Experimental nephrology最新文献

Chronic renal failure is associated with increased cardiovascular morbidity and reduced arterial elasticity. Only little information is available on the functional effects of uraemia on resistance arteries. Therefore, we studied the influence of renal failure on rat small mesenteric vessels. The responses of arterial rings were investigated in a Mulvany myograph 6 weeks after 5/6 nephrectomy or sham operation. The subtotal nephrectomy resulted in a 1.9-fold elevation of plasma urea nitrogen but was without significant effect on blood pressure. Endothelium-dependent relaxations, largely mediated via arterial K(+) channels, were preserved in the resistance vessels of uraemic rats. Endothelium-independent vasorelaxations, mediated via exogenous nitric oxide and the opening of ATP-sensitive K(+) channels, were also unchanged. However, the responses induced by isoprenaline were slightly reduced, indicating impaired relaxation via beta-adrenoceptors in experimental renal failure.

To study an involvement of glomerular endothelial cells in the development of anti-Thy-1 nephritis, we examined the expression of endothelial cell adhesion molecules during the course of this model. Ribonuclease protection assay elucidated that expression of mRNA for intercellular adhesion molecule-1 (ICAM-1) was markedly enhanced in the glomeruli with a peak at 2 h (6.5-fold, p < 0.05) after the anti-Thy-1 antibody injection when mesangial cell lysis was recognized and IL-1beta mRNA expression was induced in the glomeruli. The glomerular ICAM-1 was predominantly localized in the endothelial cells and was intensely immunostained at day 1 in the glomerular endothelial cells. In contrast, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin mRNA expression increased gradually with a peak at day 6 (2.6-fold (p < 0.05) and 4.2-fold (p < 0.05), respectively) in the glomeruli with mesangial proliferative lesion. PECAM-1 was also immunolocalized in the glomerular endothelial cells and the immunoreactivity was greatly enhanced at day 6. Glomerular expression of vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 (E-selectin) was unchanged at a low level during the course of anti-Thy-1 nephritis. Blocking of ICAM-1 by administration of anti-ICAM-1 antibody showed significant decrease in the number of polymorphonuclear leukocytes accumulating in the glomeruli by 45.7% (9.4 +/- 0.2 vs. 5.1 +/- 0.1 per glomerular cross section, p < 0.01) at 2 h. These results suggest a significant involvement of glomerular endothelial cells in the development and repair of anti-Thy-1 nephritis via direct or indirect intercellular interactions between mesangial cells and glomerular endothelial cells.

Renal proximal tubule cells are particularly vulnerable to injury following ischemia and reperfusion due to their marginal blood supply and high metabolic demand. Renal adenosine receptor (AR) modulations preserve renal function following ischemic-reperfusion injury in vivo. Numerous intracellular proteins have been shown to be pivotal in the signal transduction of adenosine-mediated protection in vivo. However, characterization of the expression and function of ARs and intracellular proteins mediating protection in human proximal tubular cells is lacking. Therefore, we studied the ARs in an immortalized human renal proximal tubular cell (HK-2) line to determine if this cell line could function as an in vitro model of AR coupling. Immunoblotting with AR subtype specific antibodies detected all 4 subtypes of ARs (A(1), A(2a), A(2b) and A(3)), several isoforms of protein kinase C (alpha, delta, and epsilon and several heterotrimeric G-protein isoforms (G(i)alpha, G(s)alpha and G(q)alpha). The A(1) and A(3) ARs inhibited forskolin- stimulated adenylyl cyclase activity. The A(1) ARs also activated 42/44-kD ERK mitogen-activated protein kinases via G(i)- and tyrosine kinase-dependent pathways. The A(2a) ARs stimulated adenylyl cyclase activity and activated the protein kinase A-->CREB pathway. Chronic (48 h) treatment with a nonselective AR antagonist (8-phenyltheophylline) upregulated A(1), A(2a) ARs and G(i)alpha. Conversely, chronic stimulation of HK-2 ARs with a nonselective AR agonist (N-ethylcarbamoyladenosine) downregulated all 4 subtypes of ARs and G(s)alpha. Based on these findings, HK-2 cells are a useful in vitro model to study the signaling cascades of AR-mediated renal protection.

The direct effects of estradiol-17beta (E(2)) on phosphate (P(i)) uptake and on DNA synthesis in the primary rabbit kidney proximal tubule cells (PTCs) have been investigated. In the present study, E(2) (>10(-9) M, over 9 days) causes an increase both in [(3)H]thymidine incorporation and the number of PTCs. The anti-estrogen tamoxifen completely prevented the E(2)-induced increase in [(3)H]thymidine incorporation, and ameliorated the stimulatory effect of E(2) on growth. E(2) (>10(-9 )M, over 5 days) also stimulated the P(i) uptake and its effect was due to the V(max) values but not to the K(m) value for P(i) uptake. Estriol and estrone also exerted significant stimulatory effects on P(i) uptake. Progesterone, tamoxifen, actinomycin D and cycloheximide prevented the E(2)-induced stimulation of P(i) uptake. In conclusion, estrogens at physiological concentrations stimulate P(i) uptake and DNA synthesis in the renal proximal tubule cells, and these effects are estrogen receptor mediated.

Background: Homozygous adenine phosphoribosyltransferase (APRT) deficiency is associated with 2,8-dihydroxyadenine (DHA) nephrolithiasis. Using whole kidney RNA from Aprt knockout mice, we previously showed that the renal deposition of DHA leads to changes in the expression of genes involved in tissue injury. To determine the cellular basis for these changes, we investigated gene expression in cultured human kidney (NHK-C) and African green monkey (BSC-1) epithelial cells exposed to DHA or calcium oxalate monohydrate (COM) crystals.

Methods: First-strand cDNAs, synthesized from mRNA isolated from treated and untreated cells, were hybridized to membrane-bound cDNA arrays containing 588 genes associated with various physiological and pathological processes. Changes in gene expression were confirmed by reverse transcription PCR.

Results: Twenty-seven percent of the array cDNAs were expressed in untreated NHK-C cells at varying levels relative to a housekeeping gene. The expression of three adhesion molecules (alpha-catenin, integrin alpha3, and integrin beta6) and platelet-derived growth factor B (PDGF-B) was elevated following exposure of NHK-C cells to DHA. Increased expression of the adhesion molecules was also observed in BSC-1 cells, but PDGF-B expression could not be detected. COM crystals also stimulated the expression of these four genes in NHK-C cells, but the expression profile was quantitatively different compared with DHA.

Conclusions: These findings suggest that DHA crystals stimulate the expression of specific genes in kidney epithelial cells and that the pathways for DHA-induced cell injury may be similar to those for COM crystals. The induction of adhesion molecules and PDGF-B may affect cell-cell or cell-matrix interactions and/or alter the actin cytoskeleton. These alterations may ultimately contribute to crystal-induced renal injury.

Polarized tubule epithelial cell functions are dependent on correct delivery of effector proteins to the target apical or basolateral plasma membrane and associated cortical cytoskeleton. PDZ (Postsynaptic density protein 95/Drosophila Disks large/Zona occludens-1) domain-containing proteins have been identified as playing a critical role in membrane trafficking and sorting of ion transporters, receptors and other signalling proteins. These scaffolding proteins coordinate the assembly of functional plasma membrane multiprotein complexes, through PDZ domain binding to a consensus amino acid motif within the carboxyl-terminus of target proteins. The organization of these proteins into submembranous complexes may facilitate downstream signalling. Although several epithelial PDZ proteins that bind to a number of important mammalian proteins have been isolated, in many cases the significance of these interactions is unclear. However, the epithelial PDZ domain-containing Na(+)/H(+) exchanger regulatory factor tethers the Na(+)/H(+) exchanger and cystic fibrosis transmembrane regulator Cl(-) channel within an apical plasma membrane signalling complex, and has been shown to regulate the activity of these proteins. This article reviews the current evidence that supports a central role for the PDZ protein in the regulation of polarized tubule cell functions, such as vectorial solute transport.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar-Kyoto (WKY) rats (RT1-l) by immunization with rat glomerular basement membrane (GBM) in adjuvant. The model in this rat strain is characterized by anti-GBM antibody production accompanied by focal necrotizing glomerulonephritis with crescent formation. The main autoantigen in humans and rats has been identified as the non-collagenous domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). By contrast, Lewis (LEW) rats with the same MHC background (RT1-l), immunized with the same antigen, develop similar levels of circulating anti-GBM antibodies, but no histological evidence of nephritis. In order to investigate the genetic basis of susceptibility to EAG, we examined the response of both F1 (WKY x LEW) and backcross (BC1; WKY x F1) rats to immunization with rat GBM. F1 animals were completely resistant to the development of EAG, while BC1 animals showed a range of responses from severe crescentic glomerulonephritis to no histological evidence of disease. The results indicate that EAG is inherited as a complex trait under the control of WKY genes unlinked to the MHC. cDNA sequence analysis of alpha3(IV)NC1 in the two parental strains was identical, indicating no predicted amino acid sequence variation in the alpha3(IV)NC1 domain between these strains. Radiation hybrid mapping, using two separate PCR amplicons from rat alpha3(IV)NC1, localized rat Col4a3 to a region of chromosome 9. Since Col4a3 (encoding the autoantigen) is a candidate for susceptibility to EAG, we screened the region of rat chromosome 9 where Col4a3 is localized, using polymorphic microsatellite markers in segregating BC1 progeny. No significant linkage was detected. These results exclude Col4a3 as a recessive susceptibility gene for EAG in the BC1 progeny.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) belongs to a superfamily of nuclear receptors, which plays important roles in lipid and glucose metabolism. However, expression of PPARgamma in extra-adipose tissues and stimulation of apoptosis by PPARgamma activators has been previously reported. We investigated the functions of PPARgamma using a clonal kidney cell line (LLC-PK1). RT-PCR revealed the expression of PPARgamma in LLC-PK1 cells. The cells accumulated fat droplets and increased beta-oxidation of free fatty acids in response to troglitazone, a ligand for PPARgamma. At physiological concentrations, ligands for PPARgamma including troglitazone, BRL49653, and 15-deoxy-delta-12,14-prostaglandin J(2) inhibited serum-deprivation-induced apoptosis of the cells. On the other hand, PPARalpha activators did not inhibit the apoptosis. Apoptosis of LLC-PK1 cells was determined by a cell viability assay, condensation of the nucleus on fluorescent and electron microscopy, and DNA fragmentation as indicated by the appearance of nucleosomal ladders on an agarose gel. Troglitazone also suppressed serum-deprivation-induced activation of Caspase 3. However, troglitazone did not suppress apoptosis induced by ATP deprivation. Anti-apoptotic effects of troglitazone were partially blocked by a phosphatidylinositol-3-kinase (PI3K) inhibitor, wortmannin, but not by other kinase inhibitors such as PD98059 and AG490. These results suggest that PPARgamma is functionally expressed in LLC-PK1 cells, and its activation inhibits apoptosis induced by serum deprivation, at least in part, through the PI3K pathway.

Myofibroblast differentiation characterizes a prominent cellular phenotype identified in experimental models of progressive kidney disease and human kidney biopsies. Mesangial cells, tubulointerstitial fibroblasts and, perhaps, tubular epithelial cells undergo myofibroblast differentiation, a process characterized by alpha-actin expression, synthesis of interstitial collagens and a growth response. Inhibition of myofibroblast differentiation could prevent kidney disease progression but may be difficult to accomplish, since inhibition of multiple signaling pathways would be required. Cell biology advances have enabled a better understanding of how information from many microenvironmental stimuli are integrated by spatial compartmentalization of extracellular receptors and cytosolic signaling molecules within specialized plasma membrane domains, such as focal adhesions and lipid rafts. We review this information and hypothesize that myofibroblast differentiation of renal cells can only proceed if the spatial arrangement of intracellular molecules, in large part determined by extracellular matrix-regulated cytoskeletal organization, permits activation of appropriate signaling pathways by soluble molecules interacting with receptors in specialized plasma membrane microdomains. If proven, this hypothesis suggests targeting key molecules within adhesion complexes and rafts (in some cases with drugs that are already clinically available) may provide more effective therapy for kidney disease progression.