bluuebunny/crossref_metadata_2025 huggingface dataset:
Visceral Glomerular Epithelial Cell DNA Synthesis in Experimental and Human Membranous Disease
Authors: Bailey Elaine , Harper Steven J. , Pringle James H. , Baker Frease , Furness Peter N. , Salant David J. , Feehally John
Published: April 2003

Background: Membranous nephropathy (MN) is a ‘non-proliferative’ glomerulonephritis. However, visceral glomerular epithelial cell (vGEC) proliferating cell nuclear antigen staining and increased glomerular histone mRNA in passive Heymann nephritis (PHN), suggest that vGECs may enter the cell cycle and undergo DNA synthesis. We used in situ hybridisation for histone mRNA, an S-phase specific marker, to investigate this possibility and identify the cellular origin of histone mRNA in PHN and MN. Methods: PHN was induced in 16 Sprague-Dawley rats. There were 8 saline/serum controls. 12 animals were sacrificed on days 5 and 10. Renal biopsies from 10 proteinuric cases with MN and matched controls were studied. Results: Day-5 Heymann animals demonstrated more S-phase cells/glomerulus than controls (0.53 ± 0.09 vs. 0.195 ± 0.045; p < 0.01). Glomerular S-phase cells were also increased in patients compared to controls (0.24 ± 0.07 vs. 0.04 ± 0.018; p < 0.03). In both experimental and human MN, the peripheral location and morphology of glomerular histone mRNA-positive cells was typical of vGECs. Conclusion: The results in PHN indicate that vGECs recently injured with antibody and complement enter into the cell cycle and undergo DNA synthesis. The S-phase vGECs in MN may indicate the persistence of immune injury. Whether or not this process leads to cell replication is open to question.
Ultrastructure of the Vacuolar Apparatus in the Renal Proximal Tubule Microinfused in vivo with the Cytological Stain Light Green
Authors: Cui Shiying , Mata Lucinda , Maunsbach Arvid B. , Christensen Erik Ilsø
Published: April 2003

To obtain insight into the basic mechanisms controlling endocytosis, we tested the effects of different perfusates containing the cytological stain light green on endocytosis and ultrastructure of the vacuolar apparatus in renal proximal tubule cells. Rat proximal tubules were microinfused in vivo for 2 min in the presence or absence of light green with the following solutions: (A) perfusates containing inorganic salts and (B) perfusates with organic components or protein. In other experiments, the tubules were first microinfused for 2 min with 0.9% NaCl in the presence or absence of light green, then 15 min later further microinfused with or without light green using either group A or B solutions in order to either aggravate or reverse possible changes. All infused tubules were fixed after 5 min with 1% glutaraldehyde and examined by electron microscopy. In tubules microinfused without light green, the endocytic vacuolar apparatus in the apical cytoplasm showed a normal ultrastructure. However, microinfusion of solutions containing light green in either inorganic salts or a low concentration of protein caused significant changes in the apical endocytic apparatus. Large endocytic vacuoles were absent, and invaginations and small endocytic vacuoles were decreased in frequency. On the other hand, the amount of dense apical tubules was significantly increased, and in some cells dense apical tubules had transformed into a cisternalike network. These changes were aggravated in tubules which received a second microinfusion of NaCl and reversed in tubules that received a second infusion of protein. Furthermore, in the tubules microinfused with light green using perfusates containing organic components or protein, the apical cytoplasm of proximal tubule cells showed an essentially normal endocytic apparatus. The present study demonstrates that microinfusion of renal proximal tubules with light green disrupted normal endocytic membrane trafficking and recycling. These changes could be prevented or reversed by microinfusion of solutions containing protein or organic components.
Gene Transfer into the Adult Kidney for Unravelling Disease Processes
Authors: Kitamura Masanori , Fine Leon G.
Published: April 2003

Insights into the pathogenesis of human disease must be based on an understanding of the molecular mechanisms that regulate the structure and function of individual organs. For this purpose, gene transfer technologies provide powerful and attractive tools. In principle, two different approaches are feasible to identify pathophysiological roles of certain genes: ‘gain-of-function’, and ‘loss-of-function’. The former examines consequences of overexpression of an exogenous gene, and the latter investigates the outcomes of inhibition of a particular molecule via antisense, decoy, ribozyme and dominant-negative strategies. Gene transfer to specific renal structures allows evaluation of in vivo effects of certain molecules on the structure and function of each nephron segment. It would also be useful for therapeutic intervention in renal diseases through introduction of therapeutically relevant genes into affected sites. This article summarizes current experience with renal gene transfer and addresses its potential impacts on the understanding of renal function in vivo.
Integrated Function of Urea Transporters in the Mammalian Kidney
Authors: Trinh-Trang-Tan Marie-Marcelle , Bankir Lise
Published: April 2003

Specific urea transporters are responsible for the rapid urea movements occurring in precise medullary structures of the mammalian kidney. Three of them, ensuring facilitated passive transports, have been cloned yet: UT2-long is responsible for the high vasopressin-dependent urea permeability of the terminal inner medullary collecting ducts; UT2-short is located along a short portion of the thin descending limbs of Henle’s loops; UT11 is expressed along the descending vasa recta. These three transporters are involved in the accumulation of urea in the medulla, participating to the corticopapillary osmotic gradient required for urine concentration in the presence of antidiuretic hormone. UT2-long enables diffusion of urea in the inner medulla, and UT2-short and UT11 enable the recycling of this urea by counter-exchange. These transporters could also be involved in nitrogen balance by modulation of their expression according to the need for urea excretion (protein-rich diet), or for nitrogen conservation (protein-poor diet). Several other urea transporters, including active transporters responsible for urea secretion or reabsorption, remain to be cloned and characterized.
Vascular Endothelial Growth Factor: How It Transmits Its Signal
Authors: Zachary Ian
Published: April 2003

Vascular endothelial growth factor (VEGF) is an essential molecule in the development and formation of mammalian blood vessels in health and disease. VEGF is also increasingly implicated in other biological processes including renal development and pathophysiology. The biological activities of VEGF in vivo and in its target cells in culture are mediated through two receptor protein tyrosine kinases, KDR/Flk-1 and Flt-1. KDR/Flk-1 is able to mediate the tyrosine phosphorylation of several cellular components as well as the generation of second messengers. Recent findings have revealed novel signaling mechanisms which may mediate the biological actions of VEGF. In contrast, the signal transduction mechanisms triggered by Flt-1 remain largely unknown.
Highly Specific Separation of Heterogeneous Cell Populations by Lectin-Coated Beads: Application for the Isolation of Inner Medullary Collecting Duct Cells
Authors: Grupp Clemens , Troche Ilka , Steffgen Jürgen , Langhans Sigrid , Cohen David I. , Brandl Lydia , Müller Gerhard A.
Published: April 2003

Conditions for the highly specific selection of a cell type by the use of lectin-coated magnetic beads are reported for the isolation of inner medullary collecting duct (IMCD) cells from a heterogeneous inner medullary cell suspension, containing both single cells and tubular fragments of variable size. The lectin Dolichos Biflorus Agglutinin (DBA), which binds in rat inner medulla exclusively to IMCD cells, was coupled via the avidin-biotin system to beads. By isolating DBA-bead-IMCD cells in a magnetic field (positive selection) from a suspension containing about 50% IMCD, a fraction of 98 ± 1% purity was obtained; recovery of cells was up to 90%. Suspensions negative on reverse-transcriptase polymerase chain reaction for vimentin as a marker of contaminating interstitial and vascular cells could be received by repeating this procedure and additional trypsinization. On the other hand, it was possible to reduce the portion of IMCD cells in the suspension by one isolation step to 1.5 ± 0.9% (negative selection). Performing this step twice resulted in virtually pure suspensions. No significant effects of this isolation technique on cell viability, growth characteristics, and biochemical parameters were observed. Therefore, this method appears to be a powerful tool for the highly specific separation of heterogeneous cell populations.
Accelerated Recovery of Glycerol-Induced Acute Renal Failure in Rats with Previous Partial Hepatectomy
Authors: Homsi Eduardo , Pires de Oliveira Dias Eliana , Figueiredo Jose Francisco , Gontijo Jose Antonio
Published: April 2003

Several studies have reported a favorable effect following the administration of growth factors during the course of acute renal failure. To evaluate the effect of an increased endogenous production of growth factors, rats underwent 70% hepatectomy before glycerol-induced acute renal failure. The renal function was then monitored 12 and 48 h after glycerol injection in conscious rats. Twelve hours after the induction of acute renal failure, a reduction in creatinine clearance and sodium reabsorption was seen in both prehepatectomized and sham-hepatectomized rats. At 48 h, there was total recovery of glomerular filtration and tubular function in the prehepatectomized rats, whereas the sham-hepatectomized rats still had reduced glomerular filtration and sodium reabsorption. In rats treated with dexamethasone before hepatectomy and studied 48 h after the induction of acute renal failure, the protective action on renal function seen in prehepatectomized rats was totally blocked. A semiquantitative histological analysis done 48 h after the induction of acute renal failure demonstrated a fourfold increase in the number of necrotic tubules in both sham-hepatectomized and dexamethasone-treated rats as compared with hepatectomized rats. It is concluded that partial hepatectomy, a maneuver used to increase the endogenous synthesis of growth factors, accelerates recovery of renal function following glycerol-induced acute renal failure.
Transgene Regulation by the Tetracycline-Controlled Transactivation System
Authors: Kitamura Masanori
Published: April 2003

The tetracycline-controlled transactivation system is one of the most powerful tools for modifiable expression of exogenous genes. This system enables selective, stringent and quantitative regulation of transgenes in vitro and in vivo. By combining with antisense genes, ribozymes or genes encoding dominant negative mutants, it is also feasible to control certain endogenous gene functions. Combination with transgenic or ‘gene knockout’ technologies enables the generation of mice in which a certain gene function is conditionally reinforced or deleted. These sophisticated methods are useful for investigation of gene function during embryogenesis and in adult organs and for well-controlled gene therapy approaches. This article briefly outlines the concept of the tetracycline-regulated gene expression systems and addresses the current experience in kidney research.
Alpha Macroglobulins and the Low-Density-Lipoprotein-Related Protein/Alpha-2-Macroglobulin Receptor in Experimental Renal Fibrosis
Authors: van Goor Harry , Diamond Jonathan R. , Ding Guohua , Kaysen GeorgeA.
Published: April 2003

In this study, we evaluated the location of non-specific proteinase inhibitors and their receptor in experimental glomerular and interstitial fibrosis. The alpha macroglobulins alpha-2-macroglobulin (α2M) and alpha-1-inhibitor 3 (α1I3) are proteinase inhibitors, including metalloproteinases and serine proteases. Using immunohistochemistry, we detected α1I3 in the glomerular mesangium in control rats. In acute and chronic fibrosis, the α1I3 protein expression was dramatically increased throughout the glomerulus and at sites of increased extracellular matrix deposition in the interstitium. The presence of α1I3 in normal and nephrotic kidneys was confirmed by Western blotting. Under chemically reducing conditions, we found that, in contrast to native α1I3, kidney-derived α1I3 has reacted upon by proteinases, thereby revealing a functional role for this macroglobulin under normal and pathological conditions. Double staining revealed that high amounts of glomerular α1I3 were present in sclerotic lesions. α2M was absent in glomeruli and interstitium from control rats, but present in small amounts in glomerular mesangial areas of acute nephrotic rats. α2M was also present in significant amounts in glomeruli from rats with chronic fibrosis. The receptor mediating the uptake of proteinase inhibitor-proteinase complexes, the low-density-lipoprotein-related protein/alpha-2-macroglobulin receptor, was found in the glomerular mesangium and tubulo-interstitium from control rats. Significant increments in receptor expression were found in glomeruli and interstitium of rats with chronic fibrosis, with a preferential localization in fibrotic areas. Interstitial staining for low-density-lipoprotein-related protein/alpha-2-macroglobulin receptor was attributed to fibroblasts, since double staining ruled out dendritic cells and macrophages. In conclusion, these studies demonstrate the intrarenal presence of plasma-derived proteinase inhibitors together with their receptors. These findings may point to a novel mechanism for renal fibrosis wherein matrix-degrading proteinases are inhibited, resulting in renal fibrosis.
Targeting Collecting Tubules Using the Aquaporin-2 Promoter
Authors: Stricklett Peter K. , Nelson Raoul D. , Kohan Donald E.
Published: April 2003

The aquaporin-2 promoter has been used to drive Cre recombinase expression in order to achieve renal collecting duct principal cell specific gene deletion. This technique requires two lines of mice: one transgenic mouse line containing a cell-specific promoter driving Cre recombinase expression and the other line, engineered using gene targeting strategies, that contains a lox-flanked target gene of interest. Mating of these two mouse lines permits cell-specific deletion of the target gene. This method could ultimately be used to obtain targeted deletion of any gene in any cell type in the kidney for which a specific promoter has been identified. The applications of this technology, as well as its strengths and weaknesses, are discussed with particular referance to the kidney.
Mesangial Matrix Modulation and Glomerulosclerosis
Authors: Fogo Agnes B.
Published: April 2003

Progressive deterioration of the kidney is common to many renal diseases. The structural injuries which lead to this progressive loss of function consist of focal segmental glomerulosclerosis and tubulointerstitial fibrosis and atrophy. These processes were previously thought to be inexorable, regardless of the primary disease. However, recent observations point to the possibility of reversal of sclerosis. Mesangial matrix accumulation is the cornerstone of glomerulosclerosis and results when matrix synthesis exceeds matrix degradation. The renin-angiotensin system appears to be one central component of this process, with links to numerous mechanisms which promote matrix accumulation. Most recently, direct induction of plasminogen activator inhibitor-1 by angiotensin has been recognized. Plasminogen activator inhibitor-1 not only promotes thrombosis, but also inhibits matrix degradation. The various mechanisms which modulate mesangial matrix accumulation and their potential reversibility are reviewed.
Glomerular Podocytes and Adhesive Interaction with Glomerular Basement Membrane
Authors: Mundel Peter , Shankland Stuart J.
Published: April 2003

Normal podocyte function requires attachment to the underlying glomerular basement membrane. Alteration or disruption of podocyte attachment occurs in many forms of glomerular injury, leading to the development of proteinuria and eventually progressive glomerulosclerosis. The inability of podocytes to proliferate and thereby recover denuded glomerular basement membrane areas may be central to the pathogenesis of certain forms of glomerular diseases.
Arg-Gly-Asp (RGD) Peptides and Peptidomimetics as Therapeutics: Relevance for Renal Diseases
Authors: Horton Michael A.
Published: April 2003

Cells interact with the extracellular matrix and other cells via cell adhesion receptors which include those of the integrin family. Since the pivotal demonstration in 1984 by Pierschbacher and Ruoslahti that cell adhesion mediated by fibronectin could be inhibited by the simple tripeptide, Arg-Gly-Asp (RGD), then number of other peptide sequences have been shown to recapitulate integrin-ligand interactions. Similarly, the function of integrins in normal renal development and physiology and changes in adhesion receptor expression in diseases, such as glomerulonephritis or renal carcinoma, have suggested that abnormal integrin function in the kidney could be susceptible to modification by integrin antagonists. This possibility has been tested in experimental acute renal failure with ‘RGD peptides’ and in modifying renal tranpslant rejection in patients by use of antibodies to various leucocyte or endothelial cell adhesion molecules. The recent development of a number of orally active, non-peptidic integrin antagonists suggests that treatment of a range of renal diseases may be susceptible to strategies that involve the blockade of integrin function or modulation of their expression.
Effect of Human Natural Xenoantibody Depletion and Complement Inactivation on Early Pig Kidney Function
Authors: Cruzado Josep M. , Torras Joan , Riera Marta , Condom Enric , Lloberas Núria , Herrero Immaculada , Martorell Jaume , Grinyó Josep M.
Published: April 2003

Preformed xenoreactive natural antibodies (XNA) and complement mediate hyperacute xenograft rejection (HXR) in pig-to-human discordant xenotransplantation. In a pig kidney-human blood xenoperfusion model, we investigated whether XNA depletion and/or human complement inactivation preserved early pig kidney function. Pig kidneys were perfused for 180 min with pig blood (AUTO group, n = 8), human blood (HETER group, n = 6), complement-inactivated human blood (COMi group, n = 5), XNA-depleted human blood (ABd group, n = 5) or complement-inactivated and XNA-depleted human blood (ABd&COMi group, n = 5). HETER kidneys were rejected after 15–30 min and showed vascular microthrombi and interstitial hemorrhages. XNA depletion and/or complement inactivation prevented HXR. The glomerular filtration rate in ABd, COMi and ABd&COMi groups was significantly lower than in the AUTO group. Also, beyond 60 min, the COMi group showed a significantly lower glomerular filtration rate than that observed in ABd and ABd&COMi groups. Kidneys from ABd, COMi and ABd&COMi groups displayed endothelial cell edema, as well as higher soluble P-selectin levels and a higher renal myeloperoxidase content than the AUTO group kidneys. COMi and ABd&COMi groups had a significantly lower renal myeloperoxidase level than the HETER group. Also, in contrast to HETER and ABd groups, these complement-inactivated groups failed to show a positive correlation between P-selectin and renal myeloperoxidase. We also investigated platelet-activating factor (PAF) as possible mediator for these functional and pathologic changes. We found that blood PAF levels were similar in HETER, ABd, COMi and ABd&COMi groups and significantly higher than in the AUTO group. Also, when PAF was added to porcine endothelial cell monolayers, morphological changes due to cytoskeleton contraction were observed, and these changes were prevented by preincubation with a PAF receptor antagonist. In conclusion, although depletion of XNA and/or complement inactivation prevent HXR, the pig kidney function is not preserved at the level of the autologous combination. The PAF overproduction observed in the xenogenic combination, which is independent of the presence of XNA and complement, may be, at least in part, responsible for early endothelial cell morphological changes still present when HXR is prevented.
Cadmium Nephrotoxicity Assessed in Isolated Rat Glomeruli and Cultured Mesangial Cells: Evidence for Contraction of Glomerular Cells
Authors: Barrouillet M.P. , Potier M. , Cambar J.
Published: April 2003

Cadmium (Cd), an important pollutant, causes severe damage at the renal tubular level. Numerous previous studies have focused upon Cd tubular nephrotoxicity. The present study of Cd-induced glomerular damage examined the vasoactive effect of Cd in freshly isolated glomeruli and mesangial cells. Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by culture for outgrowth of cells. Quantitative evaluation of glomerular and cellular contractions was performed by morphometric measurement of the area with an automatized image analyzer following different incubation times with Hanks’ balanced salt solution or Cd2+. Each glomerulus or mesangial cell served as its own control. Cd lethality was measured with microassay methods (neutral red, MTT uptake, and lactate dehydrogenase release), allowing the determination of an IC50. This ranged from 35 to 60 μM. CdCl2 induced a time-dependent contractile effect on isolated glomeruli; planar surface area decreases were 6.9% (1 μM), 7.5% (0.1 μM), and 7% (0.01 μM). The decrease started as soon as Cd was in contact with glomeruli and ended 40 min later: T5 (2%), T10 (3.5%), T20 (4.2%), T30 (6.3%), T40 (7%). Cell size reduction was 19% (1 μM), 14% (0.1 μM), and 18% (0.01 μM) and was also time-dependent. To confirm that contractile events occurred during the cell shape changes, examination of the mesangial α-actin network was performed concurrently. These results indicate that Cd contracts glomerular structures. This may, in part, explain the reduction in glomerular filtration seen in Cd nephrotoxicity.
Flow Cytometric Immunodissection of the Human Nephron in vivo and in vitro
Authors: Helbert M.J.F. , Dauwe S. , De Broe M.E.
Published: April 2003

In the present article, we show that flow cytometric immunodissection of cells immediately following their preparation from a tumor nephrectomy specimen is an accurate way of obtaining pure human primary cultures of proximal convoluted tubule origin, proximal straight tubule origin, distal tubular origin and/or collecting duct origin. By studying the expression of a panel of cell surface markers in these purified cultures, we could identify a number of markers that retain their lineage specificity in vitro. Using these appropriate stable markers, flow cytometry provides a simple yet accurate way of determining cell composition in previously unsorted (mixed type) tubular epithelial cultures in terms of proximal versus distal tubule/collecting duct subpopulations. Both subpopulations in mixed type cultures are shown to retain functional characteristics of their in vivo counterparts (glucose uptake, hormonal stimulation of adenylate cyclase) as well as cell type-specific response patterns (such as inducibility of cell adhesion and histocompatibility molecules), indicating the usefulness of studying cell responses in vitro in a cell-type-dependent way. Finally we illustrate that multi-parameter flow cytometry is a powerful tool for assessing constitutive characteristics of and/or responses by the distinct cell subpopulations present in mixed type cultures.
Protein Overload Activates Proximal Tubular Cells to Release Vasoactive and Inflammatory Mediators
Authors: Zoja Carla , Benigni Ariela , Remuzzi Giuseppe
Published: April 2003

Chronic renal diseases with highly enhanced glomerular permeability to proteins are accompanied by tubulointerstitial inflammation and scarring and progression to renal failure. As a consequence of increased glomerular permeability, proteins filtered through the glomerular capillary in excessive amount have intrinsic renal toxicity at least partially linked to their accumulation in the proximal tubular cell cytoplasm during the process of reabsorption along the nephron. Experimental evidence is available showing that protein overload per se activates proximal tubular epithelial cells in culture to upregulate genes encoding for endothelin, chemokines and cytokines. These vasoactive and inflammatory substances, formed in excessive quantities by the tubular cells, are released mainly into the basolateral compartment, a pattern of secretion that in the kidney would favor recruitment and activation of inflammatory cells into the renal interstitium and fibrogenic reaction leading to renal scarring.
The Number of Nephrons in the Mammalian Kidney: Environmental Influences Play a Determining Role
Authors: Lelièvre-Pégorier Martine , Merlet-Bénichou Claudie
Published: April 2003

Several lines of evidence, mostly derived from animal studies, indicate that changes in the fetal environment may affect the renal development. Fetal growth retardation is associated with a nephron deficit in both humans and animals. Changes in the supply of vitamin A to the fetus may be responsible for the variations in the number of nephrons in the human kidney. In utero exposure to hyperglycemia or drugs may also cause a nephron deficit.
Simultaneous Inhibition of Renal Phospholipase A2 and Glutathione Synthesis by Manoalide and DL-Buthionine Sulfoximine Induces Acute Tubular Dysfunction in Rats
Authors: Soejima Akinori , Ishizuka Shunji , Miyake Nobuyuki , Fukuoka Kazuhito , Suzuki Michihiko , Kamiya Yasushi , Nagasawa Toshihiko
Published: April 2003

We have previously demonstrated that gentamicin-induced acute renal failure is mediated by the consumption of renal glutathione (GSH) and accumulation of oxidized phospholipids in tubular epithelial cells as a result of inhibition of phospholipase A2 (PLA2) activity. Based on these results, we tested the hypothesis that the simultaneous inhibition of PLA2 and GSH synthesis induces acute renal failure similar in characteristics to gentamicin-induced acute renal failure. Male Sprague-Dawley rats kept under standard laboratory conditions were administered 3 mmol/kg of DL-buthionine sulfoximine (BSO; γ-glutamylcysteine synthetase inhibitor) and 30 μg/kg of manoalide (PLA2 inhibitor), following which significant elevations in serum creatinine and urinary lysosomal enzyme levels (elevation of N-acetyl-β-D-glucosaminidase activity) were observed. The renal tissue GSH content was reduced in the group that received both BSO and manoalide as compared with the group that received manoalide alone. The renal tissue GSH content was also reduced in the group that received BSO alone. The renal tissue concentration of 2-thiobarbituric-acid-reactive substances increased rapidly, followed by an increase in renal tissue total phospholipid concentration in the group that received both BSO and manoalide. In contrast, the activity of PLA2 in renal tissue decreased in the group that received both BSO and manoalide as compared with the groups that received BSO alone or physiological saline. In conclusion, concomitant administration of BSO and manoalide induces renal tubular damage and acute renal failure in rats, similar in characteristics to gentamicin-induced nephrotoxicity, whereas administration of BSO or manoalide alone did not. These results suggest that both inhibition of PLA2 and GSH depletion are necessary for the induction of acute renal failure.
Chronic Ureteral Obstruction in the Rat Suppresses Renal Tubular Bcl-2 and Stimulates Apoptosis
Authors: Chevalier Robert L. , Smith Christopher D. , Wolstenholme Jennifer , Krajewski Stanislaw , Reed John C.
Published: April 2003

Unilateral ureteral obstruction (UUO) results in widespread tubular apoptosis in obstructed kidneys of both adults and neonates. The oncoprotein bcl-2 inhibits many forms of apoptosis, whereas the related protein bax promotes apoptosis. To evaluate the interaction of bcl-2, bax, and apoptosis in the renal response to UUO, adult and neonatal rats were subjected to UUO or sham operation, and kidneys were harvested 14 days later. Apoptotic cells were identified by the Tunel technique, and the distribution of bcl-2 and bax was determined by immunochemistry. In both adults and neonates, tubular and interstitial apoptosis was present in the obstructed kidney, but not in intact kidneys. In both adults and neonates, there was diffuse tubular bcl-2 and bax staining of sham-operated and intact kidneys. While bcl-2 was increased in scattered nonapoptotic tubules of the obstructed kidney, there was minimal staining of dilated apoptotic tubules. These results are consistent with the premise that bcl-2 normally suppresses renal tubular apoptosis. The distribution of bax staining in tubules of the obstructed kidney overlapped that of bcl-2. We conclude that chronic UUO inhibits bcl-2 expression in selected tubules of the obstructed kidney which contributes to activation of apoptosis and progressive renal damage in either neonatal or adult kidneys. Dysregulation of apoptosis may be a response to renal injury similar to that underlying the development of cystic kidney disease or renal dysplasia.
Preferential COX-2 Inhibitor, Meloxicam, Compromises Renal Perfusion in Euvolemic and Hypovolemic Rats
Authors: Birck R. , Krzossok S. , Knoll T. , Braun C. , van der Woude F.J. , Rohmeiss P.
Published: April 2003

Nonsteroidal anti-inflammatory drugs can impair renal perfusion through inhibition of cyclooxygenase (COX)-mediated prostaglandin synthesis. We investigated the influence of the preferential COX-2 inhibitor, meloxicam (MELO), on renal hemodynamics in eu- and hypovolemic rats compared to the nonselective COX inhibitor indomethacin (INDO). The hypovolemic state was obtained in rats by three daily injections of furosemide (2 mg/kg i.p.) followed by a sodium-deficient diet for 7 days. In euvolemic rats (n = 6) neither INDO (5 mg/kg i.v.) nor MELO (1 or 2 mg/kg i.v.) influenced mean arterial blood pressure (MAP) or impaired renal (RBF) and cortical blood flow (CBF). Medullary blood flow (MBF) decreased after INDO (18%; p < 0.05), and dose-dependently after MELO (1 mg, 10%; 2 mg, 18%; p < 0.05). In hypovolemic rats (n = 6) INDO and MELO had no effect on MAP. RBF and CBF were reduced after INDO (11 or 20%; p < 0.05), but showed no changes after MELO. INDO induced a decrease in MBF (22%; p < 0.05) which was less pronounced after MELO (12%; p < 0.05). In conclusion the preferential COX-2 inhibitor MELO compromized renal perfusion in the outer medulla both in eu- and hypovolemic animals.
Virulence Factors of Escherichia coli Contribute to Acute Renal Failure
Authors: Kreft Burkhard , Pagel Horst
Published: April 2003

Background: The development of acute renal failure (ARF) significantly enhances the mortality of patients with Gram-negative septic shock. The role of specific bacterial virulence factors different from lipopolysaccharides (LPS) in the deterioration of renal function in septic shock remains to be determined. Methods: An Escherichia coli wild-type strain (536/21 WT, O6:K15:H31) was isolated from a patient suffering from a urinary tract infection. The strain expresses various virulence factors (e.g. hemolysin, fimbriae) genetically encoded by pathogenicity islands. The spontaneous deletion mutant 536/21 Del lacks the expression of these virulence factors. Isolated rat kidneys were perfused with a suspension (5 × 104/ml) of the respective strain or control perfusion medium and the renal functional parameters were analyzed. Intrarenal deposition of E. coli was detected by immunohistology and Gram staining. Results: The perfusion of the isolated perfused rat kidney with a uropathogenic E. coli wild-type strain (536/21 WT) caused an acute deterioration of renal function which was not observed in kidneys exposed to a deletion mutant of E. coli 536/21 lacking the expression of virulence factors. The glomerular filtration rate and the urine flow rate significantly decreased only in kidneys perfused with the E. coli wild-type strain, while there was no change versus controls in kidneys perfused with the deletion mutant. Conclusions: Distinctive bacterial virulence factors different from LPS such as hemolysin and the presence of different fimbriae may contribute to the development of ARF in sepsis induced by E. coli. Anti-LPS strategies may not be sufficient to reduce the risk of ARF in Gram-negative septic shock.
Cellular Localization of Aquaporin 7 in the Rat Kidney
Authors: Ishibashi Kenichi , Imai Masashi , Sasaki Sei
Published: April 2003

The cDNA for the seventh mammalian aquaporin (AQP7) was isolated from rat testis, and its expression was demonstrated at the tail of late spermatids [Ishibashi et al: J Biol Chem 1997;272:20782–20786]. AQP7 is also expressed in the kidney. The localization of AQP7 in the kidney is unknown. We examined the cellular localization of AQP7 in the kidney with Northern blot, reverse transcribed PCR, Western blot and immunohistochemistry in the rat kidney. In Northern blot, AQP7 was expressed in the cortex and the outer medulla but absent in the inner medulla of the kidney. Reverse transcribed PCR of rat nephron segments revealed the selective expression of AQP7 at the proximal straight tubules (PST). Western blot of the membrane fraction of outer medulla revealed a single band of ∼33 kDa. Immunohistochemistry of the rat kidney showed the selective expression of AQP7 at the brush border membranes of PST (S3 segment). AQP7 is now shown to be localized selectively at the brush border membranes of PST in the rat kidney. The result suggests that AQP7 may function as a pathway for transcellular water transport in PST in concert with more widely expressed AQP1 in proximal tubules. Alternatively, as AQP7 transports urea as well as water, AQP7 may function as a passive urea secretory pathway in this segment and may play a role in the formation and/or maintenance of the medullary urea concentration gradient.
Endotoxin-Induced Renal Failure
Authors: Heyman Samuel N. , Darmon David , Goldfarb Marina , Bitz Helena , Shina Ahuva , Rosen Seymour , Brezis Mayer
Published: April 2003

The pathogenesis of sepsis-induced renal failure is multifactorial and only partially understood. In these studies we evaluated intrarenal microcirculatory changes during endotoxemia and the potential role of nitric oxide (NO) and endothelin in these changes. In anesthetized rats endotoxin infusion [lipopolysaccharide (LPS), Escherichia coli serotype 0127:B8; 10 mg/kg/h] resulted in hypotension and a transient enhancement of renal blood flow, with cortical vasodilation and a loss of outer medullary vasodilatory response to hypotension. The initial cortical vasodilation was abolished by the NO synthase inhibitor NG-nitro-L-arginine methyl ester, but not by indomethacin. Direct NO measurements disclosed a gradual rise in cortical NO, despite the waning vasodilatory effect, suggesting antagonizing vasoconstrictive stimuli. In rats pretreated by LPS (1 mg/kg i.p. 1 day earlier) the renal blood flow was reduced to 55% of that of controls. Moreover, the vasodilatory response to LPS infusion was converted into profound cortical and medullary vasoconstriction. In these preconditioned rats the endothelin receptor antagonist bosentan evoked a vasodilatory response and attenuated the vasoconstrictive reaction to LPS infusion. The infusion of another LPS (E. coli serotype 0111:B4) exerted predominant and protracted renal vasodilation without hypotension. In conclusion, different LPS exert diverse systemic and renal hemodynamic responses. The 0127:B8 serotype attenuates renal medullary vasodilation during hypotension, exerts transient cortical vasodilation, and following repeated exposure induces profound renal vasoconstriction. NO and endothelin participate in LPS-induced vascular responses that may predispose to hypoxic tubular damage.
Sublytic Complement Injury Does Not Activate NF-κB, or Induce Mitogenesis in Rat Mesangial Cells
Authors: Mudge S.J. , McRae J.L. , Auwardt R.B. , Murphy B.F. , Chen C.-G. , Power D.A.
Published: April 2003

Sublytic complement injury to glomerular mesangial cells, mediated by the terminal membrane attack complex of complement (C5b-9), is a potential initiating mechanism in IgA nephropathy. Sublytic complement injury has been reported to result in the production of a variety of pro-inflammatory molecules and growth factors, including many regulated by the transcription factor NF-κB. To determine the importance of complement injury in the pro-inflammatory signalling which occurs in IgA nephropathy, we investigated NF-κB activation following sublytic complement injury to cultured rat glomerular mesangial cells (RMCs). A sublytic dose of rabbit anti-Thy 1.1 (THY) serum and normal human serum was selected based upon flow cytometry, chromium-release assay, and induction of superoxide production. No significant C5b-9-induced NF-κB activation was detected by electrophoretic mobility shift assays, luciferase activity of RMCs transfected with a NF-κB-driven luciferase reporter construct, nor by Northern blots for the NF-κB-responsive mRNA species monocyte chemoattractant protein-1 or IκBα. Furthermore, measurements of 3H incorporation following sublytic complement injury showed inhibition of mesangial cell mitogenesis in comparison to the heat-inactivated serum treatment and to THY alone. The results of this study suggest that sublytic complement injury to RMC does not directly activate NF-κB nor induce mesangial cell proliferation in mesangial cells. Other mechanisms such as IgA immune complex formation must be required to produce these events in IgA nephropathy.
Prepubertal Rats Are More Resistant to Ischemic Renal Injury and Recover More Rapidly than Adult Rats
Authors: Fernández Marta , Medina Alberto , Carbajo Eduardo , Santos Fernando , Cobo Angeles , Álvarez Jesús , Rodríguez Julián , Fernández Porfirio
Published: April 2003

Bilateral clamping of renal pedicles during 60, 75 or 90 min was used to characterize the evolution of ischemic acute renal failure (ARF) in prepubertal rats. To verify the existence of age-conditioned differences in the evolution of ARF, adult rats were exposed to 40, 60 or 75 min of clamping. After 7 days, survival rate was significantly better in young than adult rats for identical times of clamping (89 vs. 35% for 60 min and 69 vs. 35% for 75 min). Young rats largely died within the first 24 h following ischemia while the risk of death extended until the 4th day after ischemia in adult rats. Peak values of serum urea nitrogen and creatinine were observed on the 1st and 3rd day after ischemia in young and adult rats, respectively. In young rats, these markers of renal function returned to normal on days 5 and 6 whereas they remained elevated at the end of the study in adult animals. Growth curves of young rats paralleled those of sham-operated animals from the 3rd day of clamping whereas adult rats did not even reach the initial weight at the end of the study. Analysis of kidneys obtained 7 days after clamping revealed more severe histopathological lesions in adult rats as well as a higher proliferative activity (10–40 times the value of sham-operated animals versus 2–4 times the control value in young rats). Our findings indicate that kidneys from young rats are more resistant to ischemia and recover more quickly from the ischemic insult. Therefore, the experimental model of ischemic ARF is clearly different in young and adult rats.
Sensitive Analysis of Apoptosis Using Confocal Laser Scan Microscopy
Authors: Oite Takashi
Published: April 2003

Application of the TUNEL method and immunostaining cell-specific markers to a whole isolated glomerulus in combination with confocal laser scan microscopy can be used to analyze cell turnover including apoptosis within glomeruli. Furthermore, the technologies can be used to deepen our understanding of glomerular cell biology and pathophysiology at the cellular and molecular levels.
Defective Processing and Trafficking of Water Channels in Nephrogenic Diabetes insipidus
Authors: Kamsteeg E.J. , Deen P.M.T. , van Os C.H.
Published: April 2003

Nephrogenic diabetes insipidus (NDI) is a disease characterized by the inability of the kidney to concentrate urine upon stimulation with vasopressin. Mutations in the gene for aquaporin-2 (AQP2) are the cause of the autosomal recessive and autosomal dominant forms of NDI. Mutant AQP2 proteins, found in autosomal recessive NDI, were shown to be misfolded and retarded in the endoplasmic reticulum. One mutant protein leading to autosomal dominant NDI, E258K, has been analyzed in detail. It was shown that this mutant was not retarded in the endoplasmic reticulum but mainly retained in the Golgi network. Furthermore, this particular mutant was able to form heterotetramers with wild-type AQP2, in contrast to mutants found in autosomal recessive NDI. The subsequent misrouting of complexes containing wild-type and mutant AQP2 proteins explains dominant NDI.
Molecular Pathology of Renal Chloride Channels in Dent’s Disease andBartter’ s Syndrome
Authors: Thakker R.V.
Published: April 2003

Recent advances in molecular biology have characterised a new class of chloride channels that are referred to as voltage-gated chloride channels (CLCs). To date 9 such CLCs (CLC-1 to CLC-7, CLC-Ka and CLC-Kb which are respectively encoded by the genes CLCN1 to CLCN7, CLCNKa and CLCNKb) have been identified in mammals. Mutations in 2 of these, referred to as CLC-5 and CLC-Kb, have been defined in the hypercalciuric nephrolithiasis disorders of Dent’s disease and a form of Bartter’s syndrome, respectively. In addition, other forms of Bartter’s syndrome have been defined with mutations involving the bumetanide-sensitive sodium-potassium-chloride co-transporter (NKCC2) and the potassium channel ROMK. Finally, mutations of the thiazide-sensitive sodium chloride co-transporter (NCCT) are associated with Gitelman’s syndrome, in which hypocalciuria and hypomagnesaemia are notable features. These molecular genetic studies have increased our understanding of the renal tubular mechanisms that regulate mineral homeostasis.
Low Leptin mRNA Level in Adipose Tissue and Normoleptinemia in Experimental Chronic Renal Failure
Authors: Swierczynski Julian , Korczynska Justyna , Szolkiewicz Marek , Karbowska Joanna , Kochan Zdzislaw , Nieweglowski Tomasz , Kusiak Elzbieta , Rutkowski Boleslaw
Published: April 2003

Background: Anorexia and weight loss frequently accompany chronic renal failure (CRF). Although multiple metabolic changes occur during CRF, a bulk of evidence indicates that the decrease in caloric intake plays a major role in CRF-induced weight loss. Recently, it has been suggested that elevated plasma leptin concentrations could contribute to anorexia and to downregulation of leptin gene expression in CRF patients. However, in some CRF patients, plasma leptin concentrations have been found to be lower than one could expect. Thus we assumed that inhibition of leptin synthesis plays an important role in the regulation of plasma leptin concentrations in CRF patients. Methods: To test this assumption, the leptin mRNA level in rat white adipose tissue from ad-libitum-fed control (sham operated), pair-fed control (sham operated) and rats with experimentally induced CRF has been measured by Northern blotting analysis. In addition, serum leptin concentration (by radioimmunoassay) was determined in all three groups of animals. Results: The results of the present study indicate that in experimental CRF the leptin mRNA level is decreased by about 50% as compared to the sham-operated animals (ad-libitum-fed and pair-fed controls). The mean serum leptin concentration in CRF rats was essentially similar to the leptin concentration in sham-operated ones. Conclusion: The data obtained suggest that in CRF animals the serum leptin concentration might be affected not only by the decrease in leptin removal in the kidney, but also by the decrease in leptin secretion from adipose tissue. Furthermore, the results of the study suggest that leptin may be only one of many factors involved in the pathogenesis of malnutrition associated with CRF.
Intrarenal Renin-Angiotensin System Contributes to Tubular Acidification Adaptation following Uninephrectomy
Authors: Amorena C. , Damasco C. , Igarreta P. , MacLaughlin M.
Published: April 2003

Background/Aims: Reduction in renal mass by uninephrectomy induces a functional compensation in the remnant kidney. The activity of the angiotensin-converting enzyme (ACE) as well as renin mRNA in the proximal convoluted tubule (PCT) of uninephrectomized (UNx) rats increases. The aim of this work was to determine whether the increased activity of the local renin-angiotensin system (RAS) participates in the adaptation of renal function after uninephrectomy. Method: We utilized normal two-kidney (2K) and 3-week UNx rats to study the activity of the ACE in vesicles obtained from luminal membranes of proximal tubular cells and the acidification kinectics in PCTs using micropuncture techniques. Results: The converting enzyme activity was significantly larger in UNx (5.87 ± 0.69 nmol · min–1 · mg protein–1) than in 2K rats (2.43 ± 0.13 nmol · min–1 · mg protein–1; p < 0.05). The acidification rate constant (κ) in PCT of 2K rats was 0.18 ± 0.02 s–1 and in UNx rats 0.30 ± 0.04 s–1 (p < 0.001). In UNx rats, microperfusion with 10–5 M ramipril or 10–5 M losartan decreased κ to 0.19 ± 0.02 and 0.18 ± 0.02 s–1, respectively, but had no effect on 2K rats. Luminal steady-state pH (pH∞) was the same in 2K and UNx rats, and was not modified by addition of 10–5 M ramipril or 10–5 M losartan in both groups. The proximal H+ flux (JH+), calculated from pH∞ and κ, was 1.12 nmol · cm–2 · s–1 in 2K rats and, 1.77 nmol · cm–2 · s–1 in UNx rats (p < 0.001). In 2K rats, this value was not changed by 10–5 M ramipril or 10–5 M losartan, but in UNx rats JH+ decreased 25 and 30% with ramipril or losartan, respectively (p < 0.001). Conclusions: These data suggest that the increase in the local RAS activity could be an adaptive change that contributes to maintain the homeostasis of body fluids after uninephrectomy.
Interactions between Mitochondrial Proteins and Lipid Peroxidation Products in the Maintenance of the Glomerular Filtration Barrier in the in vitro Perfused Kidney
Authors: Wang Shi-Xuan , Solin Marja-Liisa , Ahola Heikki , Luimula Pauliina , Holthöfer Harry
Published: April 2003

Background: The fourth complex of the mitochondrial respiratory chain, cytochrome-c oxidase (CytC) consists of thirteen both mitochondrially and nuclearly encoded subunits, which are differently regulated in proteinuric kidneys. The effect of mitochondrial involvement on proteinuria is not known. Methods: We set up an in vitro kidney perfusion model to study the direct effect of inhibitors of the mitochondrial respiratory chain, rotenone and antimycin A, on the glomerular filtration barrier by using immunohistochemistry and Northern blotting and quantitating the resulting proteinuria. Results: Rapid onset of proteinuria and characteristic changes in CytC subunits were seen in the perfused kidneys. Urinary protein excretion increased significantly in the rotenone- and antimycin-A-treated groups during perfusion. Downregulation of CytC subunits I and IV was similarly found in the groups treated with rotenone and antimycin A, while increases in the lipid peroxidation (LPO) products malondialdehyde and 4-hydroxynonenal which reflect mitochondrial damage, were observed. Conclusions: These data show rapid changes in mitochondrial proteins and induction of proteinuria in response to exposure to mitochondrial inhibitors. Together with the concomitant increase in local LPO products, these results suggest that continuous maintenance of a proper energy balance is important to maintain the glomerular filtration barrier.