In-vitro maintenance and functionality of primary renal tubules and their application in the study of relative renal toxicity of nephrotoxic drugs

Abstract

Introduction: The renal tubules play important roles in substance re-absorption from primary urine of the kidney, drug metabolism and gluconeogenesis in fasting and are vulnerable targets of nephrotoxic chemicals. Therefore, an appropriate functional model of renal tubules would enable the study of their functionality and chemical-induced toxicity. We have developed a method to maintain primary renal tubules and sustain their biochemical functionality in culture for an extended period of time.

Methods: Primary rat renal tubules were isolated from male rat kidneys by collagenase perfusion and the tubules maintained in culture as a suspension by a gyratory culture method.

Results: The tubule fragments gradually formed renal tubule aggregates within 6days and were maintained in culture for up to 12days without apparent morphological changes. Biochemical functions including glucose release, galactose uptake and pyruvate uptake were retained for the observed period of 12days after isolation. The aggregates showed significant cytochrome P450 1A1 activity recovery from day 6 after isolation and this was maintained thereafter during the 12-day period of in-vitro culture. A new toxicity test termed the cell spreading inhibition test (CSIT) of renal tubule aggregates was developed to study the effect of toxicants on cell spreading/growth and determine the minimum concentration of each toxicant that caused cell spreading inhibition (CSI-C). The CSI-Cs of selected nephrotoxic drugs were determined as chlorpromazine (60μM), cisplatin (200μM), diclofenac (800μM), valproic acid (10mM), and gentamycin (30mM).

Discussion: The gyratory method of primary renal tubule aggregate culture can retain tubular cell functions such as glucose release, galactose uptake and allow cytochrome P450 1A1 activity to recover, which are essential for an in-vitro model. Therefore, renal tubule aggregates can be used as a model for studies of biochemical functions of renal tubules and relative renal toxicity of nephrotoxic agents.

Keywords: CSIT; Galactose; Kidney glucose; Pyruvate; Renal tubule aggregate; cytochrome P450 1A1.