In vitro formation and expansion of cysts derived from human renal cortex epithelial cells

Abstract

Acquired renal cysts derive from terminally differentiated tubular epithelium in adults as a consequence of increased epithelial cell proliferation, fluid accumulation and extracellular matrix remodelling. To understand better how human epithelial cysts may be initiated and progressively expand, cells from primary cultures of normal human adult renal cortex were dispersed in polymerized type I collagen. The transparent matrix permitted repeated observation by light microscopy of cyst formation from individual renal cells. The cyst cells reacted strongly with distal nephron histochemical markers (cytokeratin antibodies AE1/AE3, epithelial membrane antigen, and Arachis hypogaea lectin) but inconsistently or not at all to markers of proximal tubules (Tetragonolobus purpureas lectin and Phaseolus vulgaris erthroagglutinin lectin). The number of spherical, fluid-filled epithelial cysts that developed in a standardized microscope field quantified cyst initiation. Cyst progression was determined from the increase in the diameter (surface area) of cysts and represents a hyperplastic event. EGF or TGF alpha, were required in serum-free defined medium to cause cysts to develop from individual epithelial cells dispersed in the matrix; insulin was required as a co-factor. The EC50 for EGF was approximately 0.1 ng/ml, and for insulin 1 microgram/ml. Early cultures of normal cortex formed cysts more efficiently when dispersed in collagen matrix than cells passaged several times before suspension in the gel. Agonists of adenylate cyclase (PGE1, AVP, VIP, PTH, forskolin, cholera toxin), methylisobutylxanthine, and 8-Br-cAMP, though incapable of causing cyst formation alone in defined medium, enhanced cyst initiation and progression in the presence of EGF and insulin. Angiotensin II, TNF alpha, beta-estradiol, and pertussis toxin had no effect in the absence or presence of EGF and insulin. Pertussis toxin inhibited cyst initiation and expansion caused by EGF and forskolin but potentiated cyst initiation and expansion caused by EGF and PGE1. Cyst formation and expansion were inhibited by TGF beta 1 and 2-chloroadenosine. Polarized monolayers of human renal cortical cells grown on permeable membranes were used to independently quantify the effects of agonists on the net secretion of solute and water from the basolateral to the apical surface of the cells. PGE1, forskolin, and 8-Br-cAMP stimulated net fluid secretion that was sustained for several days; EGF enhanced forskolin-stimulated fluid secretion. We conclude that the formation and expansion of in vitro cysts derived from solitary human cortex cells depends on the coordinated interplay between cellular proliferation and fluid secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Simple Renal Cysts in Hypertensive Patients: Relation between Cyst Growing and Anti-Hypertensive Therapy

“…Formation and expansion of "in vitro" cysts, derived from solitary human cortex cells, depends from the coordinated interplay among cellular proliferation, fluid secretion and remodeling of extracellular matrix surrounding cysts (8)(9). The molecular mechanism and the pathogenesis of cyst formation are still unclear.…”

Urinary EGF Receptor Ligand Excretion in Patients with Autosomal Dominant Polycystic Kidney Disease and Response to Tolvaptan

“…Ligands for the EGF receptor are EGF, TGF-a, and HB-EGF. Recently, experimental studies have shown that activation of this EGF receptor by binding with the ligands EGF or TGF-a results in cyst formation in vitro (8) and in vivo (9). Inhibition of EGF receptor tyrosine kinase activity blocked tubular cyst formation in an in vitro system (14), blockade of EGF receptor resulted in attenuation of cystic disease in animal models (15,16), whereas ErbB4 knockout mice showed an accelerated cyst progression and renal function deterioration (17).…”

“…We included 27 patients with ADPKD who all completed a single-center interventional trial investigating the short-term renal hemodynamic effects of the V2RA tolvaptan that was performed between 2011 and 2013 (8). ADPKD was diagnosed using the modified Ravine criteria (9).…”

“…Patients were studied at baseline, after 3 weeks of treatment with tolvaptan (uptitration to 90/30 mg per day, split dose), and 3 weeks after withdrawal of tolvaptan. Exclusion criteria have been described previously (8). Controls were randomly chosen from the Prevention of Renal and Vascular ENd-stage Disease (PREVEND) study, a general population-based, prospective, observational cohort study in which participants visit an outpatient clinic for detailed measurement of health status (10).…”

“…Excretion of EGF receptor ligands in human ADPKD has not been investigated yet. Activation of the EGF receptor by binding with its ligands results in cyst formation in vitro and in mice (8,9). These data form a rationale for our interest in the EGF receptor ligands in human ADPKD.…”