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Kidney Week

Abstract: FR-PO1002

Cyclosporine A and Paraquat Induce Lysosomal Deacidification in Proximal Tubular Cells In Vitro

Session Information

  • CKD: Pathobiology - I
    November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms


  • da Silva Fernandes, Sylvina, Universiteit Antwerpen, Antwerpen, Belgium
  • Schreurs, Gerd, Universiteit Antwerpen, Antwerpen, Belgium

Group or Team Name

  • Lab of Pathophysiology - CINAC

Chronic interstitial nephritis in agricultural communities (CINAC) is a chronic toxin-induced nephropathy of which the underlying molecular mechanism and causal toxins are unknown. Due to the geographical distribution of CINAC cases in and around agricultural communities, we and others suspect that environmental toxins could play a role in disease development. Recently, we discovered a diagnostic lesion in CINAC renal biopsies encompassing large dysmorphic lysosomes in proximal tubular cells (PTCs). The same phenotype was found in PTCs of transplant patients receiving nephrotoxic calcineurin inhibitors (CNIs) as immunosuppressive therapy. Hence, it is hypothesized that CINAC is caused by exposure to an (unknown) environmental toxin, which affects the lysosomal system. In search for the CINAC culprit(s), we therefore aimed to optimize a in vitro assay to assess effects on PTC lysomal function.


Since impaired lysosomal acidification indicates a functional defect, the central approach to the assay consists in exposing PTCs to various toxins and quantifying acidification. HKC-8 cells were exposed to Bafilomycin A1 (Baf), an inhibitor of lysosomal acidification (positive control), Cyclosporine A (CsA, a CNI) or Paraquat (PQ, a herbicide). For each toxin a sub-toxic dose was chosen from a concentration range based on cell death quantification using Sytox Green. Lysosomal acidification was assessed by incubating cells with Lysotracker (LT) (50 nM, 30 minutes) followed by flow cytometric quantification. To correct for the size of the lysosomal compartment in these cells, a Western Blot targeting LAMP-1, a lysosomal protein, was performed.


In the optimized assay, HKC-8 cells were incubated with normal medium (Control), 10nM Baf, 25 µM CsA and 100 µM PQ for 6 hours. The mean LT Red signal, relative to control, was significantly lower (p < 0.01) in all three conditions. No significant difference in LAMP-1 abundance was found, indicating no significant contribution of lysosome to the LT signal. CsA exposure caused a significant increase in granularity as measured by flow cytometry and observed visually as clear, irregular vacuoles under brightfield microscopy.


Both CsA and PQ induce lysosomal deacidification in PTCs in vitro, but only CsA causes an increase of granules. Immunostains are planned to identify the nature of these granules.


  • Government Support – Non-U.S.