Simulating Nephrocalcinosis in a jar: An in vitro temperature-dependent model of CO₂-induced precipitation relevant to intensive aquaculture

Nephrocalcinosis (“kidney stones”) is a persistent health issue in intensive aquaculture systems, affecting fish welfare and productivity. However, its causes and risk factors remain poorly understood. Dissolved CO 2 concentrations are typically elevated (hypercapnia) in intensive aquaculture systems, causing elevated blood PCO 2 levels. Fish compensate for this hypercapnia-induced “respiratory acidosis” by elevating plasma bicarbonate (HCO 3 − ) to restore pH. However, if returned to normocapnia (normal CO 2 ), fish experience an alkalosis which we propose may induce nephrocalcinosis. We used an in vitro model to test whether a rapid rise in pH in the pre-urine, caused by an abrupt PCO 2 drop when fish are transferred from hypercapnic to normocapnic levels, could be the trigger for kidney stone formation. Synthetic fish pre-urine was sequentially subjected to: (1) elevated PCO 2 (1–3 %) to mimic hypercapnia-induced respiratory acidosis; (2) HCO 3 − addition to simulate metabolic compensation; and (3) a rapid reduction in PCO 2 mimicking events that occur routinely during fish culture and handling ( e.g. , transfer, sorting/grading, fasting, vaccination). These exposures were conducted at 6, 12, and 18 °C. Precipitation in the pre-urine, measured by turbidity, was rapid and sustained following the drop in PCO 2 (step 3 above). The magnitude of precipitation was strongly associated with pH rise (ΔpH), particularly at higher temperatures. These findings demonstrate that abrupt decreases in PCO 2 can promote mineral precipitation under physiologically relevant ionic conditions, and that elevated temperature exacerbates this effect. The results support the hypothesis that nephrocalcinosis may result, in part, from acid-base disturbances commonly encountered under intensive aquaculture conditions. • Nephrocalcinosis was modeled using an in vitro fish pre-urine precipitation assay. • Acid-base disturbances were induced by manipulating PCO 2 and bicarbonate levels. • Rapid PCO 2 drops triggered pH increases and mineral precipitation. • Precipitation was significantly enhanced at elevated temperatures.