetoprotein and secreted high levels of albumin and urea. After systemic injection, the undifferentiated cells engrafted only to the injured sites in the liver and not to the normal areas of the liver. In conclusion, omentum-induced regenerating liver yields hepatocyte-committed stem cells in culture. Such cells could prove to be useful in cell transplantation therapies.

• Foreign body-induced granulation tissue is a source of adult stem cells.

  Authors: Jilpa Patel, Krishnamurthy P Gudehithlu, George Dunea, Jose A L Arruda, Ashok K Singh

  Translational research : the journal of laboratory and clinical medicine. 04/2010; 155(4):191-9.

  In the current study, we have cultured and propagated the cells obtained from the granulation tissue that forms around perforated polyvinyl tubes placed in the subcutaneous space of normal rats. WeIn the current study, we have cultured and propagated the cells obtained from the granulation tissue that forms around perforated polyvinyl tubes placed in the subcutaneous space of normal rats. We found that these cells (called granulation tissue-derived stem cells [GTSCs]) expressed markers of embryonic pluripotent cells (Oct-4 and Nanog) and of adult stem cells (CXCR4 and Thy1.1) as well as produced high levels of vascular endothelial growth factor (VEGF) for up to 10 passages. By fluorescence-activated cell-sorting (FACS) analysis, GTSCs were positive for stem-cell surface markers CD90, CD59, and CD44 and were negative for CD45, which suggests that they were of mesenchymal origin and not of hematopoietic lineage. When incubated in specific differentiation medium, these cells transformed into adipogenic, osteogenic, and chondrogenic lineages, which shows that they were multipotent. Furthermore, after systemic injection, these cells were found in the vicinity of an injured site created in the liver but not in normal areas of the liver, which indicates their propensity to seek and engraft to an injured area in the body. We conclude that granulation tissue induced by a large foreign body is a convenient source of adult stem cells that can be maintained in culture and can be used to repair and regenerate injured tissue.

• Omentum facilitates liver regeneration.

  Authors: Ashok K Singh, Nishit Pancholi, Jilpa Patel, Natalia O Litbarg, Krishnamurthy P Gudehithlu, Perianna Sethupathi, Mark Kraus, George Dunea, Jose Al Arruda

  World journal of gastroenterology : WJG. 04/2009; 15(9):1057-64.

  AIM: To investigate the mechanism of liver regeneration induced by fusing the omentum to a small traumatic injury created in the liver. We studied three groups of rats. In one group the rats wereAIM: To investigate the mechanism of liver regeneration induced by fusing the omentum to a small traumatic injury created in the liver. We studied three groups of rats. In one group the rats were omentectomized; in another group the omentum was left in situ and was not activated, and in the third group the omentum was activated by polydextran particles. METHODS: We pre-activated the omentum by injecting polydextran particles and then made a small wedge wound in the rat liver to allow the omentum to fuse to the wound. We monitored the regeneration of the liver by determining the ratio of liver weight/body weight, by histological evaluation (including immune staining for cytokeratin-19, an oval cell marker), and by testing for developmental gene activation using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: There was no liver regeneration in the omentectomized rats, nor was there significant regeneration when the omentum was not activated, even though in this instance the omentum had fused with the liver. In contrast, the liver in the rats with the activated omentum expanded to a size 50% greater than the original, and there was histologically an interlying tissue between the wounded liver and the activated omentum in which bile ducts, containing cytokeratin-19 positive oval cells, extended from the wound edge. In this interlying tissue, oval cells were abundant and appeared to proliferate to form new liver tissue. In rats pre-treated with drugs that inhibited hepatocyte growth, liver proliferation was ongoing, indicating that regeneration of the liver was the result of oval cell expansion. CONCLUSION: Activated omentum facilitates liver regeneration following injury by a mechanism that depends largely on oval cell proliferation.

  Download

• A case of lymphangioleiomyomatosis with membranous nephropathy and likely systemic lupus.

  Authors: Ramin Sam, Samra Khalid, Carolyn Brecklin, Melvin Schwartz, George Dunea

  Clinical and experimental nephrology. 10/2008;

  Membranous glomerulonephritis is most often idiopathic, but it can be secondary to systemic lupus erythematosus, viral hepatitis, and drugs. A number of malignancies have also been associated withMembranous glomerulonephritis is most often idiopathic, but it can be secondary to systemic lupus erythematosus, viral hepatitis, and drugs. A number of malignancies have also been associated with membranous glomerulonephritis, although a causal link has not been established yet. A young patient with lymphangioleiomyomatosis is described who developed massive proteinuria and was found to have membranous glomerulonephritis on renal biopsy. There were many pathological features and some clinical features that suggested the patient also suffered from systemic lupus erythematosus.

• Determination of carbamylated hemoglobin by an enzyme immunoassay (ELISA) based on a novel antibody.

  Authors: Ashok K Singh, Krishnamurthy P Gudehithlu, Ramin Sam, Jose A L Arruda, George Dunea, Arshia Gaffari, Perianna Sethupathi

  Clinica chimica acta; international journal of clinical chemistry. 06/2008; 391(1-2):112-4.

• Stromal cells cultured from omentum express pluripotent markers, produce high amounts of VEGF, and engraft to injured sites.

  Authors: Ashok K Singh, Jilpa Patel, Natalia O Litbarg, Krishnamurthy P Gudehithlu, Perianna Sethupathi, Jose A L Arruda, George Dunea

  Cell and tissue research. 05/2008; 332(1):81-8.

  When rat omentum becomes activated by intraperitoneal injection of inert polydextran particles, these particles are rapidly surrounded by cells that express markers of adult stem cells (SDF-1alpha,When rat omentum becomes activated by intraperitoneal injection of inert polydextran particles, these particles are rapidly surrounded by cells that express markers of adult stem cells (SDF-1alpha, CXCR4, WT-1) and of embryonic pluripotent cells (Oct-4, Nanog, SSEA-1). We have cultured such cells, because they may offer a convenient source of adult stem cells, and have found that they retain stem cell markers and produce high levels of vascular endothelial growth factor for up to ten passages. After systemic or local injection of these cultured cells into rats with acute injury of various organs, the cells specifically engraft at the injured sites. Thus, our experiments show that omental stromal cells can be cultured from activated omentum, and that these cells exhibit stem cell properties enabling them to be used for repair and possibly for the regeneration of damaged tissues.

  Download

• Activated omentum becomes rich in factors that promote healing and tissue regeneration.

  Authors: Natalia O Litbarg, Krishnamurthy P Gudehithlu, Perianna Sethupathi, Jose A L Arruda, George Dunea, Ashok K Singh

  Cell and tissue research. 07/2007; 328(3):487-97.

  In order to study the mechanism by which an omental pedicle promotes healing when applied to an injured site, we injected a foreign body into the abdominal cavity to activate the omentum. One weekIn order to study the mechanism by which an omental pedicle promotes healing when applied to an injured site, we injected a foreign body into the abdominal cavity to activate the omentum. One week after the injection, we isolated the omentum and measured blood vessel density, blood content, growth and angiogenesis factors (VEGF and others), chemotactic factors (SDF-1 alpha), and progenitor cells (CXCR-4, WT-1). We found that the native omentum, which consisted mostly of adipose tissue, expanded the mass of its non-adipose part (milky spots) 15- to 20-fold. VEGF and other growth factors increased by two- to four-fold, blood vessel density by three-fold, and blood content by two-fold. The activated omentum also showed increases in SDF-1 alpha, CXCR-4, and WT-1 cells (factors and cells positively associated with tissue regeneration). Thus, we propose that an omentum activated by a foreign body (or by injury) greatly expands its milky-spot tissue and becomes rich in growth factors and progenitor cells that facilitate the healing and regeneration of injured tissue.

• Impaired integration of endothelial progenitor cells in capillaries of diabetic wounds is reversible with vascular endothelial growth factor infusion.

  Authors: Ashok K Singh, Krishnamurthy P Gudehithlu, Shreya Patri, Natalia O Litbarg, Perianna Sethupathi, Jose A L Arruda, George Dunea

  Translational research : the journal of laboratory and clinical medicine. 06/2007; 149(5):282-91.

  To understand impaired angiogenesis in diabetic wounds, polyvinyl tubes were implanted subcutaneously in rats to form a granulation tissue for 2 weeks and the granulation tissue was studied afterTo understand impaired angiogenesis in diabetic wounds, polyvinyl tubes were implanted subcutaneously in rats to form a granulation tissue for 2 weeks and the granulation tissue was studied after inducing diabetes with streptozotocin. By 1 week of diabetes, the granulation tissue was bloody and thinner than controls, its medial layer was depleted of microvessels, and the surviving vessels appeared dehisced. Vascular endothelial growth factor (VEGF) in the diabetic granulation tissue was reduced by 50% compared with control granulation tissue. After 3 days of diabetes, the diabetic tissue showed a greater degree of apoptosis in the microvessels. Chemotactic factors [stromal cell-derived factor-1alpha and chemokine receptor-4 (CXCR-4)], responsible for attracting bone marrow cells, showed equal intensity in control and diabetic tissues. As expected, progenitor endothelial CD-34 cells were observed in abundance in both the control and the diabetic granulation tissues. However, although the CD-34-positive cells appeared mostly to be integrated in the blood vessels of the control tissue, fewer such cells were present in the blood vessels of the diabetic tissues, suggesting that CD-34 failed to integrate into new blood vessels. Infusion of VEGF in the granulation tissue of diabetic rats for 1 week resulted in complete prevention of the microvascular defect compared with the contralateral granulation tissue that showed the typical diabetic changes. It was concluded that diabetes causes reduction of VEGF in the wound, resulting in loss of blood vessels by apoptosis and possible failure of CD-34 cells to integrate into the vessel structure.

• Transplanting fragments of diabetic pancreas into activated omentum gives rise to new insulin producing cells.

  Authors: Ashok K Singh, Krishnamurthy P Gudehithlu, Natalia O Litbarg, Perianna Sethupathi, Jose A L Arruda, George Dunea

  Biochemical and biophysical research communications. 04/2007; 355(1):258-62.

  To determine if pancreatic progenitor cells can be induced to form insulin producing cells in vivo, we auto-transplanted fragments of streptozotocin-induced diabetic pancreas into omentumTo determine if pancreatic progenitor cells can be induced to form insulin producing cells in vivo, we auto-transplanted fragments of streptozotocin-induced diabetic pancreas into omentum pre-injected with a foreign material. As shown previously, omentum pre-activated in this manner becomes rich in growth factors and progenitor cells. After auto-transplanting diabetic pancreas in the activated omentum, new insulin secreting cells appeared in the omentum in niches surrounding the foreign particles--a site previously shown to harbor progenitor cells. Extracts of these omenta contained measurable insulin. Four of eight diabetic animals treated in this manner became normoglycemic. This shows that new insulin producing cells can be regenerated from diabetic pancreas by auto-transplanting pancreatic fragments into the activated omentum, an environment rich in growth factors and progenitor cells.

• Bilateral emphysematous pyelonephritis in a patient with no known risk factors.

  Authors: Peter D Hart, Mohammad Vaseemuddin, Osbert Egiebor, George Dunea

  Journal of the National Medical Association. 03/2007; 99(2):179-81.

  Emphysematous pyelonephritis (EPN) is a rare life-threatening necrotizing infection of the kidney and perirenal space with gas formation. It is usually unilateral and affects patients with a riskEmphysematous pyelonephritis (EPN) is a rare life-threatening necrotizing infection of the kidney and perirenal space with gas formation. It is usually unilateral and affects patients with a risk factor such as diabetes or urinary obstruction. In the past, most patients required nephrectomy, and in bilateral cases long-term dialysis was inevitable. We present here the unusual case of a patient who developed bilateral EPN in the absence of any known risk factor. He was managed conservatively, required dialysis and bilateral nephrostomies, but eventually recovered completely.

• Idiopathic hypocomplementemic immune-complex-mediated tubulointerstitial nephritis.

  Authors: Mohammad Vaseemuddin, Melvin M Schwartz, George Dunea, Mark A Kraus

  Nature clinical practice. Nephrology. 02/2007; 3(1):50-8.

  BACKGROUND: A 42-year-old man presenting with flank pain was found to have renal failure with severe hypocomplementemia and eosinophilia. INVESTIGATIONS: Physical examination, laboratory testing,BACKGROUND: A 42-year-old man presenting with flank pain was found to have renal failure with severe hypocomplementemia and eosinophilia. INVESTIGATIONS: Physical examination, laboratory testing, renal ultrasonography, and renal biopsies. DIAGNOSIS: Acute immune-complex-mediated tubulointerstitial nephritis. MANAGEMENT: Immunosuppressive therapy with 1 mg/kg/day prednisone.

• Glomerular epithelial cells transform to myofibroblasts: early but not late removal of TGF-beta1 reverses transformation.

  Authors: Ramin Sam, Linda Wanna, Krishnamurthy P Gudehithlu, Sandra L Garber, George Dunea, Jose A L Arruda, Ashok K Singh

  Translational research : the journal of laboratory and clinical medicine. 10/2006; 148(3):142-8.

  Studies were carried out to determine whether epithelial-mesenchymal transformation (EMT), well described in renal tubular epithelial cells, also occurs in glomerular epithelial cells and whether itStudies were carried out to determine whether epithelial-mesenchymal transformation (EMT), well described in renal tubular epithelial cells, also occurs in glomerular epithelial cells and whether it is reversible. To this effect, cultured glomerular epithelial cells were incubated with TGF-beta(1) and their transformation into myofibroblasts was studied. At 4 days, the cells altered their phenotype, as shown by a change in shape, an increase in intracellular staining for alpha-smooth muscle actin (alpha-SMA), a decrease in membrane staining for cytokeratin, and an increase in matrix deposition. Changing the medium after 4 days by excluding TGF-beta(1) and adding fetal bovine serum (FBS) [as a source of epidermal growth factor (EGF) and other growth factors] caused the cells to revert to their original epithelial phenotype. By contrast, when the medium was changed in the same manner after 8 days of exposure to TGF-beta(1), the cells did not revert but remained myofibroblastic. Staining the cells for expression of EGF receptor before and after exposure to TGF-beta(1) caused this receptor, originally present on the plasma membrane, to become partly intracellular after 4 days of TGF-beta(1) exposure and completely intracellular after 8 days of TGF-beta(1) exposure. Kidney sections from 2 models of renal mass reduction were stained. Loss of the epithelial marker (podocalyxin) staining and the acquisition of alpha-SMA staining was observed in the glomeruli. It is concluded that EMT takes place in glomerular epithelial cells in vivo and in vitro. In cultured glomerular epithelial cells, the process can be reversed by early, but not late intervention. It seems that TGF-beta(1) exposure progressively downregulates the EGF receptor on the membrane, rendering the cell refractory to EGF signals critical for maintaining the epithelial phenotype.

• Lipoprotein glomerulopathy: a new apolipoprotein E mutation with enhanced glomerular binding.

  Authors: Ramin Sam, Henry Wu, Lily Yue, Ted Mazzone, Melvin M Schwartz, Jose A L Arruda, George Dunea, Ashok K Singh

  American journal of kidney diseases : the official journal of the National Kidney Foundation. 04/2006; 47(3):539-48.

  We describe a case of lipoprotein glomerulopathy, the second ever reported from the United States, in a Mexican man with a hitherto undescribed mutation in the apolipoprotein E gene (substitution ofWe describe a case of lipoprotein glomerulopathy, the second ever reported from the United States, in a Mexican man with a hitherto undescribed mutation in the apolipoprotein E gene (substitution of proline for arginine at position 147 [Arg147Pro]). In this patient, glomerular basement membranes showed double contours and circumferential mesangial extensions, suggesting that deposition of lipids could be injurious to endothelial cells. Immunofluorescence staining of thrombi was positive for apolipoprotein E and B. To study the reason for lipid deposition in glomeruli, we incubated normal human kidney sections with serum from the patient and a healthy control. Apolipoprotein E from the patient's serum showed binding to the glomerular capillary wall, but the control did not, suggesting enhanced binding of the mutated apolipoprotein E to glomerular capillaries. Apolipoprotein E genotyping by means of restriction endonuclease digestion of polymerase chain reaction-amplified genomic DNA showed it to be of the wild-type E3/E3.

• Antagonism of vascular endothelial growth factor results in microvessel attrition and disorganization of wound tissue.

  Authors: Krishnamurthy P Gudehithlu, Naila Ahmed, Henry Wu, Natalia O Litbarg, Sandra L Garber, Jose A L Arruda, George Dunea, Ashok K Singh

  The Journal of laboratory and clinical medicine. 05/2005; 145(4):194-203.

  Vascular endothelial growth factor (VEGF) is a potent growth factor that is indispensable for the development of blood vessels in the fetus and for wound healing in adults. VEGF likely plays a roleVascular endothelial growth factor (VEGF) is a potent growth factor that is indispensable for the development of blood vessels in the fetus and for wound healing in adults. VEGF likely plays a role in maintaining the blood vessels once they have been formed. It is not clear, however, whether a low tissue VEGF (caused either by disease or by systemic administration of VEGF antagonists) can cause abnormalities in preexisting blood vessels, especially of wound tissue that requires high local levels of VEGF for healing. The present study investigated the effect of VEGF antagonism on blood vessels of foreign-body granulomas (a model of wound-healing tissue). Granulomas were induced by implanting perforated polyvinyl tubes into the subcutaneous tissue of rats and allowed to develop for 14 days, at which time the implanted tubes were completely encapsulated by the subcutaneous tissue. The encapsulated granulomas consisted of 3 distinct histological layers, of which the middle layer was well perfused by a rich supply of microvessels. Morphologically, the granuloma remained "stable" after developing for 14 days. At 1 week, VEGF levels in the granuloma fluid, which is in equilibrium with the interstitial fluid, were 25 times higher than in the plasma. VEGF levels in the granuloma fluid continued to increase for up to 3 weeks, reflecting the high dependence of the wound tissue on ambient VEGF levels. After injection of the VEGF receptor antagonist in the fully formed granuloma, the preexisting blood vessels in the middle layer regressed and underwent apoptosis, accompanied by expansion of the extracellular matrix (predominately collagen I) into areas normally devoid of matrix. We conclude that wound tissue is sensitive to ambient VEGF levels, and that a low VEGF condition resulting from VEGF receptor antagonism can disrupt the healing of wound tissue.

• Decreased urinary peptide excretion in patients with renal disease.

  Authors: Ashok Singh, Krishnamurthy P Gudehithlu, Gigi Le, Natalia O Litbarg, Viorica Khalili, Jane Vernik, Peter Hart, Jose A L Arruda, George Dunea

  American journal of kidney diseases : the official journal of the National Kidney Foundation. 01/2005; 44(6):1031-8.

  BACKGROUND: Normal urine contains low-molecular-weight peptides or protein fragments that have been poorly studied, primarily because of the technical difficulty of measuring peptides in the presenceBACKGROUND: Normal urine contains low-molecular-weight peptides or protein fragments that have been poorly studied, primarily because of the technical difficulty of measuring peptides in the presence of proteins. We studied these substances in healthy subjects and patients with renal disease and varying degrees of proteinuria to understand the factors that determine their excretion. METHODS: We estimated these substances as the difference between results using the Lowry method (which detects both proteins and peptides) and those obtained using the dye-binding Bradford (Biorad) method (Biorad Laboratories Inc, Hercules, CA; which detects only proteins). RESULTS: We validated this 2-assay approach to measure peptide levels by showing that such proteins as immunoglobulin G, albumin, and lysozyme were measured equally by the Lowry and Biorad methods, whereas degraded proteins were recognized by the Lowry method only, but not by the Biorad method. We found that healthy subjects excreted less than 200 mg of protein, but 3 to 4 g of peptides/g creatinine; thus, peptides constituted approximately 95% of total protein material excreted in urine. Patients with renal disease and proteinuria had a progressive decrease in peptide excretion, ranging from 3 to 0 g/g creatinine. Twenty-five percent of nephrotic patients (18 of 72 patients) excreted very small amounts of peptides in urine (0% to 10% of total protein material). CONCLUSION: We found that healthy persons excrete substantial amounts of peptides in urine, and this excretion decreases in the presence of proteinuric renal disease. It is possible that these peptides in urine arise from the tubular degradation of filtered proteins and exocytosis of protein fragments toward the urinary side, a process that becomes increasingly impaired as proteinuria increases.

• Degradation of albumin by the renal proximal tubule cells and the subsequent fate of its fragments.

  Authors: Krishnamurthy P Gudehithlu, Alfredo A Pegoraro, George Dunea, Jose A L Arruda, Ashok K Singh

  Kidney international. 07/2004; 65(6):2113-22.

  BACKGROUND: In view of recent reports of large amounts of albumin fragments present in normal urine we investigated the mechanism of albumin handling by the proximal tubule. METHODS: We injectedBACKGROUND: In view of recent reports of large amounts of albumin fragments present in normal urine we investigated the mechanism of albumin handling by the proximal tubule. METHODS: We injected (125)I-albumin intravenously in rats and measured the excretion of intact and degraded (125)I-albumin in the urine by trichloroacetic acid (TCA) precipitation. The excretion rate of intact (125)I-albumin was compared to that obtained by routine radioimmunoassay (RIA). Human proximal tubular HK-2 cells were used to characterize the albumin receptor and study the degradation of albumin to peptides, establish their size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, and determine the direction in which the degradation products are removed from the cell. RESULTS: Following injection of (125)I-albumin intravenously into rats we recovered large quantities of (125)I-albumin fragments in urine and determined that 98% was in a highly degraded form and only 2% was intact. Only the intact albumin could be detected by RIA. We observed similar results in the urine of ex vivo kidneys perfused with (125)I-albumin. We found that (125)I-albumin was taken up by HK-2 cells via a receptor, degraded in the lysosomes and the peptides exocytosed to both the apical and basolateral sides of the cells. CONCLUSION: We conclude that normally the kidney degrades large amounts of albumin and that the degradation fragments appear in the urine. These findings are in sharp contrast with the established view that degraded albumin is completely reabsorbed into the blood stream.

• Vascular factors altered in glucose-treated mesangial cells and diabetic glomeruli. Changes in vascular factors impair endothelial cell growth and matrix.

  Authors: Ashok K Singh, Krishnamurthy P Gudehithlu, Alfredo A Pegoraro, Gogi K Singh, Khaja Basheerudin, Robert B Robey, Jose A L Arruda, George Dunea

  Laboratory investigation; a journal of technical methods and pathology. 06/2004; 84(5):597-606.

  We studied the effect of a high glucose (HG) environment on the vascular factors that are secreted by mesangial cells, and regulate endothelial growth and mesangial matrix deposition. To this effect,We studied the effect of a high glucose (HG) environment on the vascular factors that are secreted by mesangial cells, and regulate endothelial growth and mesangial matrix deposition. To this effect, we measured the vascular factors in the glomeruli of streptozotocin-induced diabetic kidneys and in mesangial cells exposed to a HG concentration. We then transferred the media of mesangial cells previously exposed to high glucose to cultured endothelial cells to study the effects on endothelial growth, matrix formation, and in vitro capillary proliferation. In 1-week diabetic kidneys, glomerular vascular endothelial growth factor (VEGF) and angiopoietin-1 were inhibited by 38 and 57%, respectively, but angiopoietin-2 was increased by 318%. We found similar results in mesangial cells exposed to HG. There was a decrease of VEGF (50% by enzyme immunoassay, 27% by mRNA), decrease of angiopoietin-1 (65% by mRNA), and a much greater increase of angiopoietin-2 (280% by immunoassay, 523% by mRNA). Compared to controls, the media of mesangial cells previously exposed to HG impaired endothelial cell growth by 61%, increased extracellular matrix by 100%, and decreased capillary formation by 90%. We conclude that high ambient glucose alters the secretion of vascular factors elaborated by mesangial cells, resulting in an expansion of the endothelial cell matrix and disruption of capillary structure.

• Handling of low-density lipoprotein by the renal tubule: release of fragments due to incomplete degradation.

  Authors: Alfredo A Pegoraro, Krishnamurthy P Gudehithlu, Ernest Cabrera, Ravi Shankar, Jose A L Arruda, George Dunea, Ashok K Singh

  The Journal of laboratory and clinical medicine. 07/2002; 139(6):372-8.

  Because the mechanism by which lipoproteins are processed and modified in the renal tubule in patients with nephrosis is not completely understood, we studied the handling of low-density lipoproteinBecause the mechanism by which lipoproteins are processed and modified in the renal tubule in patients with nephrosis is not completely understood, we studied the handling of low-density lipoprotein (LDL) in perfused rat kidneys made permeable by protamine. Protamine pretreatment increased the clearance of 125(I) LDL 25-fold compared to controls, thereby simulating a proteinuric kidney. Similar studies were also conducted in kidneys of rats made proteinuric by the induction of passive Heymann nephritis. Of the perfused iodinated LDL, 5% was localized in the cortex and lesser amounts in the medulla and urine. In the cortex and medulla, iodinated LDL was present mainly in the intact form (90%); just 10% was present in the degraded form. Using horseradish peroxidase conjugated to LDL, we demonstrated specific staining in the proximal tubules, suggesting that specific LDL receptors were present in that location. Although LDL in the tissue was present mostly in the intact form, it was 95% degraded in urine, and the degradation was inhibited by chloroquine, indicating that the lysosomes were the site of LDL metabolism. Gel chromatography and electrophoresis of iodinated LDL in the urine showed the presence of fragments in the range of 5 to 15 kD. We conclude that renal degradation of LDL is incomplete and that the incompletely degraded fragments released into the urine may be toxic to the kidney by virtue of their lipid side-chains.

• Differential effect of xanthopterin and biopterin on cell growth

  Authors: Marvin Rubenstein, Sergey Muchnik, Sandra L. Garber, Jose A.L. Arruda, George Dunea

  International Journal of Biochemistry.

  1.1. Xanthopterin inhibited proliferation of primary renal proximal tubule cells (RPTC) and LLC-PK1 cells while in a growth phase but when incubated at confluence the cells were relatively1.1. Xanthopterin inhibited proliferation of primary renal proximal tubule cells (RPTC) and LLC-PK1 cells while in a growth phase but when incubated at confluence the cells were relatively insensitive.2.2. The growth of malignant human prostate PC-3 cells was also inhibited by xanthopterin in a concentration and time dependent manner.3.3. Dunning R3327 AT-3 rat prostate tumor cells which were exposed to xanthopterin in vitro before their in vivo inoculation resulted in smaller tumours while in vivo administration of xanthopterin following implantation also resulted in smaller tumors.4.4. Xanthopterin exerts differential effects on cell growth dependent upon the cell origin and their state of proliferation.