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Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney

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Summary

Renal cortical thick ascending limbs of Henle’s loop (CAL) and distal convoluted tubules (DCT) represent sites at which much of the final regulation of urinary ionic composition, particularly that of calcium, is accomplished in both humans and in rodents. We sought in the present work to develop an efficient means for isolating parathyroid hormone (PTH)-sensitive cells from these nephron segments and to grow them in primary culture. [CAL+DCT] cells were isolated from mouse kidney using an antiserum against the Tamm-Horsfall glycoprotein which, in the renal cortex, is produced exclusively by these cells. A second antibody conjugated to coated ferrous particles permitted magnetic separation of [CAL+DCT] cells from Tamm-Horsfall negative renal cortical cells. Approximately 3 × 106 cells per kidney with a trypan blue exclusion greater than 94% were isolated by these procedures. Experiments were performed to characterize the cells after 7 to 10 days in primary culture. PTH and isoproterenol, but neither calcitonin nor vasopressin, stimulated cyclic AMP (cAMP) formation in [CAL+DCT] cells, consistent with the pattern of hormone-activated cAMP synthesis found in freshly isolated CAL and DCT segments. Alkaline phosphatase, an enzyme present dominantly in proximal tubule brush border membranes, was virtually absent from [CAL+DCT] cells but was present in Tamm-Horsfall negative cells. Similarly, Na-glucose cotransport was absent in [CAL+DCT] cells but present in Tamm-Horsfall negative renal cortical cells. Finally, transport-related oxygen consumption in [CAL+DCT] cells was blocked by bumetanide and by chlorothiazide, diuretics that inhibit sodium transport in CAL and DCT nephron segments. These results demonstrate that PTH-sensitive [CAL+DCT] cells can be isolated in relatively high yield and viability and grown in cell culture. Primary cultures of these cells exhibit a phenotype appropriate to their site of origin in the nephron.

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Dartmouth Medical School, 03756, Hanover, New Hampshire

    J. H. Pizzonia, F. A. Gesek, S. M. Kennedy, B. A. Coutermarsh, B. J. Bacskai & P. A. Friedman

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  1. J. H. Pizzonia
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  2. F. A. Gesek
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  3. S. M. Kennedy
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  4. B. A. Coutermarsh
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  5. B. J. Bacskai
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  6. P. A. Friedman
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Additional information

Experimental work reported here was supported by grants from the National Institutes of Health, Bethesda, MD GM34399, American Heart Association (grant-in-aid 88-0721), and the Hitchcock Foundation. J. H. Pizzonia was supported by a Ford Foundation Fellowship and this work constitutes partial fulfillment of the requirements for a doctoral degree at Dartmouth College. B. J. Bacskai was supported by a Pharmaceutical Manufacturers Association Foundation Advanced Predoctoral Fellowship. P. A. Friedman was an Established Investigator of the American Heart Association during the tenure of these studies.

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Pizzonia, J.H., Gesek, F.A., Kennedy, S.M. et al. Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro Cell Dev Biol - Animal 27, 409–416 (1991). https://doi.org/10.1007/BF02630961

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