Maintaining the healthy differentiated state of the renal proximal tubule should be the goal of every pet caregiver.
In all mammals, the renal proximal tubule is necessary for reclaiming vital nutrients, regulating the body’s pH homeostasis, and stimulating red blood cell production. A correctly formed barrier in the kidneys results from well functioning proximal tubules. This barrier is essential for both electrical and concentration gradients. These gradients allow for active and passive pumping across the barrier, which is vital for the kidneys to carry out their essential functions. In our research, we measured these electrical gradients and examined the effects of ION*Gut Health For Pets on these critical barriers.
MATERIALS AND METHODS
Cell culture
As starting material, primary renal proximal tubule cells were isolated and grown from approximately one gram of fresh renal cortex from mouse, rat, cow, dog, and cat. Primary human renal proximal tubule cells were purchased and grown according to manufacturer’s instructions (Lifeline Cell Technology).
Trans-Epithelial Electrical Resistance (TEER)
Cells were plated in the apical compartment of 24 collagen coated Transwell Inserts (Corning, Sigma Aldrich) at 90% confluence, and incubated for three days when the TEER values stabilized. TEER values were measured with an EVOM2 (World Precision Instruments). Briefly, electrodes were sterilized by soaking in 70% ethanol for ten minutes then conditioned by soaking in cell culture media for one hour. A background reading of an empty Transwell Insert without cells was subtracted from all values and recorded as Ohms x CM^2. Six Transwell Inserts were used for either vehicle treatment, phosphate buffered saline (VEH), or ION*Gut Health For Pets treatment added to 20% ION by adding to both apical and basolateral media for four hours.
RESULTS
A proximal tubule cell is known to be a leaky epithelium, meaning that it is characterized as having a relatively low TEER value compared to other epithelial layers. In growth media, the TEER value was found to be barely over background for all six species tested (VEH in graph). However, in all six species, a dramatic and significant increase in TEER was observed when treated for only four hours with ION*Gut Health For Pets (*p<0.01 ION vs VEH, n=6 per group).
DISCUSSION
The integrity of epithelial barriers is now considered a major component of longevity. One of the features of a disrupted epithelium is the concept of epithelial to mesenchymal transition, where cells of the confluent cell layer lose close association with their neighbors. This disruption of barrier function causes the cells to progressively become more similar to mesenchymal cells, which are then responsible for the fibrotic conversion of a tissue toward a less resilient condition. Tight junctions are among the structures within the cells that are disrupted as cells lose their identity and are the feature of an epithelial layer that mediates the TEER measured in this paper. The ability of ION*Gut Health For Pets to increase TEER and inhibit the reduced differentiation observed in these proximal tubule cell cultures may be an important component supporting overall kidney health, whole body homeostasis, and lifetime wellness.
