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Regulation of magnesium transport in the renal distal convoluted tubule

University of British Columbia

1. Hormonal regulation of magnesium uptake in mouse distal convoluted tubule cells - modulation by the Mg²⁺/Ca²⁺ sensing receptor. The distal convoluted tubule plays a significant role in renal magnesium conservation. An immortalized mouse distal convoluted tubule cell line (MDCT) has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. Various hormones are known to act on the kidney to conserve magnesium. We studied the cellular action of parathyroid hormone (PTH), insulin, vitamin D 3 and aldosterone on Mg²⁺ uptake into MDCT. MDCT cells have been shown to possess an extracellular polyvalent cation-sensing mechanism responsive to Mg²⁺, Ca²⁺, and neomycin. We determined the effect of cation-sensing on Mg²⁺ uptake into MDCT cells. PTH increased cellular cAMP concentrations by about 2 fold above basal levels in normal and Mg²⁺-depleted cells. Intracellular free Mg²⁺ concentration ([Mg²⁺]i) was determined on single MDCT cells using rnicrofluorescence with mag-fura 2. To assess Mg²⁺ uptake, MDCT cells were first Mg²⁺ depleted (0.22 mM) by culturing in Mg²⁺-free media overnight and then placed in 1.5 mM MgCl2 , and the [Mg²⁺]i was determined. [Mg²⁺]i returned to basal levels, 0.53 mM, with a mean refill rate, d([Mg²⁺ ]i)/dt of 164±5 nM/s. PTH added 5min prior to addition of 1.5 mM MgCl₂ increased the refill rate to 205+5 nM/s. PTH stimulation of Mg²⁺ uptake was not dependent on protein synthesis. Protein kinase A, protein kinase C and phospholipase C inhibition prevented PTH stimulation of Mg²⁺ uptake. Activation of the extracellular cation sensor inhibited both PTH-stimulated cAMP generation and Mg²⁺ uptake. The distal convoluted tubule possesses insulin receptors, although it is unclear what action insulin has in the distal tubule. Insulin stimulated Mg²⁺ entry in a concentration-dependent manner with maximal response of 214±12 nM/s. This was associated with a 2.5-fold increase in insulin-mediated cAMP generation (52±3 pmol/mg protein 5min). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg²⁺ uptake (169+11 nM/s). Insulin and PTH increase Mg²⁺ uptake in an additive fashion. Activation of the extracellular divalent cation sensing mechanism did not significantly change insulin stimulated Mg²⁺ uptake although it did inhibit insulin-stimulated cAMP generation. The cells of the distal convoluted tubule possess the vitamin D receptor. Treatment of MDCT cells with l,25(OH)2D3 for 16-24 h stimulated basal Mg²⁺ uptake in a concentrationdependent manner from basal levels of 164±5 nM/s to 210±11 nM/s, representing a 28±3 % change. Acute l,25(OH)2D3 treatment, for 20 min, was without effect. Pretreatment with actinomycin D or cycloheximide, transcriptional and protein, synthesis inhibitors, respectively, abolished l,25(OH)2D3-stimulated Mg²⁺ uptake, 154±18 nM/s, in Mg²⁺-depleted cells. This suggests that l,25(OH)2D3 stimulates Mg²⁺ transport through gene activation and protein synthesis. Elevation of extracellular Ca²⁺+ inhibited l,25(OH)2D3-stimulated Mg²⁺ uptake, 143±5 nM/s. Preincubation with a specific antibody to the Casr prevented the inhibition by elevated extracellular Ca²⁺ of l,25(OH)2D3-stimulated Mg²⁺ uptake, 202±8 nM/s. Blockade of Casr mRNA translation with an antisense oligodeoxynucleotide also mitigated the effects of extracellular Ca²⁺ on l,25(OH)2D3-responsive Mg²⁺ entry. Pretreatment of MDCT cells with U-73122, a phospholipase C inhibitor, also abolished inhibition of l,25(OH)2D3-stimulated Mg2 + entry, 202±5 nM/s. It is unclear what role aldosterone has on renal regulation of magnesium reabsorption. Incubation of MDCT cells with aldosterone for 16h did not change the rate of Mg²⁺ uptake (172±8 nM/s). However, aldosterone potentiated PTH-stimulated Mg²⁺ entry to 250±11 nM/s. Aldosterone also potentiated cAMP accumulation in a concentration-dependent manner. As cAMP stimulates Mg²⁺ entry in MDCT cells, it is inferred that aldosterone may stimulate Mg²⁺ uptake through signaling pathways involving cAMP. The actions of aldosterone were dependent on de novo protein synthesis, as pretreatment of the cells with cycloheximide inhibited aldosterone-potentiation of PTH stimulation of Mg²⁺ uptake. Elevation of extracellular Ca²⁺ inhibited aldosterone-potentiation of PTH-stimulated Mg²⁺ uptake, suggesting that divalent cations may inhibit the transcription processes activated by aldosterone. These studies suggest that Mg²⁺ reabsorption is under the control of a number of different hormones which interact together along with the extracellular divalent cation concentration to determine the final Mg²⁺ reabsorption rate. 2. A novel gene upregulated by magnesium depletion. Magnesium transport in epithelial cells is, in part, under gene control. Madin-Darby canine kidney (MDCK) cells upregulate magnesium transport when cultured in low magnesium media. Transcription and translation inhibitors block the upregulation of Mg²⁺ transport. PCR-differential display was used to identify one differentially expressed gene (termed the magnesium response gene, MRG), which was upregulated by Mg²⁺ depletion. Upregulation was evident after 30 min and maximum (2 fold increase) after 4 h. Re-addition of Mg²⁺ to Mg²⁺-depleted cells resulted in the return of MRG levels to control levels within 4h. MRG upregulation was specific to Mg²⁺ depletion as Ca²⁺ , K⁺ , and phosphate depletion of MDCK cells failed to upregulate the MRG. However, phosphate depletion with Mg²⁺ depletion diminished Mg²⁺ transport and inhibited the upregulation of the MRG. Five exons have been identified, which may be alternatively spliced. Two antisense oligomers complementary to the 3' and 5' end respectively inhibited Mg²⁺ entry into Mg²⁺ - depleted cells. Just 5' to the MRG mRNA sequence is a serum response element. Serum deprivation of MDCK cells blocks the upregulation of both the MRG and Mg²⁺ uptake. A human and mouse gene were identified by library screening and database searches that have similar genomic structure and sequence homology to the MDCK sequence. The mouse MRG was upregulated in MDCT cells by Mg²⁺ depletion. None of the sequences possess open reading frames suggesting that the MRG may be a noncoding mRNA. The results suggest that the MRG must be expressed for upregulation of Mg²⁺ transport in response to Mg²⁺ depletion.

Thesis/Dissertation

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2009-10-09

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http://hdl.handle.net/2429/13813

Experimental Medicine

University of British Columbia

UBCV

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