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PubMed2001

Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin.

Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of podocalyxin, we generated podocalyxin-deficient (podxl(-/)-) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl(-/)- mice, they fail to form foot processes and slit diaphragms and instead exhibit cell--cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl(-/)- mice frequently display herniation of the gut (omphalocele), suggesting that podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans.

Animals↗
PubMed2002

MICROANATOMY, ULTRASTRUCTURE, AND SYSTEMATIC SIGNIFICANCE OF THE EXCRETORY SYSTEM AND MANTLE CAVITY OF AN ACOCHLIDIAN GASTROPOD (OPISTHOBRANCHIA).

The microanatomy and ultrastructure of the excretory system of an undescribed mesopsammic gastropod of the genus Hedylopsis have been examined by means of semithin serial sections, reconstructions, and transmission electron microscopy. The functional metanephridial system comprises a monotocardian heart with a single ventricle and auricle in a spacious pericardium as well as a single, large kidney. Podocytes in the auricular epicardium represent the site of ultrafiltration and formation of the primary urine, whereas the flat epithelium of the kidney with extensive basal infoldings, large vacuoles and the apical microvillous border indicates modification of the primary filtrate. Solitary rhogocytes (pore cells) represent additional loci of ultrafiltration with an identical fine-structure as those of the podocytes (meandering slits with diaphragms covered by extracellular matrix). The presence of podocytes situated in the epicardial wall of the auricle is regarded as plesiomorphic for the Opisthobranchia and is confirmed for the Acochlidia for the first time. Kidney and rectum both open into a small, yet distinct mantle cavity. Within the Acochlidia this condition represents a plesiomorphic character only known from one further Hedylopsis species until now. Special cells (here termed microvillous pit-cells) with a presumed absorptive function are interspersed between the epithelial cells of the mantle cavity. They are mainly characterized by a prominent invagination of the apical border with densely arranged, very large microvilli. The presence of a mantle cavity that has been lost in all other acochlidian genera supports the systematic placement of the Hedylopsidae at the base of the Achochlidia.

Journal Article↗
PubMed2006

Angiotensin converting enzyme inhibition prevents development of collapsing focal segmental glomerulosclerosis in Thy-1.1 transgenic mice.

BACKGROUND: Thy-1.1 transgenic mice develop hypercellular focal and segmental glomerulosclerosis (FSGS) lesions that mimic human collapsing FSGS, in 7 days after injection with anti-Thy-1.1 antibodies. These lesions consist of proliferating parietal epithelial cells (PECs). We questioned whether the angiotensin converting enzyme inhibitor (ACE), captopril, could prevent the development of FSGS and if protection is related to the timing of drug administration. METHODS: First, we compared the effect of captopril treatment with angiotensin II-(ANGII) independent antihypertensive therapy (triple therapy). Second, we tested the effects of captopril administered over four different time intervals: days -7 to 0 (Ca-7>0), days -7 to 7 (Ca-7>7), days 0-7 (Ca0>7) and days 3-7 (Ca3>7) (day 0 being the day of injection of the antibody). RESULTS: In anti-Thy-1.1 injected control (C) mice we observed dedifferentiation and activation of podocytes, reflected by loss of ASD33 and increased expression of desmin, followed by a marked accumulation of PECs forming hypercellular lesions. PECs showed an increased expression of connective tissue growth factor (CTGF). Triple therapy or captorpil pre-treatment (Ca-7>0) had no significant effect on albuminuria or FSGS. In contrast, Ca0>7 and Ca3>7 treatment significantly lowered albuminuria and attenuated development of FSGS. The latter two treatments attenuated loss of ASD33 expression by podocytes but could not prevent increased desmin expression. In addition, these treatments reduced CTGF expression by PECs and prevented PEC proliferation. CONCLUSIONS: ACE inhibition, but not triple therapy, prevents the development of FSGS, suggesting an important role for ANGII. ACE inhibition has a protective effect even when started 3 days after the initial podocyte insult, which is probably related to the ability of ACE-inhibition to block PEC activation and proliferation.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2002

A mutant form of the Wilms' tumor suppressor gene WT1 observed in Denys-Drash syndrome interferes with glomerular capillary development.

The Wilms' tumor suppressor gene WT1 encodes a zinc finger protein that is required for urogenital development. In the kidney, WT1 is most highly expressed in glomerular epithelial cells or podocytes, which are an essential component of the filtering system. Human subjects heterozygous for point mutations in the WT1 gene develop renal failure because of the formation of scar tissue within glomeruli. The relationship between WT1 expression in podocytes during development and glomerular scarring is not well understood. In this study, transgenic mice that expressed a mutant form of WT1 in podocytes were derived. The capillaries within transgenic glomeruli were dilated, indicating that WT1 might regulate the expression of growth factors that affect capillary development. Platelet endothelial cell adhesion molecule-1 expression was greatly reduced on glomerular endothelial cells of transgenic kidneys. These results suggest that WT1 controls the expression of growth factors that regulate glomerular capillary development and that abnormal capillary development might lead to glomerular disease.

Animals↗
PubMed2003

PDGF-C expression in the developing and normal adult human kidney and in glomerular diseases.

PDGF-C is a new member of the PDGF-family and has recently been identified as a rat mesangial cell mitogen. Its expression and function in human kidneys is unknown. Localization of PDGF-C protein was analyzed by immunohistochemistry using a rabbit polyclonal antibody directed against the core-domain of PDGF-C in human fetal kidneys (n = 8), normal adult human kidneys (n = 9), and in renal biopsies of patients with IgA nephropathy (IgAN, n = 31), membranous nephropathy (MGN, n = 8), minimal change disease (MC, n = 7), and transplant glomerulopathy (TxG, n = 12). Additionally, PDGF-C mRNA was detected in microdissected glomeruli by real-time RT-PCR in cases of normal adult kidneys (n = 7), IgAN (n = 27), MGN (n = 11), and MC (n = 13). In the fetal kidney, PDGF-C localized to the developing mesangium, ureteric bud epithelium, and the undifferentiated mesenchyme. In the adult kidney, PDGF-C was constitutively expressed in parietal epithelial cells of Bowman's capsule, tubular epithelial cells (loops of Henle, distal tubules, collecting ducts), and in arterial endothelial cells. A marked upregulation of glomerular PDGF-C protein was seen in MGN and TxG with a prominent positivity of virtually all podocytes. In MC, PDGF-C localized to podocytes in a more focal distribution. In MGN, increased glomerular PDGF-C protein expression was due to increased mRNA synthesis as a 4.3-fold increase in PDGF-C mRNA was detected in microdissected glomeruli from MGN compared with normal. PDGF-C protein was additionally expressed in individual mesangial cells in TxG. Finally, upregulated PDGF-C protein expression was detected within sclerosing glomerular and fibrosing tubulointerstitial lesions in individual cases from all analyzed groups. We conclude that PDGF-C is constitutively expressed in the human kidney and is upregulated in podocytes and interstitial cells after injury/activation of these cells.

Adult↗
PubMed2003

Reversal of glomerulosclerosis after high-dose enalapril treatment in subtotally nephrectomized rats.

Interventions to block the renin-angiotensin system (RAS) halt the progression of renal lesions in renal damage models. It has recently also been reported that established glomerulosclerosis can be reversed by pharmacologic blockade of the RAS. It was the aim of this study to confirm that high doses of angiotensin-converting enzyme (ACE) inhibitors reverse established glomerulosclerosis and to extend the findings by providing quantitative information on glomerular geometry, podocytes and other glomerular cells, renal vessels and tubulointerstitial tissue. Male Sprague Dawley rats were subjected to subtotal surgical renal ablation (SNX) (n = 27) or sham operation (n = 31) and fed using a pair-feeding protocol. Eight weeks after surgery, rats were either sacrificed or allocated to two arms: enalapril treatment (48 mg/kg body wt per day administered in the drinking fluid for 4 wk) or no treatment. Renal morphology was evaluated after 8 or 12 wk, respectively, by stereology in tissue fixed by pressure-controlled perfusion. Both systolic BP and albumin excretion rate were significantly higher in SNX compared with sham-operated controls. They were significantly reduced in SNX after delayed enalapril treatment. The glomerulosclerosis (GSI), tubulointerstitial (TII), and vascular (VI) damage indices were significantly higher in all SNX groups than in sham-operated controls. At the end of the experiment (12 wk after SNX) GSI, TII, and VI were significantly lower in SNX with delayed enalapril treatment (0.77 +/- 0.18, 0.63 +/- 0.19 and 0.43 +/- 0.16, respectively) compared with untreated SNX (1.64 +/- 0.14, 1.16 +/- 0.34 and 0.67 +/- 0.29, respectively). GSI, TII, and VI were also significantly lower in SNX with delayed enalapril treatment compared with SNX sacrificed without treatment 8 wk after SNX. The same was true for glomerular volume. The number of podocytes was not affected by SNX, but podocyte volume was increased. Both indices remained unaffected by treatment. The numbers of cells within the mesangium and endothelial cells per glomerulus were significantly lower in SNX after delayed enalapril treatment compared with untreated SNX. These results strongly suggest regression of preexisting lesions, i.e., glomerular, tubular, and vascular remodeling as well as reversal of glomerular hypertrophy by ACE inhibitor treatment. The study confirms that high-dose ACE inhibitor treatment causes partial reversal of glomerular as well as interstitial lesions in subtotally nephrectomized rats.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

p38 Mitogen-activated protein kinase activation and cell localization in human glomerulonephritis: correlation with renal injury.

Activation of the p38 mitogen-activated protein kinase (MAPK) signal transduction pathway plays an important role in the inflammatory response. It was postulated that p38 MAPK is important in the pathogenesis of human glomerulonephritis and contributes to the development of renal injury. p38 MAPK activation was examined by immunodetection for dual phosphorylated p38 (p-p38) in normal human kidney and 77 renal biopsy specimens encompassing a wide spectrum of glomerulonephritides. In normal kidney, p-p38 immunostaining was restricted to the nuclei of a small number of podocytes, parietal epithelial cells, and tubular cells. There was a dramatic increase in the number of p-p38-positive cells in glomeruli and tubules in nonproliferative and proliferative glomerulonephritis and a substantial increase in the number of interstitial p-p38-positive cells in proliferative glomerulonephritis. Double immunostaining identified p38 activation in intrinsic renal cells (podocytes and endothelial and tubular cells), infiltrating macrophage and neutrophils, and myofibroblasts. Renal failure correlated with the number of p-p38-positive glomerular, tubular, and interstitial cells. Proteinuria correlated with the number of p-p38-positive tubular and interstitial cells and the number of p-p38-positive podocytes in nonproliferative glomerulonephritis. Furthermore, glomerular p38 activation correlated with segmental proliferative and necrotic lesions, and interstitial p38 activation correlated with the degree of interstitial inflammation. In conclusion, activation of p38 MAPK in intrinsic renal cells and infiltrating leukocytes correlated with renal dysfunction and histopathology, suggesting an important pathogenic role for p38 MAPK activation in human glomerulonephritis.

Enzyme Activation↗
PubMed2004

Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties.

Normal glomerular filtration depends on the combined properties of the three layers of glomerular capillary wall: glomerular endothelial cells (GEnC), basement membrane, and podocytes. Podocytes produce endothelial factors, including angiopoietin 1 (ang1), and vascular endothelial growth factor (VEGF), whereas GEnC express their respective receptors Tie2 and VEGFR2 in vivo. As ang1 acts to maintain the endothelium in other vascular beds, regulating some actions of VEGF, these observations suggest a mechanism whereby podocytes may direct the unique properties of the glomerular endothelium. This interaction was investigated by studies on the barrier properties of human GEnC in vitro. GEnC were examined for expression of endothelium-specific markers by immunofluorescence and Western blotting and for typical responses to TNF-alpha by a cell-based immunoassay. Expression of angiopoietin and VEGF receptors was examined similarly. Barrier properties of GEnC monolayers cultured on porous supports were investigated by measurement of transendothelial electrical resistance (TEER) and passage of labeled albumin. Responses to a cAMP analogue and thrombin were examined before those to ang1 and VEGF. Results confirmed the endothelial origin of GEnC and their expression of Tie2 and VEGFR2. GEnC formed monolayers with a mean TEER of 30 to 40 Omega/cm(2). The cAMP analogue and thrombin increased and decreased TEER by 34.4 and 14.8 Omega/cm(2), respectively, with corresponding effects on protein passage. Ang1 increased TEER by 11.4 Omega/cm(2) and reduced protein passage by 45.2%, whereas VEGF reduced TEER by 12.5 Omega/cm(2) but had no effect on protein passage. Both ang1 and VEGF modulate GEnC barrier properties, consistent with potential in vivo roles; ang1 stabilizing the endothelium and resisting angiogenesis while VEGF induces the high permeability to water, characteristic of the glomerular endothelium.

Angiopoietin-1↗
PubMed2004

Sidekick-1 is upregulated in glomeruli in HIV-associated nephropathy.

Infection of podocytes by HIV-1 induces unique changes in phenotype, which contribute to the pathogenesis of glomerular disease in HIV-associated nephropathy (HIVAN). The host genetic pathways altered by HIV-1 infection that are responsible for these phenotypic changes are largely unknown. For identifying such pathways, representational difference analysis was performed comparing cDNA from HIV-1 transgenic podocytes with nontransgenic controls. In this way, a gene named sidekick-1 (sdk-1) was cloned, a transmembrane protein of the Ig superfamily that is highly upregulated in HIV-1 transgenic podocytes. Sdk-1 and its ortholog, sidekick-2 (sdk-2), were recently shown to guide axonal terminals to specific synapses in developing neurons. Their presence and role in other organs, including the kidney, has not been described. The current study demonstrates developmental expression of both sdk-1 and sdk-2 and a tight spatial and temporal regulation of these genes in kidney. During nephrogenesis, sidekick expression was observed first in ureteric bud and ureteric bud-derived tissues in a pattern similar to other genes known to play important roles in branching morphogenesis. In adult murine renal tissue, sidekick proteins were seen in glomeruli at low levels, and expression of sdk-1 was greatly upregulated in diseased HIV-1 transgenic mouse kidneys. In a human HIVAN kidney biopsy, sidekick expression was increased in glomeruli in a pattern consistent with the mouse model. It is proposed that the dysregulation of sdk-1 protein may play an important role in HIVAN pathogenesis.

AIDS-Associated Nephropathy↗
PubMed2006

New insights into the pathogenesis of membranous glomerulonephritis.

PURPOSE OF REVIEW: Membranous nephropathy is one of the most common glomerulopathies. Current treatments are entirely empirical, and concept-driven therapies are dramatically lacking. This review focuses on new pathophysiologic aspects of the disease, with special emphasis on the antigenic targets of pathogenic antibodies. RECENT FINDINGS: Neutral endopeptidase - a podocyte antigen that can digest biologically active peptides - was recently identified as the target antigen of antibodies deposited in the subepithelial space of glomeruli in a subset of patients with antenatal membranous nephropathy. The mothers became immunized because they are deficient in neutral endopeptidase due to truncating mutations in the gene. Membranous nephropathy could be transferred to the rabbit by injection of mothers' immunoglobulin. Development of the renal disease was associated with anti-neutral endopeptidase IgG1. SUMMARY: Membranous nephropathy most likely is a heterogeneous disease, although a common denominator may be that podocytes provide antigenic targets for in-situ formation of glomerular immune deposits. Identification of neutral endopeptidase and additional (podocyte) antigens and characterization of their epitopes should make it possible to design more effective and better tolerated therapies. Fetomaternal alloimmunization is a novel mechanism of renal disease that may apply to other organs as well.

Complement Activation↗
PubMed2005

Characterization of the interactions of the nephrin intracellular domain.

Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.

Amino Acid Sequence↗
PubMed1985

Daunomycin-induced nephropathy in rats.

A single intravenous injection of daunomycin into rats induced severe glomerular injury with massive proteinuria. Mesangial thickening due to an increase in the matrix appeared as early as 5 weeks after injection. Focal and segmental glomerular tuft distortion developed by 10 weeks associated with a progressive mesangial change, which was accompanied by detachments of endothelial cells and podocytes from the glomerular basement membrane (GBM) resulting in obliteration of the affected tufts. After 20 weeks, the lesion ultimately progressed to cause diffuse and global glomerular obliteration. Scattered glomeruli also showed frank shrinkage with a mild obliterative change. By observing a number of isolated glomeruli in scanning electron microscopy, it was revealed that podocyte alterations were variable from case to case and foot processes remained discrete in some cases until 10 weeks, despite the presence of marked proteinuria. Anionic sites distributed throughout the GBM and on the surface of podocytes were usually preserved in proteinuric rats as far as evaluated by ruthenium red and colloidal iron stainings. Our results indicate that loss of foot processes and of glomerular anionic sites are not causative factors but consequences of proteinuria.

Animals↗
PubMed2004

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

BACKGROUND: The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. METHODS: Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. RESULTS: As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. CONCLUSION: The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Animals↗
PubMed2005

Glomerular endothelial cell differentiation.

BACKGROUND: Glomerular endothelial cells differ from most other endothelial cells in that they are extraordinarily flattened and highly fenestrated. In this differentiated form, they allow formation of glomerular ultrafiltrate at a prodigious rate. METHODS: Molecular processes that dictate the development and differentiation of glomerular endothelium are reviewed. RESULTS: During glomerular development, angioblasts already present in the metanephric blastema well before any organized angiogenic sprouts invade the capillary cleft of developing nephrons at the comma and S-shape stages in response to chemotactic and guiding cues from primitive podocytes. The angioblasts then undergo homotypic aggregation into precapillary cords as yet devoid of a lumen. Lumen development then proceeds through the loss of superfluous endothelial cells by apoptosis as well as flattening of the remaining viable endothelial cells. The final step, fenestration, is critically dependent on appropriate stimuli, most notably vascular endothelial growth factor A (VEGF-A), from differentiated podocytes. Current evidence suggests that the fenestrae of fully differentiated glomerular endothelium can be lost within hours if the VEGF-A stimulus is removed, and that the glomerular endotheliosis, loss of glomerular filtration rate (GFR) and proteinuria observed in preeclampsia are due to the circulating inhibitor of VEGF-A, soluble VEGF receptor 1 (VEGFR-1). CONCLUSION: Differentiation of the glomerular endothelium is highly dependent on podocyte-derived stimuli and their loss leads to the derangements of glomerular function in preeclampsia.

Animals↗
PubMed2005

ACE inhibitors improve nephrin expression in Zucker rats with glomerulosclerosis.

BACKGROUND: Podocyte injury is associated with many forms of human and experimental proteinuric glomerular disease. The aim of this study was to investigate the level of nephrin expression in a model of obesity and type II diabetes mellitus, the obese Zucker rat, as well as to investigate whether nephrin expression is influenced by treatment with quinapril or diltiazem, 2 drugs frequently used in type II diabetes mellitus. METHODS: Obese Zucker rats were treated with either quinapril or diltiazem at a dose of 10 mg/kg body weight per day and 100 mg/kg body weight per day, respectively, for 6 months. Real time reverse transcription-polymerase chain reaction (RT-PCR) and immunoperoxidase assays were used to assess and quantify nephrin gene expression and other markers of podocyte damage, such as desmin and synaptopodin protein. RESULTS: Quinapril treatment prevented the reduction of nephrin levels compared with the control group, while diltiazem treatment did not prevent the reduction. Similar results were obtained when other phenotypic markers, such as desmin, were assessed. Similarly, synaptodin showed this tendency, although it did not achieve statistically significant differences. CONCLUSION: The podocyte phenotypic changes assessed in a model of obesity and type II diabetes mellitus were corrected by an angiotensin-converting enzyme (ACE) inhibitor. These results could be associated with an improvement in the slit diaphragm, and therefore, in the maintenance of the filtration barrier. Diltiazem did not achieve similar results.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2006

MafB is essential for renal development and F4/80 expression in macrophages.

MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly, these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells, GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly, F4/80 expression in macrophages was suppressed in the homozygous mutant, although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells, MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages, whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation, renal tubule survival, and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages.

Animals↗
PubMed2005

Localization of the GLUT8 glucose transporter in murine kidney and regulation in vivo in nondiabetic and diabetic conditions.

Kidney disease is a major complication of diabetes, and poor glycemic control is associated with the development of diabetic nephropathy. The precise mechanisms that lead to diabetic kidney disease still remain largely unknown and are under current investigation. Because glucose transporters in the kidney play an important role in the local maintenance of intracellular glucose and plasma glucose homeostasis, the tissue distribution and regulation of glucose transporter GLUT8, a new member of the glucose transporter family with important functions in cellular survival, were examined. To understand the normal regulation of GLUT8 expression in response to metabolic signals, fasting and feeding conditions were studied. Additionally, GLUT8 expression was studied using two different models of insulin resistance, GLUT4-/- and db/db mice. GLUT8 was localized to glomerular podocytes and tubular epithelial cells in the distal portion of the nephron. Expression of GLUT8 in the kidney was influenced by plasma glucose levels in vivo. Podocytes in kidneys of diabetic db/db mice express higher levels of GLUT8 compared with nondiabetic db/m mice. Because podocytes play an important role in glomerulosclerosis development and high levels of glucose have been shown to induce apoptotic cell death in various kidney cells, these data may provide further insight into the pathogenesis of glomerulosclerosis and diabetic nephropathy.

Animals↗
PubMed2005

Mechanical force-activated phospholipase D is mediated by Galpha12/13-Rho and calmodulin-dependent kinase in renal epithelial cells.

The renal glomerulus, the site of plasma ultrafiltration, is exposed to mechanical force in vivo arising from capillary blood pressure and fluid flow. Studies of cultured podocytes demonstrate that they respond to stretch by altering the structure of the actin cytoskeleton, but the mechanisms by which physical force triggers this architectural change and the signaling pathways that lead to generation of second messengers are not defined. In the present study, we found that in renal epithelial cells [podocytes and Madin-Darby canine kidney (MDCK) cells], application of mechanical force to the cell surface through fibronectin-coated ferric beads and exposure of the cells to magnetic force lead to Rho translocation and actin cytoskeleton reorganization. This application of force recruited Rho and filamentous actin (F-actin) to bead loci and subsequently stimulated phospholipase D (PLD), a downstream effector of Rho. Using MDCK cells that stably express regulators of G protein-signaling (RGS) proteins [RGS4 attenuates Galpha(i) and Galpha(q), and the p115RhoGEF-RGS domain (p115-RGS) attenuates Galpha(12/13)] to define the signaling pathway, we found that mechanical force induced Galpha(12/13)-Rho activation and increased F-actin to stimulate PLD activity. The activation can be partially prevented by the C(3) exoenzyme. Pretreatment of the cells with chemical inhibitors of several kinases showed that calmodulin-dependent kinase is also involved in stretch-induced PLD activation by a separate pathway. Taken together, our data demonstrate that in cultured podocytes and MDCK cells, mechanical force leads to actin cytoskeleton reorganization and PLD activation. The signaling pathways for PLD activation involve Galpha(12/13)/Rho/F-actin and calmodulin-dependent kinase.

Actins↗