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. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: J Biochem Mol Toxicol. 2022 Apr 10;36(7):e23068. doi: 10.1002/jbt.23068

Regulation of Renal Calbindin Expression During Cisplatin-Induced Kidney Injury

Blessy George 1,*, John T Szilagyi 1,**, Melanie S Joy 2,3,4, Lauren M Aleksunes 1,5
PMCID: PMC9296602  NIHMSID: NIHMS1793909  PMID: 35403300

Abstract

Since the discovery of calbindin release into urine during renal injury, there has been growing interest in the utility of this protein as a biomarker of nephrotoxicity. However, little is known about the intrarenal regulation of the release and expression of this calcium-regulating protein during kidney injury. We sought to characterize the time-dependent expression and excretion of the protein calbindin in the distal tubule in comparison to kidney injury molecule-1 (Kim-1), a protein in the proximal tubule, in mice treated with cisplatin. Urine, blood, and kidneys were collected from male C57BL/6 mice treated with vehicle or cisplatin (20 mg/kg, i.p.). Urinary concentrations of calbindin and Kim-1 were elevated by 11.6-fold and 2.5-fold, respectively, within 2 days after cisplatin. Circulating creatinine and blood urea nitrogen levels increased in cisplatin-treated mice by 3 days, confirming the development of acute kidney injury. Time-dependent decreases in intrarenal calbindin protein were observed on days 3 and 4 and a 200-fold up-regulation of calbindin (CALB1) and KIM-1 mRNAs was observed on day 3. These data suggest that early loss of calbindin protein into the urine along with declines in renal calbindin levels initiates a compensatory induction of mRNA expression at later time points (days 3 and 4). Understanding the regulation of calbindin during cisplatin nephrotoxicity further enhances its utility as a potential urinary biomarker of kidney damage. The results of the current study support the combined use of a proximal (Kim-1) and distal tubule (calbindin) marker to phenotype acute kidney injury secondary to cisplatin administration.

Keywords: calbindin, cisplatin, kidney injury molecule, nephrotoxicity, acute kidney injury

Introduction

Calbindin is a 28 kDa cytosolic calcium-binding protein that is also known as calbindin-D28k. Calbindin protein is enriched in the kidneys, intestines, brain, and pancreas (1). Within the kidneys, calbindin is expressed in the distal tubules and early portions of the collecting ducts in rodents, canines, sheep, and humans (2,3). The primary function of this protein is to regulate calcium homeostasis. Calbindin has four affinity sites for the binding and sequestration of free calcium ions (4,5). In response to calcium binding, calbindin undergoes conformational changes that lead to interaction with various signaling proteins including myo-inositol monophosphatase-1 and caspase-3 (6-8). Calbindin knockout mice fed a high calcium diet exhibit a 2- to 3-fold increase in urinary calcium levels without significant changes in serum calcium concentrations (1), revealing an in vivo interplay between calcium and calbindin in the kidneys.

Since its discovery, there has been interest in identifying the physiological and pathophysiological mechanisms that regulate calbindin expression. Calbindin was initially identified as a vitamin D responsive protein in the intestines of chicks (9), and confirmed in other species including rodents. Administration of 1,25-dihydroxyvitamin D3 (active vitamin D) to vitamin D-deficient rats increased calbindin mRNA expression in the intestine and kidneys without changing vitamin D receptor (VDR) mRNA levels (10). Other studies revealed that renal calbindin gene transcription was up-regulated following treatment of rats with 1,25-dihydroxyvitamin D3 (11). Numerous other factors, including parathyroid hormone, calcium, and glucocorticoids, can alter the expression of calbindin (12-16). In addition, damage to the kidneys has emerged as a means by which calbindin is excreted into urine. This observation has lead to the proposition that calbindin may be a useful biomarker of nephrotoxicity resulting from exposure to xenobiotics including ketoprofen, cyclosporine A, gentamicin, and ochratoxin A (2,17-20).

Cisplatin is widely prescribed to treat solid tumors. However, its clinical utility is limited by dose-dependent acute kidney injury (AKI) in up to one-third of cancer patients (21). Our laboratories have been interested in the ability of the chemotherapeutic drug cisplatin to elevate urinary concentrations of calbindin in oncology patients as an indicator of subclinical kidney injury (22-24). Clinical studies have shown that calbindin protein increased 8- to 23-fold from baseline in the urine of patients receiving chemotherapy containing cisplatin even in the absence of a rise in serum creatinine (SCr) (25,26). Notably, circulating calcium concentrations were reduced in patients receiving cisplatin treatment. In non-human primates, urinary calbindin levels have also been reported to increase up to 7-fold following cisplatin administration (27). Likewise, cisplatin-treated rats exhibit a 3- to 10-fold rise in urinary calbindin protein (28,29). Concentration-dependent increases in calbindin protein level within media and cell lysates have also been reported in human proximal tubule HK-2 cells undergoing apoptosis following treatment with cisplatin (28). Taken together, these data demonstrate the consistent release of calbindin into urine in response to cisplatin treatment. However, there has been little investigation into the expression and regulation of calbindin within the kidneys in response to drug-induced toxicity. The current study sought to assess time-dependent changes in renal expression and urinary calbindin excretion in cisplatin-treated mice in comparison to kidney injury molecule-1 (Kim-1), a widely used biomarker of proximal tubular toxicity.

Materials and Methods

Chemicals.

Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Animal Treatment.

Male C57BL/6 mice (9-week old) were purchased from Charles River Laboratories (Wilmington, MA). For these mechanistic studies, investigations were limited to males as there are sex differences in rodent susceptibility to cisplatin toxicity (30). Cisplatin was dissolved in saline after heating to 50°C. Groups of mice (n=5-11) were injected i.p. with 5 mL/kg of saline vehicle or 20 mg/kg of cisplatin after overnight fasting. The doses of cisplatin used in this study were similar to those used clinically and are routinely used to induce acute kidney injury in mice (31). Mice were fed standard diets (Rodent Diet 20, PicoLab, St. Louis, MO) containing 0.81% calcium and Vitamin D3 2.3 IU/g. Mice were placed into metabolism cages for collection of urine and quantification of urinary calbindin, kidney injury molecule-1 (Kim-1), creatinine, and urine output (24 h periods between 0 and 4 days). Water and food (beginning at four hours after injection) were provided ad libitum throughout the remainder of the study period. Kidneys and trunk blood were collected between 2 to 4 days after cisplatin treatment at necropsy. Blood was collected in heparinized microcentrifuge tubes and used to isolate plasma. Kidneys were fixed in zinc formalin and stored at room temperature or snap frozen in liquid nitrogen and stored at −80°C. The Rutgers University Institutional Animal Care and Use Committee approved these studies.

Plasma and Urine Analytes.

Blood urea nitrogen (BUN) and plasma and urine creatinine levels were quantified as indicators of renal injury (Thermotrace, Melbourne, Australia; Pointe Scientific, Canton, MI) on a SpectraMax M5 (Molecular Devices, San Jose, CA). Quantification of calbindin protein in mouse urine and plasma was performed using an ELISA kit (OKEH00768, Aviva Biosystems, San Diego, CA) following manufacturer recommendations with a dilution of 1:2 for urine and 1:15 for plasma. Kim-1 protein concentrations in the urine (dilution 1:15) were also quantified using an ELISA kit (MKM100, R&D Systems, Minneapolis, MN) following manufacturer recommendations.

RNA Isolation and mRNA Quantification.

Total RNA from kidneys was isolated using an RNAzol-based method (Sigma-Aldrich, St. Louis, MO). The concentration of total RNA in each sample was quantified using a Nanodrop spectrophotometer and purity confirmed by 260/280 and 260/230 nm ratios (Thermo Fisher Scientific, Waltham, MA). cDNA was generated using the SuperScript first-strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA). The mRNA expression of mouse calbindin (CALB1), KIM-1, vitamin D receptor (VDR), Na+/Ca2+ exchanger (NCX1), transient receptor potential cation channel subfamily M member 6 (TRPM6), transient receptor potential cation channel subfamily V member 5 (TRPV5), and plasma membrane Ca2+ ATPase (PMCA1) was quantified by qPCR using SYBR Green (Thermo Fisher Scientific, Waltham, MA) to detect amplified products in a ViiA7 RT-PCR system (Thermo Fisher Scientific, Waltham, MA) in 384-well plates. Supplemental Table 1 includes primer sequences used for each gene. CT values were converted to ΔΔCT values by comparing with a reference gene, β-actin.

Western Blot Analysis.

Kidneys were homogenized in sucrose-Tris buffer (pH 7.5) containing 2% protease inhibitors. At each time point, there were n=3-5 mice for saline controls and n=5-6 cisplatin-treated mice. Protein concentrations were determined by a bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA). Western blot analysis was performed by loading homogenate protein (10 μg for calbindin, beta-actin or 30 μg for Kim-1) and electrophoretically resolved using polyacrylamide gels (pre-cast 4-12% gradient Bis-Tris) alongside MagicMark™ XP molecular weight standards (ThermoFisher Scientific, Waltham, MA) and transblotted overnight at 4°C onto polyvinylidine fluoride membranes (EMD MilliporeSigma, Burlington, MA). Membranes were blocked with 5% non-fat dairy milk in PBS with 0.5% Tween-20 for 1 h. The following antibodies were diluted in 2% non-fat dairy milk in PBS with 0.5% Tween-20: calbindin (ab49899, 1:1000, 30 kDa), Kim-1 (ab190696, 1:10000, 48 kDa), and beta-actin (ab8227, 1:2000, 42 kDa) (Abcam Inc, Cambridge, MA). Primary antibodies were probed using anti-rabbit HRP-conjugated secondary antibody (Sigma Aldrich, St. Louis, MO) for 1 h and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA). Detection was performed with a FluorChem imager (ProteinSimple, Santa Clara, CA) and semi-quantified using AlphaView SA version 3.4 (ProteinSimple, Santa Clara, CA).

Immunohistochemistry.

For immunohistochemistry, kidneys were embedded in paraffin and 5 μm thick sections prepared. After deparaffinization, tissue sections were quenched in 3% H2O2 (10 min, room temperature). Tissue sections were then blocked with an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) followed by 5% goat or donkey serum. After 2 h of blocking at room temperature, tissues sections were incubated with primary antibodies to calbindin (ab49899, 1:500; abcam) or Kim-1 (AF1817, 1:500, R&D Systems) for 16 h at 4°C. Then, tissue sections were washed and incubated with biotinylated anti-rabbit (for calbindin staining) and anti-goat (for Kim-1 staining) secondary antibodies for 60 min at room temperature (Vector Laboratories, Burlingame, CA). Tissue sections were then stained using a 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA). After counterstaining with hematoxylin, tissue sections were dehydrated and imaged by light microscopy (VS120-S5, Olympus, Center Valley, PA).

Statistical Analysis.

GraphPad Prism version 6 (GraphPad Software Inc., La Jolla, CA) was used for statistical analysis. Differences among groups (n=5-8) were evaluated by Student’s unpaired t-test (for comparison of two groups) or one-way analysis of variance followed by Newman-Keuls multiple comparison tests (for comparison of three or more groups). Where warranted, control groups from different time points were combined after confirming there were no statistical differences and presented as time 0. Statistical significance was set at P<0.05.

Results

Cisplatin-Induced Nephrotoxicity in Mice.

Cisplatin caused time-dependent renal injury in mice. Compared to saline-treated mice (designated as day 0), BUN and creatinine levels increased 2.8-fold and 1.4-fold, respectively, on day 3 (Figures 1A and 1B). Urinary output was also significantly decreased at this time point (Figure 1C). By day 2 after cisplatin administration, the kidneys exhibited minimal injury to proximal tubules affecting less than 10% of cells (31). By days 3 and 4, the kidneys progressed to mild-to-moderate injury with up to 40% of cells undergoing apoptosis or necrosis in response to cisplatin treatment (31).

Figure 1. Renal Injury Markers in Mice Treated with Cisplatin.

Figure 1.

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(A) Blood urea nitrogen (BUN) and (B) serum creatinine (SCr) concentrations were quantified in C57BL/6 mice between 2 to 4 days after treatment with saline vehicle (day 0, black bars) or cisplatin 20 mg/kg (gray bars). Urine output (C) was assessed in C57BL/6 mice between 2 to 4 days after treatment with saline vehicle (day 0) or cisplatin 20 mg/kg i.p. Urine volume was quantified from mice in metabolic cages for 24 h and was normalized to body weight. Data are presented as mean ± SE. *P<0.05, compared to saline-treated mice (designated as day 0).

Increased Urinary Kim-1 and Calbindin Concentrations During Cisplatin Nephrotoxicity.

In response to cisplatin, urinary concentrations of Kim-1 were significantly increased by up to 2.5-fold on days 1 and 2 and returned to baseline levels by day 4 (Figure 2A). By comparison, urinary concentrations of calbindin on day 2 were significantly elevated over 11-fold in cisplatin-treated mice compared to saline-treated mice (Figure 2A). To further characterize the dynamics of calbindin regulation, circulating levels within plasma were also quantified. By day 4, plasma concentrations of calbindin were significantly decreased in cisplatin-treated mice to levels 50% of control (Figure 2B).

Figure 2. Urinary and Plasma Calbindin and Kim-1 Concentrations in Mice Treated with Cisplatin.

Figure 2.

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Urine was collected in 24-h intervals for 4 days using metabolic cages days 1 through 4 following treatment with saline vehicle (day 0) or cisplatin 20 mg/kg. Urinary Kim-1 (A) and calbindin and plasma calbindin (B) concentrations were quantified by ELISA. Data are presented as individual values and mean ± SE. *P<0.05, compared to saline-treated mice (designated as day 0).

Enhanced Kim-1 and Reduced Calbindin Protein Expression During Cisplatin Nephrotoxicity.

The time-dependent expression of calbindin and Kim-1 proteins in total kidney homogenates was examined (Figure 3). In cisplatin-treated mice, Kim-1 protein expression increased in a time-dependent manner with maximal elevation on day 4. In contrast, calbindin protein expression decreased in cisplatin-treated mice with maximal loss (60% of control levels) on day 4.

Figure 3. Intrarenal Expression of Calbindin and Kim-1 Protein in Mice Treated with Cisplatin.

Figure 3.

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Expression of calbindin and kidney injury molecule-1 (Kim-1) proteins in kidney from C57BL/6 mice treated with saline vehicle (black bars) or cisplatin 20 mg/kg (gray bars) were assessed on days 2 through 4 using western blotting. A. Blot from day 4 are shown (n=5 saline control and n=5 cisplatin). B. Protein expression was semi-quantified and normalized to beta-actin loading control and presented as mean ± SE. *P<0.05, compared to saline-treated mice (designated as day 0).

Immunohistochemical analysis revealed Kim-1 staining was not detected in the kidneys of saline-treated mice (Figure 4). In mice treated with cisplatin, Kim-1 staining was strongest on day 4 and largely observed in dilated and damaged proximal tubules. In contrast, calbindin staining appeared in the cytoplasm of distal tubules and collecting ducts in both saline- and cisplatin-treated mice (Figure 4). Staining was observed both in the cortical and medullary regions of the kidney. There was large variability within each group and inconsistent staining throughout tissue sections and as a result, no trend for time-dependent differences in calbindin staining could be ascertained in cisplatin-treated mice.

Figure 4. Immunohistochemical Localization of Calbindin and Kim-1 Proteins in Mice Kidneys Treated with Cisplatin.

Figure 4.

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Kidneys collected from saline and cisplatin (20 mg/kg) treated mice on day 4 were fixed in zinc formalin and then subjected to routine tissue processing and paraffin embedding. Sections (5 μm) were prepared and stained with antibodies against Kim-1 (A) and calbindin (B) as indicated in the Materials and Methods. Antibody binding was visualized using a Vectastain DAB kit (brown staining) and counterstained with hematoxylin (blue staining). Kim-1 staining localized to proximal tubules wherase calbindin staining localized to distal tubules. Images were acquired at 4X and 10X magnification.

Up-Regulation of Kim-1 and Calbindin Genes During Cisplatin Nephrotoxicity.

To dissect the regulation of calbindin expression during kidney injury (Figure 5), mRNA profiling of CALB1 as well as other calcium transporters and vitamin D-related genes was performed (Figure 6). In response to cisplatin treatment, CALB1 and KIM-1 mRNA expression in kidneys exhibited time-dependent increases on days 3 and 4, with no change on day 2 (Figure 6). Notably, KIM-1 mRNA expression was 250-fold higher in the kidneys of cisplatin-treated mice on day 3 compared to vehicle controls. CALB1 mRNA was significantly up-regulated by 200-fold on day 3 in response to cisplatin. The VDR receptor is expressed throughout the nephron with the greatest levels in distal tubules. Notably, VDR mRNA expression was lowest in cisplatin-treated mice on day 2 compared to saline-treated mice and remained reduced through 4 days. The mRNA expression of relevant calcium and magnesium channels was also quantified (Figure 6). Expression of the apical calcium import channel TRPV5 was increased 1.5-fold compared to saline-treated mice on day 3. In contrast, mRNA expression of basolateral calcium export channels, NCX1 and PMCA1, were either down-regulated (up to 80% of control) or unchanged, respectively in cisplatin-treated mice on days 2 through 4. The apical magnesium channel TRPM6 was also maximally down-regulated (70% of control) on day 3 in cisplatin-treated mice.

Figure 5. Localization of Calcium Transport Proteins in Distal and Collecting Tubules.

Figure 5.

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Sodium-calcium exchanger (NCX1) and plasma membrane Ca2+ ATPase 1 (PMCA1) are localized on the basolateral membrane of distal tubules in the kidney and transport calcium into the blood. Transient receptor potential cation channel subfamily V member 5 (TRPV5) and transient receptor potential cation channel subfamily M member 6 (TRPM6) are localized on the apical side of distal tubules in the kidney and transport calcium and magnesium, respectively, into the cytoplasm from the lumen. Calbindin-D28k protein binds calcium ions and transports them through the cytoplasm. Adapted from (36).

Figure 6. Intrarenal mRNA Expression of Calbindin and Related Genes in Kidneys of Mice Treated with Cisplatin.

Figure 6.

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mRNA expression of calbindin (CALB1), kidney injury molecule-1 (KIM-1), vitamin D receptor (VDR), sodium-calcium exchanger (NCX1), transient receptor potential cation channel subfamily V member 5 (TRPV5), transient receptor potential cation channel subfamily M member 6 (TRPM6), and plasma membrane Ca2+ ATPase 1 (PMCA1) was quantified using total kidney RNA from vehicle-treated control (day 0, black bars) and 20 mg/kg cisplatin-treated mice (gray bars) between 2 and 4 days. Data are presented as mean ± SE. mRNA data were normalized to saline-treated mice and reference gene (beta-actin). *P<0.05 compared to saline-treated mice (designated as day 0).

Discussion

There is growing interest in novel biomarkers, including Kim-1 and calbindin, for their ability to detect early kidney injury in response to cisplatin treatment. In the current study, we investigated the time course of calbindin expression in cisplatin treated mice in order to understand its dynamic regulation during nephrotoxicity. Following a single high dose of cisplatin (20 mg/kg), mice exhibited time-dependent elevations in traditional kidney injury markers including increased BUN and circulating creatinine, and decreased urine output. These were accompanied by up-regulation of Kim-1 protein, a well-characterized kidney injury biomarker predominantly localized to proximal tubules. Urinary concentrations of calbindin protein were elevated by day 2 following cisplatin administration. This rise was accompanied by a subsequent decrease in plasma and renal tissue calbindin concentrations. By comparison, a substantial up-regulation of CALB1 mRNA was observed in a coordinated fashion with KIM-1 mRNA as well as calcium- and vitamin D-related genes in renal tissue. The results of the current study suggests the utility of combined use of a proximal (Kim-1) and distal tubule (calbindin) marker to phenotype acute kidney injury secondary to cisplatin.

The ability of cisplatin to enhance the urinary excretion of calbindin in this study was consistent with previous reports in cisplatin-treated Sprague-Dawley rats (28,29), cynomologous monkeys (27) as well as oncology patients (25,26). However, it should be noted that two prior studies have reported a decrease in urinary calbindin protein following cisplatin (6-7 mg/kg) administration in Sprague-Dawley and WKY rats (3,18). The decline in urinary calbindin occurred after histopathological changes were observed and therefore may not reflect the acute response observed in the aforementioned studies (28,29). Interestingly, in the current study, we observed a decrease in plasma calbindin concentrations following cisplatin treatment. This contradicts previous reports in Sprague-Dawley rats (18) and oncology patients (26) that have elevated circulating levels in response to cisplatin. It is not clear whether the contrasting reports of circulating calbindin are due to differences across the species evaluated or other experimental design variables such as diet.

To date, there are no studies assessing the in vivo renal expression of calbindin protein over time following cisplatin-induced injury. In our study, we quantified calbindin expression and demonstrated for the first time a time-dependent decrease in total kidney calbindin expression compared to saline-treated mice. Immunohistochemical localization of calbindin in this study was consistent with findings from other reports. Calbindin was found to be localized to distal tubules and collecting ducts in rats and sheep (2,3). In our study, there was large variability in calbindin staining within control and cisplatin-treated groups. A lack of correlation between Kim-1 and calbindin intra-renal protein expression in the current study makes it difficult to ascertain time-dependent or treatment-dependent changes. One previous study examined the expression of calbindin by immunohistochemical staining in the kidneys of WKY rats following cisplatin-induced kidney injury (3). In that study, cisplatin induced pathological damage primarily to the proximal tubules, which did not overlap with calbindin-stained regions. Additionally, the overall decrease in immunohistochemical staining of calbindin in the kidneys of cisplatin-treated rats (3) are in alignment with our western blot results. These data suggest the potential origin of urinary calbindin protein is from distal tubules and that release into urine is stimulated in response to cisplatin injury. This finding is particularly appealing as it demonstrates the utility of a more specific distal tubule injury marker to understand the entire tubule spectrum of cisplatin tubular toxicity.

The degree and timing of the renal mRNA induction of CALB1 and KIM-1 were similar following cisplatin administration. The induction of CALB1 and KIM-1 mRNAs on day 3 occurred alongside histopathological damage and changes in BUN and SCr, indicating a potential transcriptional response to kidney injury. While the mRNA up-regulation of KIM-1 coincided with an induction in protein expression, up-regulation of CALB1 mRNA did not result in increased protein levels at the time-points evaluated; rather, expression was reduced. We speculate that the up-regulation of CALB1 mRNA in cisplatin-treated mice is likely a compensatory or adaptive response to the 1) decrease in tissue levels following continued loss into the lumen of the nephron and/or 2) loss of calcium reabsorption earlier in the nephron with the necrosis and sloughing of proximal tubules into the lumen. Notably, we have previously noted a decrease in circulating calcium concentrations in patients receiving cisplatin-based chemotherapy that coincided with elevated urinary calbindin levels (25).

Expression of calbindin in the kidneys of rodents is known to be regulated by vitamin D receptor signaling (10). Since vitamin D-mediated regulation of calbindin is relatively well-characterized (11,32-35), we quantified changes in VDR mRNA as well as several downstream target and co-regulated genes including sodium-calcium exchanger (NCX1), transient receptor potential cation channel subfamily V member 5 (TRPV5), transient receptor potential cation channel subfamily M member 6 (TRPM6), and plasma membrane Ca2+ ATPase 1 (PMCA1). Localization of the proteins encoded by these genes in the distal tubule and early collecting duct and their role in Ca2+ and Mg2+ reabsorption is shown in Figure 5 (adapted from (36)). Unexpectedly, only TRPV5, was up-regulated in cisplatin-treated mice, while mRNA levels of the other cation-related genes were decreased or unchanged. The up-regulation of TRPV5 may be secondary to calcium loss at the proximal tubule. Although this does not preclude vitamin D receptor involvement in regulating CALB1 expression, the down-regulation of various related downstream target genes suggests a more complicated mechanism underlying calbindin regulation during kidney injury. There is an interesting relationship between TRPV5 and calbindin. A prior study demonstrated that mouse kidneys revealed a tendency for TRPV5 and calbindin to colocalize (37). In vitro, calbindin has been shown to bind TRPV5 in renal cells (37). Moreover, cells lacking TRPV5 had impaired translocation of calbindin to the plasma membrane (37). In vivo, knockdown of TRPV5 has been shown to down-regulate CALB1 mRNA in naïve mice (38). Finally, TRPV5 and calbindin are co-regulated by various hormones such as parathyroid hormone (12), vitamin D (39), and testosterone (14). The concurrent mRNA up-regulation of the apical transporter TRPV5 and the calcium-binding protein, calbindin, alongside the down-regulation of NCX1 on the basolateral surface suggest that, during kidney injury, renal cells adapt to restore intracellular calcium concentrations. Further studies to determine urinary, intracellular, and plasma calcium concentrations in this model would help to inform this hypothesis.

Conclusion

In summary, we demonstrate a time-dependent increase in calbindin urinary protein coinciding with a decrease in intrarenal calbindin protein expression and an up-regulation of renal CALB1 mRNA in a mouse model of cisplatin-induced kidney injury. Although these data support the utility of urinary calbindin as a cisplatin-induced kidney injury marker and suggest distal tubules as the source of urinary calbindin, it also raises new questions regarding the transcriptional regulation of Calb1 and its role in calcium signaling in renal tubules during cisplatin AKI. The study also demonstrates using a distal tubule biomarker (calbindin) in addition to a proximal tubule biomarker (Kim-1) to more fully phenotype acute kidney injury secondary to cisplatin.

Supplementary Material

1

Acknowledgements

This work was supported by the National Institute of General Medical Sciences [Grant GM123330] and the National Institute of Environmental Health Sciences [Grants ES005022, ES007148], National Cancer Institute [Grants CA072720, CA046934] and the National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK093903], components of the National Institutes of Health.

Abbreviations:

AKI

acute kidney injury

Kim-1

kidney injury molecule 1

PMCA1

plasma membrane Ca2+ ATPase 1

NCX1

sodium-calcium exchanger 1

TRPM6

transient receptor potential cation channel subfamily M member 6

TRPV5

transient receptor potential cation channel subfamily V member 5

VDR

vitamin D receptor

Footnotes

Conflict(s) of Interest

The authors do not have any conflicts to declare.

Data Availability Statement

The datasets generated during the current study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1793909-supplement-1.pdf (105.3KB, pdf)

Data Availability Statement

The datasets generated during the current study are available from the corresponding author upon reasonable request.

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