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Calcium Exchange Rates in Rat Kidney Tubule Cells Affected by Insulin and pH

B.W. Brazier

Bendigo Kangan Institute, Bendigo, Victoria, Australia

Abstract: Calcium exchange rates in rat kidney tubule are measured by measuring the loss of Ca⁴⁵ isotopes from tubule cells when the tubules are placed in Ca⁴⁵ free media that was adjusted to different pH sand when different additives of sulphate, acetate, glutamate and /or insulin were included. The exchange rates showed decrease with low pH and glutamine but increased with addition of acetate and insulin. The effect of insulin is considered in relation to its observed effect on protein induced postprandial calciuria.

Keywords: hypercalciuria, insulin, calcium exchange rates, gluconeogenesis, ammoniogenisis

Citation: B.W. Brazier, Calcium Exchange Rates in Rat Kidney Tubule Cells Affected by Insulin and pH. American Journal of Food and Nutrition, 4 (5). 115-126. January 2016. doi:10.12691/ajfn-4-5-1

1. Introduction

In a previous experiment at this laboratory plasma insulin has been linked with dietary protein induced hypercalciuria [1] Brazier (2016a). The postprandial calciuria was observed to show considerable variation between the test subjects. This variation appeared to correlate with plasma insulin levels. In another experiment [2] Brazier (2016b) where dietary fat was tested for variation in calciuria the main correlation was found to be between insulinemia and calciuria. This was also observed by [3] Allen et. al. (1981) and [4] Wood and Allen (1983) and [5] Howe (1990) who indicate that insulin may be involved in increasing calciuria. However in the experiments looking at the effect of dietary protein,the indication wasthat plasma insulin reduced calciuria. Another study by [6] Brazier (2016c) showed that plasma insulin had an inverse relationship with calciuria. The effect of insulin could be because it causes increased protein anabolism thus reducing sulphate production and less acid excretion. The reduced calciuria could also be the result ofinterplay between insulin and the renin-angiotensin system or a direct effect of insulin on calcium transports the kidney tubules.

Urinary sulphate has been implicated in reduced calcium reabsorption [7] Wachman and Bernstein (1960) and reports have indicated a correlation between the sulphur content of dietary protein and the increase in excreted calcium [8] Walser and Browden, (1958, [9] Whiting and Draper, 1981, [10] Zemel et. al. 1981). Whether sulphate had a direct effect on reabsorption or if the effect was a result of reduced pH was not determined. However pH has been shown to significantly reduce membrane transport of calcium in kidney tubules [11] Lemann et. al., (1961), and [12] Studer and Borle, (1979). When protein derived sulphate reduces the pH of urine the tubules are stimulated to produce ammonia to neutralise the acid. This experiment will test the hypothesis that this ammoniogenesis uses mainly glutamine for deamination thereby producing α-ketogluterate as a by-product. The α-ketogluterate is then usually removed by gluconeogenesis. The gluconeogenesis then uses up ATP making less ATP available for Ca++ absorption. If the cells are exposed to insulin the gluconeogenesis is inhibited and the α-ketogluterate is redirected into the citric acid cycle and extra ATP is produced. This would allow an increase in Ca++ absorption. Thus insulin would have a direct effect of increasing calcium reabsorption thus causing less calciuria.

The involvement of insulin and calciuria may be significant as indicated by several report of the difference in occurrence of osteoporosis in subjects with type I and type II diabetes mellitus. E.g., [13] Leidid-Buckner and Ziegler (2001). They report that people with type I diabetes exhibit low bone density (i.e. less calcium) and people with type II diabetes have normal or greater bone density (i.e. more calcium). [14] Osteoporosis Australia (2014) suggest that although people with type II diabetes are more likely to have bone fractures than normal people this is probably due to increased falls and inactivity even though they have normal bone density. The observations by Brazier [1] and [2] that some young health individuals with no apparent pre-diabetes had exaggerated insulin responses. If that insulin influenced calciuria it could be a contributor to idiopathic hypercalciuria and nephrolithiasis. [21] Worcester (2008) This experiment attempts to examine the possibility that insulin effects fraction reabsorption by looking at a series of Ca⁴⁵ isotope exchange measurements using desaturation analysis to determine if the gluconeogenesis stimulated by reduced pH is the causative factor in reduction of calcium reabsorption and to see if insulin has a direct action on this process. The mechanism of the effect of pH on calcium membrane transfer rates has been considered by [11] Lemann (1967) who suggested that metabolic acidosis has a direct effect on the metabolism of the renal tubular cells.

To test the above hypothesis the effect of various glutamine levels are tested to determine if the effect of pH is due to the ATP drain involved in ammoniogenesis /gluconeogenesis. This was described in a similar manner by [15] Silva et. al. (1980) who showed that sodium transport was inhibition by ammoniogenesis linked gluconeogenesis. The effect of a combination of insulin and glutamine is also compared with control tubules to see if insulin directly effecting renal calcium metabolism. Although [16] Janda (1969) observed increased calcium uptake when insulin was added to kidney slices, the use of isotope exchange studies here allow measurement of transmembrane movement under steady state conditions which should be a better measure of calcium reabsorption than calcium uptake into tissue slices as done by Janda [16]. This is because tissue uptake does not differentiate between throughput and intercellular accumulations.

Kidney tubules are sometime reported as being insulin insensitive because they have a fast acting enzyme to degrade insulin. But [17] Mahler and Szabo (1967) have shown that insulin insensitive tissues can react to insulin if insulin degradation mechanisms are impaired. The method used in these experiments involved transferring tubules cells to fresh media every ten minutes so as to be similar to the continuous flow of in vivo conditions and thereby minimise the effect of insulin enzymic degradation.

2. Method

The exchange rate between the cytosol of isolated kidney tubules and incubation medium were calculated from Ca⁴⁵ desaturation curves after the method of [18] Uchikava and Borle (1978). The kidneys from two 150-200 g male Sprague-Dawley rats were buttered through a 170-mesh sieve in 0.15 M Saline at 5°C and the mixture filtered through an 80-mesh sieve as described by [19] Price (1979). The homogenate was centrifuged at 20 g at 5 °C for 1 minute and the supernatant discarded. The tubules were washed four times in phosphate buffer then divided into 2 cm³ aliquots to provide about 2 mg protein per ml. The tubules were allowed to equilibrate in incubation media for one hour at 37 ° C before labelling for 60 minutes with 1 μCu, Ca⁴⁵cm³. The cells were washed then suspended in non- radioactive media for 5 minutes then centrifuged and suspended in fresh media each 10 minutes for 250 minutes. Each supernatant collected was retained and the fluxed radioactivity measured using a refrigerated scintillation counter and PPO toluene scintillant as described by [20] Borle (1975). The specific activity of the tubule cells initially and after each washing was calculated by the method of Borle (1975) to produce the radioactive desaturation curves similar shown in Figure 4A to 4E.

For each experiment media was prepared and refrigerated the day before. On the day of each experiment, rats were killed at about 0800 hrs. It took two hours to isolate the tubules then the process involving saturation with Ca⁴⁵ and then the subsequent desaturation involved continuous activity as each test tube had to be handled separately as media was transferred into the scintillant. The desaturation stage was finished by 1700 hrs. The scintillation counting was then done overnight.

The method of analysis used by [21] Uchikawa and Borle (1978) involved plotting the percentage Ca⁴⁵ remaining in the sells against time and drawing by hand a line of best fit. Values of cell content and time were then read off at 10 minute intervals and used for the mathematical analysis of the curves.

The assumption was made that the calcium pool sizes are: S1 indicates the interstitial space, S2 indicates the cytosol and S3 the mitochondria, and P20 and P30 indicate exchange between medium and compartments S2 and S3 respectively. S2, S3 P20 and P30 are calculated using the formulae used by [21] Uchikawa and Borle (1978) and using the desaturation equations:

[picture] | (1)

[picture] | (2)

[picture] | (3)

Figure 1. Step one in determining the curvilinear line of best for the multi term exponential curve for the experimental data set

Analysis of each desaturation curve involved three stages. First a line of best fit was drawn using the last few points on the graph. The gradient of this line of best fit was used to calculate the P10 from the third term of equation (1) as in Figure 1 and using the formulae in Table 3. A second curve is then produced by subtracting the third term from the equation. The last few points on this second curve was used to produce a new line of best fit and its gradient used to calculate P20as in Figure 2. This value was used to remove the second term from Equation (2) above to produce a curve of equation(3) A third line of best fit was then used to calculate P30 as per Figure 3. As this method allows some human interpretation of the original data in drawing each line of best fit by hand, in this experiment the raw data was used each time by a computer to calculate a least squares line of best fit. After obtaining values for P01, P02 and P03 a new theoretical cure was produced was produced. The goodness of fit of the theoretical curve to the original experimental results was then determined by the computer programed using a Chi Square test. If the Chi Square value was less 10, the choice of points used to determine the three components of the curves were varied and a new theoretical curve produced and tested with Chi Squared. This was repeated over and over until a resulting theoretical curve was produced that had a close fit with the original data.I.e. with a Chi Squared value of less than 10. The values of P20 were then used to indicate the Ca⁴⁵ exchange rate from the cytosol to the medium for each set of test conditions.

Figure 2. Step two in determining the curvilinear line of best for the multi term exponential curve for the experimental data set

Figure 3. Step three in determining the curvilinear line of best for the multi term exponential curve for the experimental data set

The media used for each experiment are shown in Table 1. In each set of experiments two media variations were compared against each other using kidney tubules from the same batch of homogenate. For each pair of media the experiment was repeated six times. In each experiment there were three 4 ml aliquots of media/cell preparation for each media type. Cells and media were incubated in 10 ml centrifuge tubes while bubbling oxygen through the media from 1 mm diameter tubes to provide aeration and agitation. The significance of the differences between pairs of media tested was determined by the paired student t- test.

Table 1. Media Used in Calcium Desaturation Experiment

Table 2. Sets of Media used in Paired Desaturation Experiments

2.1. Cell Potassium and Cell Viability

Atomic absorption was used to measure the cell potassium and calcium content of tubules before and after desaturation experiments

3. Results

3.1. Exchange Rates

The rates of exchange of calcium between the incubation medium and the metabolic compartments and the size of the calcium pools in each compartment is shown in Tables 4 A to E for each pair of media used. The assumption was made that the calcium pool sizes are: S1 indicates the interstitial space, S2 indicates the cytosol and S3 the mitochondria, and P20 and P30 indicate exchange between medium and compartments S2 and S3 respectively. S2, S3 P20 and P30 are calculated using the formula used by [20] Borle (1975).

3.2. Calculations

Calculation of P01, P02 and P03 were done using the results from the graphical analysis and the formulae in Table 3.

Table 3. Solutions for calculation of calcium fluxes (P10,P20 and P30) and pool sises (S1,S2 and S3) And rate cosnstants from Ca⁴⁵ desaturation curves

Example desaturation curves for pairs of media are shown in as Figure 4Aa and Figure 4Da. The Curves for different pH, different sulphate levels, and different glutamine levels looked similar to the pH graphs shown in Figure 4Aa. The graphs regarding different Insulin levels and acetate levels show significant differences for high and low levels and look similar to the insulin graphs shown in Figure 4Da.

Each of the experiments in Table 2 were repeated 6 times for each of the sets of media. Desaturation curves for the six repeated experiments for each pair of media are averaged to give a pairs of graphs similar to those shown in Figure 4Aa to 4Ea. These averaged results do not show significant differences when the standard errors are compared for each time period. However when the exchanged rates are calculated for each experiment separately and the results compared using the t-test, significant difference are observed for all pairs of media except high and low sulphate. Assuming a steady state of efflux equals influx the low pH medium showed a significant decrease (P>O.1) in calcium exchange (P20) rate indicating less tubular calcium exchange with the medium compared to high pH medium (Table 4A and Figure 4A). With the P20 in Table 4C the inclusion of glutamine with the low pH medium decreased the calcium exchange rate even more, and the addition of acetate produced a greater exchange rate revering the effect of low pH and made equal to the pH 7.2 medium,as shown in Table 4E. The presence of insulin with the glutamine/pH 6.2 also increased the cytosol/medium exchange rate as shown in Table 4D. However, the different rates of cytosol/medium exchange for the high and low sulphate experiment were not significant (Table 4B). The paired student t-test test was used to determine the significance of the difference between the groups of results for the P20 exchange rates.

The inclusion of glutamine in the low pH medium reduced the calcium exchange rates further but not as much as the difference between high and low pH. Glutamine was included in the media (pH 6.2) used for the insulin experiment (Table 4D) and it can be observed that insulin masks the effect of glutamine.

4. Potassium and Total Cell Calcium

Measurements of cellular potassium and calcium show no loss of potassium of total calcium from cells.

Table 4A/1. Intracellular Calcium Pool Sizes and Exchange Rates in Renal Tubules Incubated in Media with pH6.2 or with pH 7.2

Table 4A/2. Difference in Paired Calcium Exchange Rates (P20) for Media with pH6.2 and pH 7.2

Figure 4A.

Table 4 B/1. Paired Results from Analysis of Ca⁴⁵ Desaturation Curves When Renal Tubules are incubated in Media with High or Low Sulphate

Table 4B/2. Differences in Paired Calcium Exchange Rates (P20) Between Tubules Incubated in High or Low Sulphate Media

Figure 4B. Average desaturation curves for media with high sulpahrte and low sulphate

Table 4C/l. Paired Results from Analysis of Ca⁴⁵ Desaturation Curves When Renal Tubules are incubated in Media With or Without Glutamine

Table 4C/2. Differences in Paired Calcium Exchange Rates (P20) Between Tubules Incubated in Media With or Without Glutamine

Figure 4C. Average Desaturation Curves for media with or without Glutamine

Table 4D/1. Paired Results from Analysis of Ca⁴⁵ Desaturation Curves When Renal Tubules are incubated in Media With or Without Insulin

Table 4D/2. Differences in Paired Calcium Exchange Rates (P20)Between Tubules Incubated in Media With or Without Insulin

Figure 4D. Average Desaturation Curves for media with or without Insulin

Table 4E/1. Intracellular Calcium Pool Sizes and Exchange Rates in Renal Tubules Incubated in Media With or Without Acetate

Table 4E/2. Differences in Paired Calcium Exchange Rates (P20) Between Tubules Incubated in Media With or Without Acetate

Figure 4E. Average Desaturation Curves for media with or without Acetate

The changes in Ca⁴⁵ exchange rate indicate either increase or decrease over results for the previous results of each set of media conditions. These changes are indicated in Table 5 and Figure 5.

Table 5. Percentage change of Ca⁴⁵ exchange rates

Figure 5. Percentage change of Ca⁴⁵ exchange rates

5. Discussion

The results of experiment 4A comparing high pH with low pH (Table 4A) indicate that reduced pH has a pronounced effect on the plasma membrane transport of calcium. Experiment Bshowed that the presence of sulphate had no significant effect (Table 4B). It has been suggested that metabolic acid affects calcium transport by changing the transferase stereochemistry, [12] Studer and Borle (1979) and it has also been suggested that sulphate may inhibit Calcium uptake by complexing with the Ca2+ ions [8] Wlaser and Browden (1958), the latter doesnot seem to occur in this study. Metabolic sulphate probably has it effect by increasing plasma acid concentration and it is the pH drop rather than the sulphate that causes the reduced calcium exchange. However the changes caused by inclusion of glutamine (Table 4C) and acetate (Table 5E) in the incubation media indicates that the reduction in calcium absorption is caused by a metabolic change in thetubule cells rather than physical changes caused by sulphate or H+. Inclusion of glutamine probably causes increased ammoniogenesis, reduced renal pH also produces increased ammoniogenesis as referred to by [23] Brosnan (1978). However the mechanism by which ammonia production causes the change in Ca exchange is not yet established. Although [24] Nissin et. al (1991) and [25] Nissim et. al. (1990) have suggested that intracellular acidity stimulates ammoniogenesis by inhibiting citrate synthetase and stimulation of glutamase. The indications in this experiment are that glutamine reduced Ca absorption and that insulin increases calcium absorption the effect of glutamine may have been because of more ammoniogenesis connected to gluconeogenesis thereby reducing the availability of ATP for calcium uptake. The observation that insulin greatly increased Ca exchange can be explained by insulin inhibition gluconeogenesis and making ATP more ready available. But both effects connected to ammoniogenesis. These possibilities are considered in a future experiment using larger quantities of material so that ammonia production can be measured in single time point analysis of cellular calcium efflux.

The observation that acetate with insulin increased calcium exchange rate even more that insulin alone could be due to either more energy being made availability through the citric acid cycle or due to an inhibition of ammoniogenesis as described by [26] Tannen (1978) or both. Measurements of intra cellular potassium indicate that the cells remained viable throughout the experiment. The greatly reduced exchange rate of calcium by the tubules incubated at 30Cindicate that the process is an active biological process and not just diffusion.

References

[1]   | Brazier BW (2016a)Hypercalciuriaeffect of high protein diets, American Journal Nutrition and Food Science, Vol. 4, No. 1, 20-29.
[2]   | Brazier BW (2016b) ‘Effect of the level of dietary fat and fat type on postprandial calciuria and of insulin’ American Journal Nutrition and Food Science’, Vol. 4, No. 2, 55-62
[3]   | Allen, L.H., Block, G.D., Wood, Rl. and Bryce, G.F. (1981), 'The role of insulin and parathyroid hormone in the protein induced calciuria of man', Nut.Res., 1981, 1: 3-11.
[4]   | Wood, RJ. and Allen, L.H. (1983) 'Evidence for insulin involvement in arginine and glucose- induced hypercalciuria in the rat', J.Nutr., 113(8): 1561-67.
[5]   | Howe, J.C. (1990) 'Postprandial response of calcium metabolism in postmenopausal women to meals varying in protein level/source', Metabolism, 39(12): 1246-52.
[6]   | Brazier BW. (2016).’Effect of serum insulin on calciuria following protein meals in humansAmerican Journal Nutrition and Food Science. 2016, Vol. 4, No. 3, 63-67.
[7]   | Wachman, A and Bernstein, D.S. (1960) 'Diet and osteoporosis', Lancet, 1: 958-59.
[8]   | Walser, M. and Browden, A (1959) 'Ion association II the effect of sulfate infusion on calcium excretion', J.Clin.Invest., 38: 1404-10
[9]   | Whiting, S.I.and Draper, H.H. (1981) 'Diet acidity and bone composition', J.Nut., 111: 1720- 26.
[10]   | Zemel, M.B., Schuette, S.A, Hegsted, M. and Linkswiler, H.M. (1981) 'Role of the sulfurcontaining amino acids in protein-induced hypercalciuria in men', J.Nut., 111(3): 545-52.
[11]   | Lemann, J., Litzow, J.R and Lennon, E.J. (1967) The effect of treatment of acidosis on calcium balance in patients with chronic azotemic renal disease, In.Clin.Invest., 46(2) 280-6.
[12]   | Studer, RK. and Borle, AB. (1979) 'Effect of pH on the calcium metabolism of isolated rat kidney cells', J.Membrane BioI., 48(4): 325-4.
[13]   | Leidid-Buckne,G and Ziegler, R (2001) ‘Diabetes mellitus a risk for osteoporosis?’. Experimental and Clinical Endocrinology & Diabetes 109 (suppl 2): 493-514.
[14]   | Osteoporosis Australia (2014),’Diabetes and osteoporosis Consumer Factsheet’, 1st Edition 08/14.
[15]   | Silva, P., Ross, B. and Spokes, K. (1980) 'Competition between sodium reabsorption and gluconeogensis in kidneys of steroid-treated rats', Am.J.Physiol., 238(4): F290-5.
[16]   | Janda, s. (1969) 'Mechanism of calcium transport in kidney cortex slices', Physiologia Bohemoslovaca, 18(5): 413-23.
[17]   | Mahler, RJ. andSzabo, O. (1967) 'Insulin action upon insulin-insensitive tissue following impaired degradation', Proc.Soc.Exp.Biol.Med., 125: 879-82.
[18]   | Uchikawa, T.and Borle, AB. (1978) 'Kinetic analysis of calcium desaturation curves from isolated kidney cells.', Am.J.Physiol., 234(1): R29-R338.
[19]   | Price, RG. (1979) Isolation of Kidney glomerculi and tubular fragments and cell populations, In: Reid, E., Cell Populations Methodological Surveys (B) Biochemistry: Vo1.9, Ellis Horwood, Chichester.
[20]   | Borle, AB. (1975) 'Methods of Assessing Hormone Effects on Calcium Fluxes in vitro, in Methods' in Enzymology, Hormone Action, Part D, J.G. Hardman and B.W. O'Malley (eds.) New York, Academic Press, vo1.39, pp.513-573.
[21]   | Elaine M. Worcester, and Fredric L. Coe, (2008),‘New Insights into the Pathogenesis of Idiopathic Hypercalciuria’. SeminNephrol. Mar; 28(2): 120-132
[22]   | Brazier BW, (2016c) ‘Calcium exchange rate data’ http://barry-bwb2.tripod.com/calcium_exchange.html
[23]   | Brosnan, J.T., McPhee, P., Hall, B. and Parry, D.M. (1978) 'Renal glutamine metabolism in rats fed high protein diets', Am.J.Phvsiol., 235 (3), pp.E261-5.
[24]   | Nissim, 1., Nissim, I. and Yudkoff, M. (1991) 'Adaptation of renal tricarboxylic acid cycle metabolism to various acid-base states: study with [3-13C, 5-15N] glutamine', Miner- Electrolvte-Metab., 17(1): 21-31.
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