Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity

1990 ◽  
Vol 39 (8) ◽  
pp. 1335-1345 ◽  
Author(s):  
Pieter J. Boogaard ◽  
J.Paul Zoeteweij ◽  
Theo J.C. Van Berkel ◽  
Joost M. Van't Noordende ◽  
Gerard J. Mulder ◽  
...  
Keyword(s):  
Primary Culture ◽  
In Vitro Model ◽  
Rat Kidney ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Normal Rat ◽  
Vitro Model

Isolation and Culture of Proximal Tubular Cells from Porcine Kidney

1993 ◽  
Vol 21 (4) ◽  
pp. 457-465
Author(s):  
Marieke Kruidering ◽  
Frans A. Prins ◽  
Emile de Heer ◽  
Gerard J. Mulder ◽  
J. Fred Nagelkerke
Keyword(s):  
Primary Culture ◽  
Single Cells ◽  
Proximal Tubular Cells ◽  
Isolated Cells ◽  
Serum Free ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Vitro Model ◽  
Serum Free Conditions

Porcine proximal tubular cells (PPTC) were isolated from kidneys obtained from slaughterhouse pigs. After disruption of the connective tissue by collagenase, purification was achieved by filtration and centrifugation on a discontinuous density gradient. Single cells and clusters of 10–40 cells were obtained, having a viability of 93–99%. More than 81% of the single cells showed γ-glutamyltranspeptidase (GGT) activity and more than 95% showed non-specific esterase (NE) activity, marker enzymes for proximal tubule cells. One kidney yielded 1 x 107 single cells and 3x107 cells in clusters. Cells were kept in primary culture on plastic or collagen-coated dishes. In the presence of 10% serum, confluency was reached within four days. The monolayers could be kept in culture for four days after confluency, in serum-free conditions. When seeded in serum-free conditions, PPTC did not reach confluency, but the cells could be kept in culture for at least 16 days. The cells displayed epithelial morphology, i.e. cobblestone shape, dome formation, microvilli, basal infoldings and abundant mitochondria. PPTC in primary culture still displayed NE activity, while 80% of the cells showed GGT activity. In conclusion, the isolated cells are of proximal tubular origin, reach confluency in 3–4 days in the presence of 10% serum, and can be kept as monolayers in serum-free conditions for four additional days and may provide a suitable in vitro model for long-term nephrotoxicity studies.

scholarly journals A Non-Targeted Capillary Electrophoresis-Mass Spectrometry Strategy to Study Metabolic Differences in an In Vitro Model of High-Glucose Induced Changes in Human Proximal Tubular HK-2 Cells

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 512 ◽  
Author(s):  
Samuel Bernardo-Bermejo ◽  
Elena Sánchez-López ◽  
María Castro-Puyana ◽  
Selma Benito-Martínez ◽  
Francisco Javier Lucio-Cazaña ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
In Vitro Model ◽  
Proximal Tubular Cells ◽  
Extracellular Medium ◽  
Metabolic Alterations ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Vitro Model

Diabetic nephropathy is characterized by the chronic loss of kidney function due to high glucose renal levels. HK-2 proximal tubular cells are good candidates to study this disease. The aim of this work was to study an in vitro model of high glucose-induced metabolic alterations in HK-2 cells to contribute to the pathogenesis of this diabetic complication. An untargeted metabolomics strategy based on CE-MS was developed to find metabolites affected under high glucose conditions. Intracellular and extracellular fluids from HK-2 cells treated with 25 mM glucose (high glucose group), with 5.5 mM glucose (normal glucose group), and with 5.5 mM glucose and 19.5 mM mannitol (osmotic control group) were analyzed. The main changes induced by high glucose were found in the extracellular medium where increased levels of four amino acids were detected. Three of them (alanine, proline, and glutamic acid) were exported from HK-2 cells to the extracellular medium. Other affected metabolites include Amadori products and cysteine, which are more likely cause and consequence, respectively, of the oxidative stress induced by high glucose in HK-2 cells. The developed CE-MS platform provides valuable insight into high glucose-induced metabolic alterations in proximal tubular cells and allows identifying discriminative molecules of diabetic nephropathy.

scholarly journals Apoptosis as a mechanism of the albumin-induced kidney damage in childhood nephrotic syndrome

2018 ◽  
pp. 25-30
Author(s):  
Ie.A. Burlaka ◽  
I.V. Bagdasarova
Keyword(s):  
Nephrotic Syndrome ◽  
Primary Culture ◽  
Kidney Damage ◽  
In Vitro Studies ◽  
Kidney Cells ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Nephrotic Children

It was shown previously on in vivo studies that proteinuria-induced effects play a crucial role in renal damage in chronic kidney disease (CKD). However, an initial mechanism of irreversible kidney damage in pediatric diseases characterized by chronic proteinuria, i.e. nephrotic syndrome, remains to be unclear. The aim of our work was to study the initial mechanism of kidney cells apoptosis development in nephrotic children. Methods.An examination of renal biopsies of 53 patients (aged 10 to 15 years) with nephrotic syndrome hospitalized in Pediatric Nephrology unit of the Children Clinical Hospital №7 (Kyiv, Ukraine) done. In vitro studies of albumin toxicity performed on rat proximal tubular cells in primary culture (RPTC). Results. Our study showed that albumin overload in nephrotic children leads to high levels of apoptosis. Its distribution and level varies regarding the level of focal segmental glomerulosclerosis (FSGS). The progression of sclerosis as a sign of irreversible kidney damage is accompanied by gradual increase in expression of proapoptotic factor Bax. In vitro studies on rat proximal tubular cells in primary culture (RPTC) showed that excessive albumin uptake into rat primary renal cells causes an almost immediate mitochondrial accumulation of the apoptotic factor Bax. We hypothesize that this might be initial pathway leading to kidney cells apoptosis in childhood nephrotic syndrome. Conclusions. We show thatoverexpression of apoptotic factor Bax has a place in children with nephrotic syndrome. Thus, chronic influence of albumin is a factor predisposing disturbances in system controlling apoptosis in this cohort of patients. Our data demonstrate that there is a dependence between the Bax overexpression level and the stage of CKD. We show the topologic difference between the Bax levels and FSGS degree. This is an indication thatdevelopment of glomerular and tubule-interstitial disorders under the influence of proteinuria occurs in specific range. In vitro data demonstrate that albumin overload causes mitochondrial Bax translocation that could be an initial factor in apoptotic pathway activation.

Inhibition by insulin of cellular autophagy in proximal tubular cells of rat kidney

1983 ◽  
Vol 244 (2) ◽  
pp. E109-E114 ◽  
Author(s):  
U. Pfeifer ◽  
M. Warmuth-Metz
Keyword(s):  
Volume Fraction ◽  
Rat Kidney ◽  
Insulin Dose ◽  
Ringer Solution ◽  
Numerical Density ◽  
Partial Recovery ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Cellular Autophagy ◽  
Proximal Tubular

Adult male Sprague-Dawley rats were injected intraperitoneally with 5 U insulin/kg body wt (45 animals). As determined by quantitative electron microscopy, the volume fraction and the numerical density of autophagic vacuoles (AV) in proximal tubular cells decreased within 10 min by 46 and 26%, respectively. A partial recovery of the AV volume fraction was observed 20 and 30 min after the injection contrary to our previous findings with liver (J. Cell Biol. 78: 152-167, 1978). In an additional experiment (12 animals) it was shown that an insulin dose of 0.5 U but not of 0.05 U/kg body wt reduced the AV volume fraction to an extent similar to that of 5 U. To eliminate possible secondary effects, Ringer solution containing 0.8 microM insulin was dropped intravitally for 15 min to one pole of the decapsulated kidney and Ringer solution without additions to the other pole (8 animals). After intravital fixation, the AV volume fraction and numerical density in proximal tubular cells was found to be reduced under the influence of insulin by 22 and 36%, respectively. This data shows that insulin inhibits the process of cellular autophagy in proximal tubular cells of the kidney.

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