Introduction
Our previous studies have suggested a role for renomedullary interstitial cells (RMICs) and renal medullary hyaluronan (HA) in water homeostasis. The present study utilizes cultured rat RMICs to examine the relationship between osmolality, oxygen tension and the extracellular amount of HA in the culture and the cellular immunoreactivity of CD44, a HA binding protein.
Methods
Cells were isolated from male Sprague-Dawley rats. Homogenous cell populations were usually reached at passage 10 and used in subsequent experiments until passage 20. The osmolality of the media was changed from 330 mOsm kg-1 H2O according to 230 mOsm kg-1 H2O,630 mOsm kg-1 H2O 930 mOsm kg-1 H2O and 1230 mOsm kg-1 H2O, respectively. Immunohistochemical stainings and flow cytometry was used to localize and quantify CD44 and a standard radiometric assay was used to quantify hyaluronan.
Results
During isotonic (330 mOsm kg-1 H2O) and normoxic (20% O2) conditions, the supernatant from sub-confluent RMICs contained 120±37 pg·104 cells-1·48 h-1 of HA. During hyper-osmolal conditions (630 mOsm kg-1 H2O), the content of HA in the supernatant was decreased by 42% and during hypo-osmolal conditions (230 mOsm kg-1 H2O) it was increased by 100%. At hypoxic and and iso-osmolar conditions (5%O2 and 1%O2, 330 mOsm kg-1 H2O) the HA content of the supernatant was decreased by 56 and 48%, respectively, compared with normoxic, iso-osmolal conditions. Immunostaining and flow cytometric analysis for CD44 demonstrated an increased expression of the receptor on sub-confluent cells with increasing osmolality. The increase from isotonic conditions to 630, 930 and 1230 mOsm kg-1 H2O was 5, 142 and 212%, respectively. Low oxygen tension (5% O2) decreased the intensity of CD44 immunofluorescence by 31%.
Conclusions
These data indicate that cultured RMICs produce HA and show positive immunoreactivity to CD44. In the supernatant of RMICs, HA content decreases under hyperosmotic and hypoxic conditions. Conversely, CD44 immunoreactivity increases under hyperosmotic conditions. These results may explain our previous in vivo findings of a decreased renal papillary HA content during anti-diuresis and increased content during water diuresis. The results are in support of an important role for RMICs in renal water handling.
References
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