Experimental nephrology最新文献

Background/aims: Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. The aim of the work outlined here was to examine the effects and mechanisms involved in the modulation of renal proximal tubular type-IV collagen and fibronectin turnover by glucose.

Methods: The effect of glucose on type-IV collagen and fibronectin generation was studied by exposure of primary cultures of human renal proximal tubular cells (HPTC) to elevated D-glucose concentrations. Subsequently the mechanism of modulation of fibronectin generation was examined in a polarised system utilising the porcine proximal tubular cell line LLC-PK1 grown on porous tissue culture inserts.

Results: Incubation of confluent growth-arrested HPTC with 25 mM D-glucose led to the accumulation of both type-IV collagen and fibronectin. This increase was not dependent on new gene transcription for either protein. Exposure of HPTC to 25 mM D-glucose also led to the induction of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) and also gelatinase A. There was, however, a net decrease in overall gelatinolytic activity. Incubation of confluent monolayers of LLC-PK1 cells grown on tissue culture inserts with 25 mM D-glucose on either their apical or basolateral aspect led to fibronectin accumulation seen only in the basolateral compartment. Under these experimental conditions, we can demonstrate polyol pathway activation, and furthermore the increase in fibronectin concentration in response to glucose was inhibited by the aldose reductase inhibitor sorbinil. Fibronectin accumulation was also demonstrated following both apical and basolateral addition of 1 mM sorbitol, but not following the addition of 25 mM galactose to either aspect of the cells.

Conclusions: These data demonstrate that the glucose-induced accumulation of type-IV collagen and fibronectin was associated with alterations in the degradative pathway of these matrix components. In addition fibronectin generation in response to glucose was non-polar in terms of application of glucose, but polar in terms of fibronectin accumulation. The mechanisms of glucose-induced modulation of fibronectin were mediated by polyol pathway activation, and more specifically related to the metabolism of sorbitol to fructose.

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

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.

Antisense oligodeoxynucleotides offer the potential to block the expression of specific genes with the goal of altering the phenotypic behavior of the cell. Antisense technology has attracted special interest as potential therapeutic agents for the treatment of genetic disorders, viral infections, and most recently proliferative diseases such as glomerular kidney disease. This technique has recently been used for in vitro and in vivo studies in renal cells. The use of antisense technology has been applied in vitro to help define both the normal mechanisms of specific ion transport and function and the pathobiological processes leading to glomerular proliferation and matrix formation. Most promising are the recent uses of antisense technology in vivo that have been used to treat the damaged peritoneum and alter glomerular remodeling in experimental animal models. It is hoped that widespread use of antisense will not only provide new insight into the normal regulatory behavior of the kidney cells but also allow one to develop therapeutic strategies to treat kidney disease.

The complexity and heterogeneity of the human nephron with regard to cell types make well-defined in vitro systems of renal cells valuable for studies of the pathogenetic mechanisms involved in nephrotoxicity. In our laboratory renal proximal tubule cells (PTC) and collecting duct cells (CDC) have been isolated, cultured and characterized from cadaver kidneys (postmortem time <24 h) for use in studies of renal cytotoxicity induced by therapeutics and bacteria. PTC seeded at 10(6) cells/ml formed confluent monolayers within 7 days. Histochemical markers were used to determine the origin of the cell cultures. Cells were negative for factor VIII, positive for cytokeratin and gamma-glutamyltransferase (GGT), and bound winged pea lectin. CDC, isolated from the renal papillae, formed monolayers within 14 days of seeding. CDC were negative for factor VIII and GGT, positive for cytokeratin and bound peanut lectin. PTC and CDC isolates and cultures exhibited typical epithelial cell ultrastructure: cell junctions, intermediate filaments, microvilli, and numerous mitochondria. The morphological and histochemical evidence confirms that PTC and CDC can be isolated and cultured for use in in vitro studies.

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 erythropoeitin, 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.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenosine monophosphate dependent, low-conductance chloride channel found on the apical plasma membrane of secretory epithelia. Surprisingly, since cystic fibrosis patients have no kidney phenotype, CFTR is highly expressed in the kidney, present from 12 weeks of gestation in the human metanephric kidney. As well as the mature, full-length, 165-kD wild-type protein (WT-CFTR) associated with renal tubule plasma membranes, intracellular, partially glycosylated forms are also seen in normal kidneys. In addition, a kidney-specific splice variant of CFTR translates a cytoplasmic truncated protein (TNR-CFTR), apparently associated with a specific small endosomal population, and is predominantly expressed in the renal medulla. WT-CFTR and TNR-CFTR show different patterns of developmental regulation, WT-CFTR being the major form expressed early in metanephric development when it is localized at the apical plasma membrane of developing collecting tubules. By contrast, TNR-CFTR expression increases with gestational age, reaching adult levels at 23 weeks. Evidence suggests that WT-CFTR plays a role in chloride secretion into the apical lumen of normal distal tubules. In autosomal dominant polycystic kidney disease, normally targeted CFTR at the apical plasma membrane in association with mislocalized Na-K-ATPase may result in abnormal fluid secretion into cysts. Similar colocalization of WT-CFTR and Na-K-ATPase at the apical plasma membranes is found in collecting tubules during development when it is speculated to play a role in the initiation of opening of the tubule lumen.

We investigated acute and chronic effects of hyperosmolality on mRNA and protein expressions of Na-K-ATPase alpha and beta 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 alpha(1) mRNA. In contrast, dehydration decreased alpha(1) subunit mRNA and protein and beta(1) protein expressions without changing beta(1) mRNA. These data show (1) that acute hyperosmolality decreases Na-K-ATPase activity in IMCD without changing alpha(1) and beta(1) mRNA and (2) that 2 days of dehydration decreased Na-K-ATPase activity by reducing alpha(1) and beta(1) proteins. Thus, the mechanisms for the inhibition of the Na-K-ATPase activity in IMCD is different between acute and chronic exposure to hyperosmolality.

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.