bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Adhesion Molecules for Gene Delivery
Authors: Hart Stephen
Published: April 2003

Integrins are heterodimeric membrane glycoproteins important for interaction of cells with the extracellular matrix and with each other. However, integrins are also exploited as receptors by a number of bacterial, viral, and eukaryotic pathogens for cell binding and entry. This well-conserved pathway into the cell has now been exploited in the development of synthetic gene delivery systems for gene therapy. Peptides, such as the conserved arginine-glycine-aspartic acid (RGD) motif, provide convenient integrin-binding ligands for targeting polycationic complexes formed electrostatically with plasmid DNA. This review describes the development, advantages, and potential applications of such systems in gene therapy.
Regulation of the ROMK Potassium Channel in the Kidney
Authors: Wald H.
Published: April 2003

ROMK is a gene encoding inwardly rectifying adenosine triphosphate regulated K+ channels. Alternative splicing of ROMK exons yields several different transcripts, ROMK 1–3, that are differentially expressed along the nephron. Cloned ROMK channels expressed in Xenopus oocytes exhibit properties similar to those of the native low-conductance K+ secretory channels in cortical collecting duct and medullary thick ascending limb, as manifested by use of the patch-clamp technique. These similarities between the cloned and native channels suggest that ROMK represents the low-conductance secretory K+ channels in the kidney. We studied the role of dietary K+ and aldosterone in the regulation of ROMK mRNA expression in the rat kidney. K+ deficiency downregulated ROMK mRNA in cortex and medulla. Adrenalectomy markedly downregulated cortical ROMK, while it increased it in the medulla. In adrenalectomized rats K+ deficiency decreased ROMK mRNA in cortex and medulla similarly to intact rats. Na-K-ATPase subunits α1 and β1 were regulated in parallel to the regulation of ROMK. In the medulla ROMK mRNA correlated highly with serum K+ and with the α1 and β1 subunits of Na-K-ATPase. These results show that cortical ROMK expression is regulated by aldosterone and K+, while the medullary ROMK mRNA is regulated by serum K+, irrespective of aldosterone.
The Calcium Channel Blocker Nitrendipine Attenuates Renal and Glomerular Hypertrophy in Diabetic Rats
Authors: Nielsen Birgitte , Grønbæk Henning , Østerby Ruth , Ørskov Hans , Flyvbjerg Allan
Published: April 2003

The efficacy of the calcium channel blocker nitrendipine in preventing early renal and glomerular hypertrophy and increased urinary albumin excretion (UAE) was studied in experimental diabetes in rats, starting treatment at the onset of diabetes. Female Wistar rats were randomised into four groups: diabetic and non-diabetic rats were given either placebo or nitrendipine (250 mg/kg) in the diet for 8 weeks. After 8 weeks the kidneys in the diabetic animals had increased significantly compared to the non-diabetic controls. In the diabetic nitrendipine-treated animals renal and glomerular hypertrophy was significantly smaller than in the diabetic placebo-treated group (p < 0.05). After an initial increment within the first week, the UAE remained constant throughout the study period in the diabetic nitrendipine-treated animals, while a steady increase was seen in the diabetic placebo-treated group (p < 0.05). No differences were seen in systemic blood pressure between calcium- channel-blocker-treated groups and placebo-treated groups. In conclusion, administration of nitrendipine to diabetic rats for 8 weeks had a significant inhibitory effect on renal and glomerular hypertrophy, and showed a tendency towards a reduction in UAE (p = 0.06) without affecting metabolic control or systemic blood pressure.
Transfer of Genetically Engineered Cells to the Glomerulus
Authors: Kitamura Masanori
Published: April 2003

In the rat, cultured cells injected into the renal circulation are entrapped in the glomerulus. This peculiar property allows to create chimeric glomeruli in which genetically engineered cells are populated. Using glomerular cells engineered in vitro, it is feasible to generate glomeruli that produce recombinant gene products. This approach would be useful for identification of local function of a certain gene product in the glomerulus and for therapeutic intervention in glomerular disease. Transfer of activated leukocytes to the glomerulus is useful to elucidate pathologic actions of infiltrating cells on the glomerular structure and function. Use of leukocytes in which certain gene function is selectively reinforced or deleted should enable to disclose exact roles of leukocyte-associated genes in glomerular pathophysiology. Transfer of engineered leukocytes also allows to investigate how resident cells modulate the activity of infiltrating cells in normal and pathologic circumstances. This article summarizes current experience with adoptive transfer of engineered cells to the glomerulus and addresses its potential application to kidney research.
Application of Genetically Engineered Tubular Epithelial Cells in Kidney Disease
Authors: Yokoyama Hitoshi , Naito Takero , Wada Takashi , Rubin Kelley Vicki
Published: April 2003

We constructed an ex vivo gene transfer system to deliver cytokines into the kidney and circulation using genetically modified renal tubular epithelial cells (TEC). TEC were infected with recombinant retroviruses expressing macrophage (Mϕ) growth factors using a retroviral Moloney murine leukemia virus-based MFG vector. These infected TEC have the capacity to secrete stable and sustained amounts of cytokines for prolonged periods (>4 months) in vitro and in vivo (>28 days). Implanting genetically modified TEC secreting Mϕ growth factors under the kidney capsule initiates severe local renal injury in mice with a deficiency in Fas (Faslpr gene). This system offers a novel and powerful approach to probe for the impact of sustained cytokine expression in the progression of kidney destruction.
What Makes Cells Grow Larger and How Do They Do It? Renal Hypertrophy Revisited
Authors: Preisig Patricia
Published: April 2003

Hypertrophy, defined as an increase in cell size without an increase in cell number, occurs in a number of conditions, including compensatory renal growth, diabetes mellitus, protein feeding, chronic metabolic acidosis, and chronic potassium deficiency. In vitro cell culture studies have been used to characterize the mechanisms involved in the development of hypertrophy. Two mechanisms have been identified and characterized. One mechanism involves regulation of processes that are also associated with the initial events of the hyperplastic growth process, and is referred as a cell cycle-dependent mechanism. The other mechanism occurs independently of these particular cell cycle processes, but involves regulation of protein degradation by lysosomal enzymes. This latter mechanism is referred to as a cell cycle-independent mechanism. In vivo studies suggest that both compensatory renal hypertrophy following uninephrectomy and diabetes mellitus-induced hypertrophy involve the cell cycle-dependent mechanism.
Acute and Chronic Effects of Hyperosmolality on mRNA and Protein Expression and the Activity of Na-K-ATPase in the IMCD
Authors: Takayama Masanobu , Nonoguchi Hiroshi , Yang Tianxin , Machida Kenji , Terada Yoshio , Owada Akira , Tomita Kimio , Marumo Fumiaki
Published: April 2003

We investigated acute and chronic effects of hyperosmolality on mRNA and protein expressions of Na-K-ATPase α and β isoforms and Na-K-ATPase activity in the rat inner medullary collecting duct (IMCD). Incubation of IMCD in hypertonic medium for 30 min reduced the Na-K-ATPase activity by 50%. The Na-K-ATPase activity of dehydrated rats measured in isotonic medium was decreased, and incubation in hypertonic medium did not further decrease the activity. Incubation of IMCD in hypertonic medium for 6 h did not change α1 mRNA. In contrast, dehydration decreased α1 subunit mRNA and protein and β1 protein expressions without changing β1 mRNA. These data show (1) that acute hyperosmolality decreases Na-K-ATPase activity in IMCD without changing α1 and β1 mRNA and (2) that 2 days of dehydration decreased Na-K-ATPase activity by reducing α1 and β1 proteins. Thus, the mechanisms for the inhibition of the Na-K-ATPase activity in IMCD is defferent between acute and chronic exposure to hyperosmolality.
Gene Regulation of Atrial Natriuretic Peptide A, B, and C Receptors in Rat Glomeruli
Authors: Itoh Kazuko , Nonoguchi Hiroshi , Shiraishi Naoki , Tomita Kimio
Published: April 2003

Background and Methods: Atrial natriuretic peptide (ANP) has three types of receptor. We investigated the gene regulation of three types of ANP receptors (ANPR-A, B, and C) in rat glomeruli using reverse transcription coupled with competitive polymerase chain reaction (PCR). Results: Competitive PCR revealed that ANPR-C mRNA expression was most abundant (ANPR-C > A >> B) in glomeruli from control rats among mRNA expressions of three receptors, which were 20- to 15,000-fold higher than those in inner medullary collecting ducts. Two days’ dehydration caused reversible decreases of ANPR-A, B, and C mRNAs by 50–80%. To determine the mechanisms of down-regulation of mRNA expression, isolated glomeruli were incubated in isotonic or hypertonic solution. Hyperosmolality induced by NaCl, mannitol or raffinose caused significant increases of ANPR-A, B, and C mRNA expression. Hypertonicity by urea showed smaller effects. ANP stimulated the expression of ANPR-A, B, and C mRNA in vitro. Conclusion: These results indicate that dehydration caused reversible decreases of ANPR-A, B, and C mRNA expression in glomeruli, and these decreases were not caused by increased plasma osmolality but probably by lower circulating levels of ANP.
Renal Fibroblast Culture
Authors: Grupp Clemens , Müller Gerhard A.
Published: April 2003

The interstitial cells in the kidney are not a homogeneous cell population but consist of different cell types like fibroblasts, dendritic cells or lymphocyte-like cells. Fibroblasts are the most abundant interstitial cell type. They are regarded as the most important cells for the production and degradation of extracellular matrix and are assumed to play a pivotal role in renal interstitial fibrosis, which correlates directly with the decrease in excretory renal function. Renal fibroblasts also have endocrine activity: cortical fibroblasts are supposed to synthesize erythropoetin, and inner medullary fibroblasts are involved in the regulation of water and electrolyte homeostasis. A powerful tool for the further elucidation of fibroblast function are studies on cultured cells. Different techniques for the isolation of fibroblasts have been reported, including the cultivation of fibroblasts from outgrowths of minced tissue and the selective removal of contaminating epithelial cells by various methods. Several aspects have to be considered while culturing fibroblasts. Fibroblasts in culture exhibit distinct morphologic and biochemical features depending on their site of origin, state of differentiation and culture conditions. Their identification in culture exclusively by morphological criteria is therefore critical especially in mixed cultures with other cell types. Unfortunately, a constitutively expressed, specific marker for all fibroblasts is still not available. Since myofibroblast formation is considered as a key event in renal interstitial fibrosis, the transformation of fibroblasts to myofibroblasts is of special interest. Studies on cultured fibroblasts provide an effective tool to examine factors that affect this transformation and regulate the production and degradation of extracellular matrix. In addition, this technique can be used for further characterization of the endocrine activity of cultured fibroblasts. A better understanding of the biology of fibroblasts is essential to develop therapeutic strategies for the treatment of renal tubulointerstitial fibrosis, the pathologic equivalent of progressive renal failure.
Renal Tubular Cells Cultured from Genetically Modified Animals
Authors: Friedlander Gérard , Runembert Isabelle , Vrtovsnik François , Terzi Fabiola
Published: April 2003

The culture of renal tubular cells from genetically modified animals opens the opportunity of biochemical, cell biology and physiological studies under strictly controlled conditions. Either primary cultures or cell lines can be used. Through two examples of primary cultures of proximal tubular cells obtained from knock-out mice, important information about the function of proteins were obtained. Mice lacking vimentin, an intermediate filament normally reexpressed in tubular cells during regeneration and culture, have a normal tubular function under basal conditions. Proximal cells grown from these animals exhibit a defect in sodium-glucose cotransport activity, most likely related to alterations in the dimer/monomer ratio of the transporter in the apical membranes. These alterations may be important in terms of tubular function during the recovery phase following acute tubular necrosis. The situation is strikingly different with regard to mice lacking HNF-1, a transactivator involved in the transcription of multiple genes. These animals suffer from severe Fanconi syndrome related to decreased expression of proximal transporters including isoforms of sodium-glucose (SGLT2) and sodium-phosphate (NPT1) cotransporters. Whereas transport defects are observed in isolated tubules, they are no longer apparent in cultured proximal cells because the expression of these isoforms is suppressed under culture conditions. These observations illustrate the interest and limits of the in vitro models for studying renal function in transgenic animals.
Role of Endonucleases in Renal Tubular Epithelial Cell Injury
Authors: Ueda Norishi , Shah Sudhir V.
Published: April 2003

The study of cell death has emerged as an important and exciting area of research in cell biology. Although two kinds of cell death, apoptosis and necrosis, are recognized, one of the major advances in our understanding of cell death has been the recognition that the pathways traditionally associated with apoptosis may be very critical in the form of cell injury associated with necrosis. Renal tubular epithelial cell injury from ischemia or toxins has generally been regarded as a result of a necrotic form of cell death. We briefly describe recent evidence indicating apoptotic mechanisms including endonuclease activation in renal tubular injury and some mediators (oxidants, caspases and ceramide) which regulate this process. The pathway that is followed by the cell is dependent on both the nature and severity of insults, and it is likely that the cascades that lead to the apoptotic or necrotic mode of cell death are activated almost simultaneously and may share some common pathways.
Effects of Changes in Dietary Intake of Sodium and Potassium and of Metabolic Acidosis on 11β-Hydroxysteroid Dehydrogenase Activities in Rat Kidney
Authors: Thompson Anna , Bailey Matthew A. , Michael Anthony E. , Unwin Robert J.
Published: April 2003

Background/Aim: Glucocorticoid activity is modulated by NADP+- and NAD+-dependent isoforms of the enzyme 11β-hydroxysteroid dehydrogenase (11βHSD) which convert glucocorticoids to their inactive metabolites. The NAD+-dependent isoform, 11βHSD2, is present in the distal nephron where it confers aldosterone specificity on mineralocorticoid receptors. The objective of this study was to establish whether renal 11βHSD activities are affected by changes in sodium and potassium balance and by metabolic acidosis. Methods: Renal 11βHSD activities were measured ex vivo from rats fed normal and high- and low-potassium diets and a low-sodium diet or given 1.5% NH4Cl to drink. Results: Rats maintained on high-potassium and low-sodium diets exhibited 59% (p < 0.01) and 28% (p < 0.05) decreases, respectively, in NAD+-dependent renal 11βHSD activity (relative to rats fed control diet) with no changes in NADP+-dependent cortisol oxidation. Short-term (3 day) and longer-term (10 day) metabolic acidosis also decreased NAD+-dependent 11βHSD activity by 50 and 52%, respectively, without affecting NADP+-dependent cortisol oxidation. The low-potassium diet had no detectable effect on renal 11βHSD activities. Conclusion: These results suggest that adaptations to a high-potassium or a low-sodium diet and to metabolic acidosis involve decreases in renal 11βHSD2 activity, enhancing the access of glucocorticoids to renal corticosteroid receptors.
Antioxidant Enzyme Gene Expression in Rats with Remnant Kidney Induced Chronic Renal Failure
Authors: van den Branden Christiane , Ceyssens Bart , De Craemer Dirk , De Bleser Pieter , Hellemans Karine , Geerts Albert , Verbeelen Dierik
Published: April 2003

Reactive oxygen intermediates play a role in chronic renal injury and glomerulosclerosis. We investigate changes in renal cortex antioxidant enzyme gene expression in the rat remnant-kidney model of chronic renal failure and compare the new data to enzyme activities published earlier. Antioxidant enzyme gene expression is evaluated by Northern blot analysis of cortex mRNA, using cDNA probes for catalase, copper/zinc-containing superoxide dismutase, and glutathione peroxidase. Catalase gene expression decreases during development of renal failure; this decrease is accompanied by decreased catalase activity during the glomerulosclerosis phase of the remnant-kidney model. Copper/zinc superoxide dismutase and glutathione peroxidase gene expression remain at a normal level during progression of the model, whereas their activities show a temporary decrease in the early remnant kidney. In the remnant-kidney model, catalase seems to be more vulnerable to reactive oxygen intermediates than superoxide dismutase and glutathione peroxidase. Our results show that antioxidant enzyme activity and gene expression do not change in the same direction at all times during disease development and that all antioxidant enzymes do not respond in the same way.
Phenotypic Modifications of Human Mesangial Cells by Extracellular Matrix: The Importance of Matrix in the Contractile Response to Reactive Oxygen Species
Authors: Iglesias-de la Cruz M. Carmen , Ruiz-Torres M. Piedad , De Lucio-Cazaña F. Javier , Rodríguez-Puyol Manuel , Rodríguez-Puyol Diego
Published: April 2003

The progression of chronic renal diseases is characterized by the accumulation of extracellular matrix proteins in the glomerulus. The present experiments were designed to analyze the effect of hydrogen peroxide on the contractile and proliferative phenotypes of human mesangial cells grown on different culture substrates: plastic, collagen type I, and collagen type IV. Contraction was analyzed by measuring planar cell surface area and myosin light chain phosphorylation, whereas proliferation was studied by [3H]thymidine incorporation. No changes were detected in the proliferation rate of human mesangial cells grown on different culture substrates, neither under basal conditions nor in the presence of fetal calf serum or H2O2. Cells grown on plastic or collagen did not contract in the presence of H2O2, but cells grown on collagen I elicited a significant contraction with H2O2. Platelet-activating factor induced contraction of human mesangial cells on the three culture substrates. The different contractile responses observed were not due to different degradation rates of H2O2. The present experiments support the importance of extracellular matrix in the response to exogenous stimuli and point to the possibility that patients with changes in the mesangial matrix as a result of chronic renal diseases may have an increased susceptibility to the pathological actions of reactive oxygen species.
Sodium Restriction Decreases AP-1 Activation after Nephron Reduction in the Rat: Role in the Progression of Renal Lesions
Authors: Terzi Fabiola , Burtin Martine , Hekmati Mehrak , Jouanneau Chantal , Beaufils Hélène , Friedlander Gérard
Published: April 2003

Renal hyperplasia and hypertrophy are early events after nephron reduction which precede progressive destruction of the remnant kidney. Restriction of dietary sodium content was shown to reduce renal lesions following nephron reduction. AP-1 is a transcription factor, resulting from heterodimerization of fos and jun proteins, which mediates the effects of mitogenic growth factors. To elucidate the role of AP-1 in growth processes involved in renal deterioration, we evaluated whether restriction of dietary sodium content (0.25 vs. 0.50% sodium w/w) affected AP-1-DNA binding and hyperplasia in the remnant kidney after nephron reduction (70% nephrectomy). Cell proliferation, evaluated by PCNA immunostaining, increased progressively from day 7 to day 60 in glomeruli, proximal and distal tubules and loops of Henle of nephrectomized (Nx) rats compared to control sham-operated (C) animals. AP-1-DNA binding activity increased 7 and 14 days after surgery, but it was reduced below C values at day 60. c-fos and c-jun expression were also reduced in Nx rats at day 60. Sodium restriction significantly reduced the number of PCNA-stained cells in glomeruli and tubules at days 14 and 60, but not at day 7, whereas it decreased AP-1 activation at all times of the study. This effect was associated to a marked reduction of renal lesions in Nx rats. In conclusion, we showed that, after nephron reduction, the beneficial effect of sodium restriction was associated with a reduction of hyperplasia and AP-1 activation, but that the latter did not parallel delayed cell proliferation rate in remaining nephrons. Thus, we propose that different transduction pathways are involved in cell proliferation after nephron reduction, according to the time of evolution of renal lesions.
Inhibiting Albumin Glycation Ameliorates Diabetic Nephropathy in the db/db Mouse
Authors: Cohen Margo P. , Masson Nicole , Hud Elizabeth , Ziyadeh Fuad , Han Dong Cheol , Clements Rex S.
Published: April 2003

Albumin modified by Amadori glucose adducts stimulates the expression of extracellular matrix proteins by glomerular mesangial and endothelial cells, and has been mechanistically linked to the pathogenesis of diabetic nephropathy. To test the hypothesis that inhibiting the formation of glycated albumin might beneficially influence the development of kidney disease in diabetes, we treated diabetic db/db mice for 12 weeks with a low-molecular-weight compound (EXO-226) that impedes the condensation of free glucose with lysine ε-amino groups in albumin. Administration of EXO-226 (3 mg/kg) twice daily by gavage normalized the plasma concentration of glycated albumin within days after initiation of treatment and maintained glycated albumin within the normal range throughout the study, despite persistent and severe hyperglycemia. Urine albumin excretion, which was markedly increased at the start of the study (age 12 weeks), was significantly reduced in treated diabetic animals compared with their untreated diabetic littermates. The fall in creatinine clearance that was observed in untreated diabetic animals was prevented in diabetic littermates that received treatment. Compared with the nondiabetic controls, the amount of glomerular mesangial matrix was threefold greater in untreated diabetic mice; in contrast, the mesangial matrix fraction was only 1.5 times that of nondiabetic controls in the treated diabetic animals, representing a reduction in mesangial matrix accumulation of more than 50%. EXO-226 also reduced the overexpression of mRNA encoding for α1 (IV) collagen in renal cortex of db/db mice. We conclude that normalization of plasma glycated albumin concentrations with the glycation inhibitor EXO-226 ameliorates the glomerular structural and functional abnormalities associated with diabetic nephropathy in the db/db mouse.
Inhibition with Antisense Oligonucleotide Suggests that IκB-α Does Not Form a Negative Autoregulatory Loop for NF-κB in Mesangial Cells
Authors: Auwardt Russell B. , Mudge Stuart J. , Chen C.-G. , Power David A.
Published: April 2003

The IκB proteins are important in the regulation of the NF-κB/Rel group of transcription factors which are pivotal in the inflammatory response. IκB-α is itself upregulated by activation of NF-κB and is postulated to be part of a negative feedback loop. This role of IκB-α has been challenged, however, by recent evidence that demonstrates (1) continued activation of NF-κB in mesangial and endothelial cells despite the resynthesis of IκB-α protein and (2) that inhibition of the transactivating activity of NF-κB by corticosteroids can be dissociated from a rise in IκB-α protein. We investigated the role of IκB-α in mesangial cells using a phosphorothioate antisense oligonucleotide directed against the translational start point of IκB-α. If IκB-α does function as a negative feedback inhibitor in these cells, then reducing IκB-α levels should lead to an increase in NF-κB activity. We first demonstrated that IκB-α protein resynthesis following stimulation could be specifically reduced. We then showed that NF-κB DNA binding was not increased with antisense treatment following stimulation. Finally, NF- κB-dependent gene signalling after stimulation (determined through an NF-κB luciferase reporter and upregulation of the mRNA of known NF-κB-responsive genes MCP-1 and IκB-α) was reduced rather than increased. These data suggest that IκB-α does not form a negative autoregulatory loop for NF-κB in mesangial cells and may actually reduce NF-κB activity. This may have relevance to therapies directed at inhibition of NF-κB activity in mesangial cell diseases.
The Genetics of SystemicLupus erythe matosus
Authors: Roberton Cheri A. , Vyse Timothy J.
Published: April 2003

Background: There is a genetic predisposition to human systemic lupus erythematosus (SLE). The genes that contribute to susceptibility are, for the most part, unknown. The introduction of new gene mapping techniques has opened the way to explore lupus genetics on a genome-wide basis. Methods: Microsatellites are simple sequence repeats widely distributed throughout eukaryotic genomes. They exhibit length variation. This polymorphism can be exploited to provide a panoply of genome-wide markers. Thereby, loci linked with lupus have been mapped in lupus-prone mouse strains and in recently published studies in multi-case human families. Results: More than 20 non-MHC (major histocompatibility complex) loci have now been linked with murine lupus. Nine non-MHC loci have been corroborated in human SLE. Some of the mouse intervals are syntenic with human loci raising the tantalizing possibility of common suspectibility genes. Although we await the results of formal gene identification, functional studies in back-cross and congenic analyses indicate that, in the mouse at least, disease genes act at multiple levels in disease development. Conclusions: A large number of genes are involved in the pathogenesis of SLE. The data also suggest that even the MHC contribution is multiple. Having mapped disease loci, geneticists now face the task of closing down on the actual aetiological alleles and demonstrating how they might operate. This undertaking will add significantly to our understanding of disease development.
Polycystin Expression in the Kidney and Other Tissues: Complexity, Consensus and Controversy
Authors: Ong Albert C.M.
Published: April 2003

PKD1, the major gene mutated in autosomal dominant polycystic kidney disease, was identified in 1994, and fully sequenced in 1995. The protein which it encodes, polycystin-1, is the first member of a new family of proteins, whose functions presently remain unclear. This review seeks to highlight the difficulties researchers studying polycystin-1 have faced and to summarize the current areas of consensus and controversy between different groups, particularly with regard to the expression pattern, subcellular location and biochemical characterization of polycystin-1. Where relevant, more recent data regarding polycystin-2, the protein encoded by PKD2, will also be discussed.
Cell-Specific Expression of the IP3 Receptor Gene Family in the Kidney
Authors: Hayashi Matsuhiko , Monkawa Toshiaki , Saruta Takao
Published: April 2003

Background/Aim: The localization of inositol trisphosphate (IP3) receptor isoforms, types 1–3, in the kidney and their role in the regulation of the intracellular calcium concentration – [Ca2+]i – are discussed. Methods: Immunohistological studies with isoform-specific antibodies were performed to reveal the localization of IP3 receptor isoforms. To examine the role of IP3 receptor type 1 in the glomeruli, the responses of [Ca2+]i to hormonal stimuli were examined in IP3 receptor type 1 knockout mice. Results: In the immunohistological study, type 1 receptor was present in arteries, afferent arterioles, and mesangial cells. Double staining with antibodies against aquaporin 2 and IP3 type 2 receptor revealed that type 2 receptor was localized mainly in the intercalated cells. The type 3 receptor showed characteristic intracellular localization in the collecting duct cells of the cortex to the outer medulla. Immunostaining of type 3 receptor was most intense in the cytoplasm on the basolateral membrane side and was not seen on the apical side. The responses of [Ca2+]i to angiotensin II and endothelin in the glomeruli were markedly attenuated in IP3 receptor type 1 knockout mice. Conclusions: The three isoforms of the IP3 receptor showed distinctive localization in the kidney, and the type 1 receptor plays a major role in the regulation of [Ca2+]i in the glomeruli. The physiological significance of the cell-specific localization, however, remains to be determined.
Retinoids and Renal Development
Authors: Burrow Christopher R.
Published: April 2003

Although it has long been appreciated that retinoids play an essential role in kidney organogenesis, it has only recently been recognized that even mild fetal vitamin A deficiency syndromes can result in a reduction in nephron number. Recent studies have also begun to define the cellular and molecular events associated with retinoid actions in the fetal kidney and have demonstrated the essential function of retinoids in branching growth of the ureteric bud. Importantly, characterization of the renal developmental effects of RAR α/β2 double homozygous mice combined with metanephric organ culture studies have together shown that one essential function of retinoid action in the developing kidney is the maintenance of c-ret expression in the tips of the ureteric bud. However, many other potential retinoid target genes including midkine, sonic hedgehog, Hox d-11, matrix metalloproteinases, and tissue inhibitors of metalloproteinases appear to play important roles in renal development and might be important downstream mediators of retinoid effects in the developing kidney. It can, therefore, be anticipated that important new insights into fetal kidney development will be forthcoming in the near future, as the essential target genes affected by retinoid signal transduction are progressively elucidated.
Renal Expression of Two ‘Thyroid-Specific’ Genes: Thyrotropin Receptor and Thyroglobulin
Authors: Sellitti Donald F. , Akamizu Takashi , Doi Sonia Q. , Kim Ginny H. , Kariyil Joseph T. , Kopchik John J. , Koshiyama Hiroyuki
Published: April 2003

Numerous renal abnormalities accompany thyroid disease, most of which have been ascribed to the effects of thyroid hormone on renal metabolism. In the present report, we investigate the renal expression of the nominally thyroid-specific proteins, thyroid-stimulating hormone (TSH) receptor (TSHR) and thyroglobulin (Tg), as potential links between renal and thyroid function. The expression of TSHR has been identified in several extrathyroidal tissues, but its presence in the kidney remains controversial. We have used reverse-transcriptase polymerase chain reaction and DNA sequencing to demonstrate the presence of TSHR transcript in human and mouse kidney, in a primary culture of human kidney, and in a green monkey kidney epithelioid cell line. Furthermore, human kidney cells responded to TSH with a 2.5- fold increase in intracellular cyclic adenosine monophosphate, suggesting the presence of functional TSHR protein. Comparison of renal expression of TSHR in a bovine growth hormone transgenic mouse model of progressive glomerulosclerosis with control mice suggested increased TSHR transcript in the renal cortex of transgenic animals. TSHR transcript was also detected in mouse mesangial cells in vitro which responded to TSH with significant increases in the formation of three-dimensional hillhocks. Polymerase chain reaction also confirmed the presence of Tg transcript in human and mouse kidneys and in mouse mesangial cells, but no effect of either TSH or cyclic adenosine monophosphate on Tg transcript levels could be discerned. Immunofluorescent staining with a monoclonal anti-Tg antibody identified positive staining in the cytoplasm of mesangial cells. These data suggest that the kidney is capable of expressing the thyroid-specific genes, TSHR and Tg, which could conceivably mediate effects of thyroid disease in the kidney.
Improved Recovery following Posttransplant Acute Renal Failure in Rat Renal Isografts with an Oral Endothelin-A Receptor Antagonist
Authors: Braun Claude , Vetter Stephan , Conzelmann Tobias , Schaub Meike , Kirchengast Michael , van der Woude Fokko J. , Rohmeiss Peter
Published: April 2003

Background: Delayed renal function after transplantation is a strong predictor of long-term graft survival. As an increased expression of endothelin (ET) has been demonstrated during ischemia/reperfusion injury, we hypothesized that ET-A receptor blockade could improve the recovery of acute renal failure in a rat model of isogeneic kidney transplantation. Methods: Kidneys of Fisher (F344, RT11v1) rat donors flushed with cooled University of Wisconsin solution were transplanted into bilaterally nephrectomized Fisher rats. Recipient animals were treated orally either with vehicle or the selective ET-A receptor antagonist LU135252 (30 mg/kg/day p.o.) for 14 days. Unilaterally nephrectomized Fisher rats not subjected to ischemia served as controls. No immunosuppression was given. On days 2, 6 and 14, metabolic studies were performed to evaluate endogenous creatinine clearance, fractional sodium excretion, and urinary endothelin excretion. Kidneys were harvested at the end of the experiment for determination of renal ET content and immunohistochemical assessment. Results: Urinary ET excretion was increased in vehicle-treated isografts compared to uninephrectomized controls after 14 days. Treatment with LU135252 resulted in a significant improvement in creatinine clearance and fractional sodium excretion to the level of uninephrectomized rats after 14 days. Isografts treated with selective ET-A receptor blockade demonstrated a marked reduction in cell surface markers for macrophages/monocytes, T cells, MHC-II, and ICAM-1. Conclusion: Treatment with the selective ET-A receptor antagonist LU135252 accelerates recovery of renal function after isogeneic renal transplantation and attenuates cellular graft infiltration. This effect could have major implications for the treatment of patients undergoing renal transplantation, as an improved initial renal function may delay the onset of chronic allograft rejection.
Isolated Juxtaglomerular Apparatus as a Tool for Exploring Glomerular Hemodynamics: Application of Microperfusion Techniques
Authors: Arima Shuji , Ito Sadayoshi
Published: April 2003

The balance of the vascular tone between afferent and efferent arterioles (AAs and EAs, respectively) is a crucial determinant of glomerular hemodynamics. Thus, it is important to study the mechanisms that control their vascular tone to understand renal physiology and pathophysiology. In order to directly study the mechanisms that regulate their vascular tone, we have developed several in vitro microperfusion preparations of these arterioles, which have the advantage of allowing us to observe the arteriolar diameter directly in the absence of systemic hemodynamic and hormonal influences. In the AA but not EA, we have directly demonstrated the presence of two intrinsic mechanisms, namely the myogenic response and macula densa-mediated tubuloglomerular feedback, that play an important role in the control of vascular tone. We also found that both mechanism-induced constrictions of AAs can be modulated by endogenous nitric oxide. In addition, several humoral factors (such as angiotensin II or prostaglandins) play an important role in the control of AA tone. On the other hand, angiotensin II is one major factor that controls the vascular tone of the EA. We have found that the vasoconstrictor effect of angiotensin II on EAs is modulated by prostaglandins produced by the upstream glomerulus. Thus, this may be a mechanism whereby the glomerulus controls its own capillary pressure by releasing prostaglandins and thereby adjusting the resistance of the downstream EA. These varying mechanisms regulating AA and EA tone play an important role in the precise control of glomerular hemodynamics.
Disorders of the Epithelial Na+ Channel in Liddle’s Syndrome and Autosomal Recessive Pseudohypoaldosteronism Type 1
Authors: Oh Young S. , Warnock David G.
Published: April 2003

The epithelial Na+ channel (ENaC) is the key step in many Na+-absorptive epithelia, such as kidney and distal colon, that controls the overall rate of transepithelial Na+ transport. ENaC is composed of three homologous subunits, α, β, and γ. The α subunit is the key subunit for the formation of a functional ion channel, while the β and γ subunits can greatly potentiate the level of expressed Na+ currents. ENaCs belong to the recently identified DEG/ENaC supergene family, sharing the same basic structure with cytoplasmic amino and carboxy termini, two transmembrane regions, and a large extracellular loop. The human ENaC genes have been cloned, and using genetic linkage analysis the involvement of ENaC gene mutations in two distinct human diseases, Liddle’s syndrome and autosomal recessive pseudohypoaldosteronism type 1 (PHA-1), has been demonstrated. In Liddle’s syndrome, gain-of-function mutations in the β or γ ENaC subunits have been found; all identified mutations so far reside in the carboxy terminus of the protein, either deleting or modifying the functionally important PY motif. In PHA-1, loss-of-function mutations in the α, β, or γ subunits have been found; these mutations either truncate a significant portion of the structure or modify an amino acid that plays an important role in channel function. In this review, our current understanding about ENaC and the pathophysiology of Liddle’s syndrome and PHA-1 caused by ENaC mutations will be discussed.
Defects in Processing and Trafficking of Cystic Fibrosis Transmembrane Conductance Regulator
Authors: Kunzelmann Karl , Nitschke Roland
Published: April 2003

In most epithelial tissues Cl– transport relies on the cystic fibrosis transmembrane conductance regulator (CFTR) which has dual function as a Cl– channel and as a regulator of other ion channels. More than 900 different mutations in the CFTR gene are the cause for defective transport of Cl– and Na+ and impaired secretion or absorption of electrolytes in cystic fibrosis. However, the CFTR mutation ΔF508 is the most common reason for the frequently inherited disease among the Caucasian population. Maturation and processing of ΔF508-CFTR is defective which leads to expression of only very little but functional CFTR in the cell membrane. Understanding the processing and trafficking of CFTR may give a clue to the question as to how the expression and residual function of ΔF508-CFTR can be enhanced, and may lead to the development of new pharmacological tools for the treatment of cystic fibrosis.
Mechanisms of Renal Calcium Transport
Authors: Friedman Peter A.
Published: April 2003

The kidneys play a key role in the integrated regulation of calcium homeostasis. Calcium absorption takes place throughout the nephron. Proximal tubules, thick ascending limbs of Henle’s loop, and distal tubules are the major sites of calcium absorption. The mechanisms of absorption vary significantly from one segment to another, as does the extent of hormonal regulation. At one extreme is the considerable reabsorption by proximal tubules that proceeds primarily, if not entirely, by a paracellular pathway that is not regulated by hormones or drugs. In thick ascending limbs, calcium absorption occurs through a combination of transcellular and paracellular routes. The active, transcellular component is regulated by parathyroid hormone (PTH) and calcitonin, whereas the passive, paracellular route is governed by the extent of concomitant sodium absorption. At the other extreme is the distal tubule, where calcium absorption is entirely transcellular and is regulated by PTH, 1,25(OH)2vitamin D3, calcitonin, and by calcium-sparing drugs such as thiazide-type diuretics. The present review focuses on recent insights into the mechanisms of transcellular calcium movement and highlights the discovery of an epithelial calcium channel, ECaC, that may mediate calcium entry in distal tubules.
Severely Impaired Urine-Concentrating Ability in Mice Lacking the CLC-K1 Chloride Channel
Authors: Uchida Shinichi , Marumo Fumiaki
Published: April 2003

To analyze the physiological functions of CLC-K1 in vivo, we generated mice lacking CLC-K1 by targeted gene disruption. Homozygous mutant Clcnk1–/– mice produced ∼5 times more urine than Clcnk1+/– and Clcnk1+/+ mice. After 24-hour water deprivation, Clcnk1–/– mice became severely dehydrated and lethargic. Intraperitoneal injection of the V2 agonist, deamino-Cys1, D-Arg8 vasopressin, induced an increase in urine osmolarity in Clcnk1+/– and Clcnk1+/+ mice from ∼1,000 to ∼3,000 mosm/kg H2O, whereas the increase in Clcnk1–/– mice was only from ∼600 to ∼840 mosm/kg H2O, indicating nephrogenic diabetes insipidus in Clcnk1–/– mice. These results clearly established that CLC-K1 plays a major role in the urinary-concentrating mechanisms.
Molecular Characteristics of Phosphate Transporters and Their Regulation
Authors: Hernando Nati , Forster Ian C. , Biber Jürg , Murer Heini
Published: April 2003

A key process in overall Pi-homeostasis is renal proximal tubular reabsorption of inorganic phosphate (Pi), which involves secondary active sodium/phosphate (Na+/Pi) cotransport reabsorption at the brush border membrane. Among the two different molecularly identified Na+/Pi cotransporters, the type-IIa Na+/Pi cotransporter (NaPi-IIa) accounts for up to 70% of brush border membrane transport. Regulation of renal Pi reabsorption centers around brush border membrane insertion and retrieval of transporter protein under the influence of hormonal and nonhormonal factors. Immunohistochemical and fluorescence techniques have provided new insights into the tissue distribution and the regulation processes. The intrinsic electrogenicity of NaPi-IIa, has allowed detailed studies of the transport kinetics of NaPi-IIa and, combined with mutagenesis methods, structure-function information at the protein level is emerging.
Genetic Models of Hypertension in Experimental Animals
Authors: Yagil Yoram , Yagil Chana
Published: April 2003

Genetic animal models are central to ongoing efforts to elucidate the pathophysiology and genetic basis of hypertension. The rat is the leading species in experimental hypertension. Several rat models of hypertension are available for research, including inbred strains, congenic lines, transgenic animals and recombinant inbred strains. Each of these models has been designed to express different phenotypes, including spontaneous hypertension, salt sensitivity, stress sensitivity and susceptibility to end-organ damage. All these models have been extremely useful in the search for the physiological mechanisms that underlie hypertension, but some of them have been specifically designed for detecting the hypertension genes. This latter task is extremely complex in spontaneous hypertension, but genetic animal models may simplify the task by enabling to focus on specific phenotypes. Despite intensive efforts over nearly 3 decades, the genetic basis of hypertension has not been unveiled so far in the rat or in other species. Recent dense mapping of the rat genome, the development of new strategies and technologies in molecular genetics including differential gene expression, expressed sequence tags and DNA biochips render hope that the formidable task of identification of new candidate genes in hypertension will move another major step forward. Once these genes are identified, their function and role in hypertension will have to be determined, utilizing functional genomic strategies and bioinformatics. Finally, the findings in genetic animal models of hypertension will have to be extrapolated to humans by homology and syntenic mapping strategies.
Nephrotoxicity of Angiotensin Inhibition during the Perinatal Period
Authors: Miyazaki Yoichi , Ichikawa Iekuni
Published: April 2003

As the only ex utero mechanism for the removal of nitrogenous waste, the mammalian kidney achieves some 50-fold increase in urine production during the perinatal period when the placental circulation becomes no longer available as a functional dialyzer. This urine is efficiently removed from the kidney by the renal pelvis, a smooth muscle structure unique to mammals, which develops during the perinatal period. We found that mutant mice completely devoid of angiotensinogen or its type 1 receptor, as well as wild-type neonates given an ACE inhibitor, fail to develop a renal pelvis or a ureteral peristaltic movement. These structural and functional defects in the urinary tract are followed by severe obstructive injury of the renal parenchyma. The ability of angiotensin to directly induce the pelvis is demonstrated in an organ culture system, in which treatment with angiotensin induces the characteristic smooth muscle layer in the wild type, but not in homozygous null mutants. Upregulation of both renal angiotensin content and type 1 receptor at the renal hilum are also demonstrated in the wild type during the transition from intra- to extra-uterine life. By inducing the timely development of the renal pelvis, angiotensin thus facilitates the removal from the renal parenchyma of the urine that promptly increases at birth, thereby effectively preventing a buildup of intrarenal pressure and a consequent development of dysmorphic kidney.
Microchimerism and Tolerance following Intrauterine Transplantation and Transfusion for α-Thalassemia-1
Authors: Hayward A. , Ambruso D. , Battaglia F. , Donlon T. , Eddelman K. , Giller R. , Hobbins J. , Hsia Y.E. , Quinones R. , Shpall E. , Trachtenberg E. , Giardina P.
Published: April 2003

A fetus homozygous for α-thalassemia-1 was given haploidentical paternal CD34 cells at 13, 19 and 24 weeks’ gestation and supported through pregnancy by blood transfusion. The fetal hematocrit ranged between 27 and 47% and between one half and three quarters of this hemoglobin was of recipient (Bart’s) type. Intrauterine growth proceeded normally and no significant fetal hydrops was detected. Tests for donor HLA antigens, and α-globin DNA, were negative on fetal blood samples drawn before birth. A positive signal for α-globin DNA was obtained from cord blood and from marrow obtained at 3 months of age, suggesting that some donor stem cells had persisted in the recipient. The infant’s blood mononuclear cells showed little proliferative and no cytotoxic response to the donor while responses to a third party were present. Additional paternal CD34 cells given at 3 months age did not reduce transfusion dependency in the subsequent 6 months. Our results show that repeated transfusions can support an α-thalassemia-1 fetus through pregnancy, in this instance without significant birth defects or apparent hypoxic tissue injury. The donor stem cells did not have a survival advantage compared with endogenous stem cells, but appeared to survive in the recipient as judged by the persistence of an α-globin DNA signal. In vitro studies of alloreactivity suggest tolerization of the host to the donor’s MHC disparity. Future efforts will focus on exploiting this tolerance to improve the level of donor chimerism.