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*
Chair of Nephrology, Second University of Napoli, Italy
Institute of Cellular Signalling, University of Nijmegen, The
Netherlands
Institute of Physiology, Albert Ludwigs University, Freiburg,
Germany.
Correspondence to Dr. Giovambattista Capasso, Chair of Nephrology, Second University of Napoli, Policlinico Nuovo, Building 17 Via Pansini 5, 80131 Napoli, Italy. Phone: +39 081 566 6652; Fax: +39 081 566 6652; E-mail: gcapasso{at}unina.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have shown that chronic metabolic acidosis (CMA) induces an increase in urinary calcium excretion (22). The reasons for the hypercalciuric effect of CMA have not been elucidated. It has been hypothesized to be due to stimulated release of calcium salts from the bone to buffer the excess of hydrogen ions (23); in addition, it has been proposed that CMA directly inhibits distal tubule Ca2+ reabsorption (24).
Recently, using competitive reverse transcription (RT)-PCR, we have established a method that allows us to semiquantify the gene expression of selected proteins at the level of isolated and well-identified single tubule segments of the nephron. In this article, we report a detailed description of the technique used. In addition, we present data demonstrating that CMA is associated with a significant upregulation of calbindin D28k mRNA expression at the level of rat distal tubules and with a 17% increase of protein abundance as measured by Western blot experiments using calbindin D28k-specific antibodies. It is concluded that the hypercalciuric effect induced by ammonium chloride loading cannot be attributed to a defect in the efficiency of distal tubular calbindin D28k.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ketoglutaric acid, 1.3
Ca2+ -gluconate, and 14 NaHCO3, pH 7.4. Small pieces of
the renal cortex were cut under the stereomicroscope and incubated for 30 min
at 37°C in the microdissection solution containing 0.5 mg/ml collagenase,
continuously bubbled with 95% O2 + 5% CO2. After the
digestion, the tissue was washed with ice-cold, collagenase-free dissection
solution containing 1 g/L albumin. The identification and microdissection of
DT were done freehand under a stereo microscope (Wild M8, Heerbrugg,
Switzerland). DT were identified for their appearance among the superficial
tubules of the renal cortex. The use of collagenase eased the tubular
dissection, thus allowing us to harvest enough material (five to seven tubule
segments, corresponding to 1.5 to 3 mm tubule length) for the molecular
biology experiments.
Whole Kidney Clearance
In similar groups of animals, GFR and urinary calcium excretion were
measured. The animals received food and drinking water up to the time of the
study. They were anesthetized intraperitoneally with Inactin (Sigma Aldrich,
St. Louis, MO), using a dose of 120 mg/kg body wt, tracheostomized, placed on
a thermoregulated table (37°C), and prepared for renal clearance. In
brief, the right carotid artery was catheterized to record BP and take blood
samples for measurements of acid-base parameters, inulin, and calcium
concentrations. The left jugular vein was cannulated with polyethylene PE-50
tubing and used for intravenous infusion via a syringe pump (Braun, Melsungen,
Germany). Inulin (Inutest, Fresenius Pharma, Graz, Austria) was infused
through the jugular vein at a rate of 0.75 mg/min per 100 g body wt in
isotonic saline (6 to 8 µl/min per 100 g body wt). The surgical procedure
included also the bladder catheterization with PE-50 tubing. After a 60-min
equilibration period, the first of three 30-min urine collections began.
Arterial blood samples (100 µl) were taken at the start and end of each
collection period.
RNA Purification
Total RNA was purified from isolated distal tubules. After dissection, the
tubules were transferred into 350 µl of lysis buffer containing guanidinium
isothiocyanate and ß-mercaptoethanol. The lysate material was loaded on a
silica gel membrane (Qiagen, Chatsworth, CA) that binds specifically the RNA.
The membrane was washed three times, and the RNA was eluted in 50 µl of
diethylpyrocarbonate 1 g/L (DEPC-water). Possible contamination from genomic
DNA was removed by incubation with 5 U of ribonuclease-free deoxyribonuclease
I (Promega, Madison, WI), for 30 min at 37°C. The enzyme was inactivated
by heating at 70°C for 5 min; thereafter, a second purification run was
performed as detailed before. The concentration and purity of RNA was
determined by measuring its absorbance at 260 and 280 nm, using a GeneQuant
RNA/DNA calculator (Pharmacia Biotech, Freiburg, Germany).
Reverse Transcription
cDNA was synthesized starting from equal amounts of total RNA, using 200 U
of Moloney murine leukemia virus reverse transcriptase (Life Technologies,
Gaithersburg, MD), 0.5 µg of oligo(dT)12-18 (Life Technologies),
10 mM dithiothreitol, and 2.5 mM dNTP (Pharmacia) in a total volume of 20
µl. Before the addition of reverse transcriptase, the reaction mixture was
incubated at 65°C for 3 min to allow the primers to anneal to the poly(A)
tail of mRNA. cDNA was synthesized at 37°C for 1 h. Controls, incubated as
above, but without addition of reverse transcriptase, were included in each
run.
Polymerase Chain Reaction
PCR reactions were performed in a total volume of 50 µl in the presence
of 10 pmol of each oligonucleotide primer, 200 mM dNTP, 5 µl of 10 x
PCR buffer, 1.5 mM MgCl2, and 1.25 U Taq polymerase. PCR
was performed using the following primers: sense,
5'-GATGCCAGCAACTGAAGT-3'; antisense,
5'-GGCCTAAGCATAGACTTT-3'. The expected size of the PCR product was
732 bp. Samples were first denatured at 95°C for 3 min, and followed by 33
cycles consisting of denaturing at 95°C (1 min), annealing at 60°C (30
s), and extension at 72°C (1 min). After amplification, PCR products were
subjected to size separation by agarose gel electrophoresis (18 g/L).
Internal Standard Preparation
Total RNA (200 ng) purified from isolated DT was submitted to RT using the
oligo(dT)12-18 primer. Then the cDNA was amplified using the
following primers: sense, 5'-GATGCCAGCAACTGAAGT-3'; linker
antisense, 5'-GCATAGACTTTAATTCTCTATATGCAG-3'. The expected size
was 646 bp. PCR was performed as described previously, and the PCR product was
reamplified a second time using the normal antisense primer: sense,
5'-GATGCCAGCAACTGAAGT-3'; antisense,
5'-GGCCTAAGCATAGACTTT-3'. This product was analyzed by agarose gel
electrophoresis (18 g/L), and it was recovered from the gel using the agarose
gel DNA extraction kit (Boehringer Mannheim, Mannheim, Germany). The
concentration was determined by measuring the absorbance at 260 and 280
nm.
Quantification of Calbindin D28k mRNA
A competitive PCR was performed using as templates the internal standard of
646 bp and the cDNA obtained from 100 ng of total RNA, extracted from isolated
DT. The primers yielded a product of 732 and 646 bp for wild-type and internal
standard, respectively. Six to seven competitive PCR were performed by
addition of decreasing amounts (from 158.3 to 7.5 amol) of the competitive
template to replicate reactions containing identical amounts of DT cDNA. A
progressive decrease of the competitive template PCR product (646 bp)
corresponds to a progressive increase of the wild-type template PCR product
(732 bp). The PCR products were separated on 18 g/L agarose gel and stained by
ethidium bromide. The gel was photographed, and the quantification of the
fluorescence intensity of PCR products was performed using NIH Image 1.60
software. The amount of calbindin D28k mRNA was calculated using a log-log
scale plot of the ratio of PCR products versus the known amount of
internal standard used in the competitive PCR reactions. Fluorescence data
were multiplied by 732/646 to correct for the differences in molecular weight.
When the wild-type and competitive PCR products were equivalent, the amount of
wild-type present in the starting material was equal to the known starting
amount of internal standard. Results are expressed in attomoles (amol) of
calbindin D28k per nanogram (ng) of total RNA.
Western Blot Analysis
Small pieces of external cortex (80 to 100 mg) were frozen at -80°C and
then disrupted with a potter homogenizer at 4°C in 50 mM Tris-HCl buffer
containing (in µg/ml): 50 4-(2-aminoethyl)-benzene-sulfonyl fluoride, 2
leupeptin, and 2 aprotinin. After centrifugation at 10,000 x g
for 15 min at 4°C, the supernatant was collected and stored at -80°C
until use. Before electrophoresis, the samples were centrifuged again at
10,000 x g for 15 min at 4°C, and the protein concentration
of the supernatant was determined by Bradford assay. To evaluate the abundance
of calbindin D28k, 10 µg of proteins obtained from control and treated
groups were diluted in 5 x loading buffer (10 g/L sodium dodecyl sulfate
[SDS], 20% glycerol, 2% 2-mercaptoethanol, 10 mM Tris-HCl, pH 6.8), boiled for
5 min, and separated on two 10% SDS-polyacrylamide gel electrophoresis (PAGE).
After electrophoresis, the first gel was stained with Coomassie blue and the
second gel was submitted to electroblotting to transfer the proteins on a
polyvinylidene difluoride membrane that was washed with phosphate-buffered
saline. After the wash, the membrane was incubated with blocking buffer (2 g/L
high-purified casein, 1 g/L Tween 20 in phosphate-buffered saline) for 1 h and
then probed for 1 h with a rabbit polyclonal anti-calbindin D28k antibody
diluted 1:1000 in 10 ml of blocking buffer. Secondary goat anti-rabbit IgG +
IgM alkaline phosphatase conjugate was diluted 1:5000 in 5 ml of blocking
buffer and added for 1 h after wash two times in 20 ml of blocking buffer. The
last washing was performed three times with 20 ml of blocking buffer and
finally detection was obtained with a CSPD® chemiluminescent substrate
(Tropix, Bedford, MA). Calbindin D28k abundance in control and acidotic groups
was quantified by densitometric analysis.
Statistical Analyses
Inulin and total calcium concentrations in plasma and urine were measured
by the anthrone and colorimetric method (Diacron), respectively, while plasma
ionized calcium was detected by an ionized calcium analyzer (Nova 7). Arterial
blood pH, CO2, and calculated plasma bicarbonate were measured with
a blood-gas analyzer (ABL 300; Radiometer, Copenhagen, Denmark). GFR was
calculated using a standard inulin clearance equation, while fractional
calcium excretion (FECa), i.e., the urinary calcium
excretion expressed as % of filtered calcium, was calculated according to the
following formula:
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Distribution of D28k mRNA along the Nephron
Figure 1 shows the
distribution of calbindin D28k mRNA along the nephron (representative of three
experiments). It is clear that the mRNA is present only at the level of the
distal tubule, while it is absent from the glomerulus, the proximal tubule,
the thick ascending limb, and the medullary collecting duct. The identity of
PCR products was confirmed by: (1) the size of the PCR products,
separated by agarose gel electrophoresis and ethidium bromide staining; and
(2) their direct sequence. Moreover, since the distal tubule is a
very difficult segment to dissect, to be completely sure that we had isolated
distal tubules, we searched for the thiazide-sensitive NaCl cotransporter mRNA
(rTSC1) that has been demonstrated to be expressed only in this segment
(26). As illustrated in
Figure 2, the tubules that
expressed calbindin D28k mRNA showed the presence of rTSC1 mRNA. In the same
experiments, we also checked that the PCR products were not due to material
carryover from genomic DNA contamination. To this end, negative controls were
included in the RT reaction; no PCR products were obtained when reverse
transcriptase was omitted from the RT reaction
(Figure 2) (representative of
three experiments).
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Figure 3A shows the typical gel of competitive PCR (representative of five experiments). The addition of decreasing amounts of internal standard (646 bp) resulted in a corresponding increase of the wild-type template products (732 bp). Figure 3B shows the corresponding log-log plot. The amount of wild-type mRNA is calculated when the PCR products of internal standard and wild type are equated (ratio = 1). In Figure 3C, we have reported the results obtained by competitive RT-PCR starting from known amounts of total RNA. The data show that in the range of total RNA used, there was a highly significant linear correlation versus the mRNA for calbindin D28k.
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Absolute Quantification of Calbindin D28k mRNA
To examine whether calbindin D28k mRNA increases in chronic metabolic
acidosis under in vivo conditions, we measured calbindin D28k mRNA
expression by competitive RT-PCR in NH4Cl-treated and control rats.
The quantification was performed on a small amount (about 5 to 7 DT) of
starting material. The total RNA was purified simultaneously from acidotic and
control rats, and the competitive RT-PCR reactions were performed starting
from the same amounts of total RNA (about 100 ng). For each experiment, the
acidotic and control rats were studied in parallel. The results of the
competitive RT-PCR reactions are showed in
Figure 4. Calbindin D28k mRNA
abundance was 0.89 ± 0.21 amol/ng total RNA in DT of CMA rats
(n = 5) compared with 0.30 ± 0.12 amol/ng total RNA of control
rats (n = 5) (mean ± SEM) (P < 0.05).
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Calbindin D28k Protein Abundance as Measured by Western Blot
Calbindin D28k protein abundance was determined by Western blot analysis
using a rabbit polyclonal D28k antibody. In
Figure 5A, using slices of
renal outer cortex, it is shown that this antibody recognized a 28-kD protein
(compared with correspondent Coomassie blue-stained SDS-PAGE). Results
obtained by densitometric analysis demonstrated that in control animals,
calbindin D28k abundance was 2416 ± 166 (integrated optical density
units) (n = 7) in controls, while it increased (17 ± 5%) to
2823 ± 254 (n = 7) (P < 0.05) in acidotic rats
(Figure 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Competitive RT-PCR Technique
PCR is a very useful technique to examine renal tubular function. Because
of its incredible sensitivity, it is the ideal method to detect nucleic acids
from few cells. This property, combined with the tubular microdissection,
allows the detection of the distribution of the specific RNA along a very
heterogeneous structure such as the nephron. However, two main problems are
associated with the use of this technique: the poor reproducibility and the
intrinsic difficulty to obtain quantification data of gene modulation. The
first complication is clearly related to the method itself: The final yield of
a PCR reaction is connected to the exponential amplification of starting
template; small variance in the amplification effectiveness will result in a
massive difference of the final product, particularly if the quantity of the
starting material is very low. On the other hand, to obtain data on the
abundance of selected RNA, most authors have used other RNA as a standard. The
intensity of the gene expression is measured as a relative ratio factored by
the expression of another gene that is supposed to be unaffected by the
experimental maneuver. ß-actin and GAPDH have been commonly used for this
purpose. This method is clearly flawed by the assumption that the expression
of the reference gene is not altered. A way to overcome these two limitations
of PCR technique is to coamplify, in the same test tube, a reference template
(internal standard) that will differ from the wild-type template by a short
deletion, but shares the same primer sites. Under these conditions, the two
templates will compete for the same primers and will amplify at the same rate
(competitive PCR). The two amplified products can be distinguished because of
their different lengths. This method has been introduced for the first time by
Diviacco et al. (32),
and it has been used by other investigators to quantify the gene expression of
other renal transport proteins
(25). In the present article,
we have used this approach to quantify the mRNA abundance of calbindin D28k
starting from well-identified cortical distal tubules.
Figure 3A shows that the
addition of decreasing amount of internal standard to fixed amount cDNA wild
type leads to a progressive decrease of the competitive template PCR product
(646 bp) and to a progressive increase of the wild-type template PCR product
(732 bp). The ratio between the final amplification products for the two
templates for each point displays a linear relationship
(Figure 3B), allowing the
measurements of unknown calbindin D28k mRNA abundance.
Metabolic Acidosis-Induced Hypercalciuria and Calbindin D28k
Expression
The clearance studies (Table
1) clearly show that after ammonium chloride loading, the
fractional calcium excretion is increased. These results may indicate that at
some point along the nephron, Ca2+ transport is inhibited by
metabolic acidosis. However, the tubular segment and the mechanism(s)
responsible for the hypercalciuric effect of metabolic acidosis have not yet
been identified.
Several authors have shown that metabolic acidosis alters vitamin D metabolism. Although in humans ammonium chloride loading has been associated with an increase in the serum concentration of 1,25-(OH)2-D3 (33), in the rat it has been repeatedly demonstrated that CMA reduces circulating 1,25-(OH)2-D3 levels by inhibiting renal proximal tubule 25-hydroxyvitamin D3—1-hydroxylase (1-OHase) (34), the key enzyme responsible for the conversion of 25-(OH)-D3 to 1,25-(OH)2-D3, and by enhancing the activity of the renal 24-hydroxylase (35), the enzyme partially responsible for the degradation of 1,25-(OH)2-D3. Therefore, it is clear that the enhancement of calbindin D28k mRNA, described herein, cannot be attributed to a vitamin D3 effect, since there is general agreement that in the rat this substance is decreased during CMA.
Another consequence of acidosis is an increase in ionized calcium probably related to the lower blood pH. Our findings are in close agreement with those of Cunningham et al. (35), showing that after 6 d of NH4Cl administration, while total calcium was comparable in control and acidotic rats (as in our present experiments), there was a significant increase in plasma ionized calcium. This finding has been confirmed by other authors (7,36). The increase in plasma ionized calcium may be responsible for the altered vitamin D metabolism. Indeed, when it was blocked with ethyleneglycol-bis(ß-aminoethyl ether)-N,N'-tetra-acetic acid, a calcium chelator, the effect of metabolic acidosis on vitamin D metabolism disappeared (36). Our present results, showing a significant increase in mRNA for calbindin and, to a lesser extent, in calbindin D28k protein abundance, indicate that this protein may be involved in the effort to compensate the concomitant hypercalciuria. It is therefore tempting to speculate that the increase in filtered load of ionized calcium may enhance the distal calcium load, leading to an increased calcium flux through the luminal membrane, and thus stimulating calbindin D28k synthesis. This hypothesis is very attractive and suggests that luminal Ca2+ activity may be responsible for the stimulation of the calbindin gene. In all cases, it is evident that the hypercalciuria induced by ammonium chloride loading cannot be explained by a downregulation of calbindin D28k along the distal tubule, since we have clearly shown that under this condition, calbindin D28k expression is upregulated both at the mRNA and protein level. From a clinical point of view, this effect will help to reduce the hypercalciuria generated by metabolic acidosis, thus mitigating the consequence of this acid-base disorder on calcium metabolism.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase in the rat kidney. J Clin Invest
70: 135
-140, 1982
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