Chronic Exposure of Renal Progenitor Cells (HRTPT) to As (III) Implicates Microfibril Associated Protein 5 (MFAP5) in the Activation of Carcinoembryonic Antigen Related Cell Adhesion Molecules (CEACAM 5 & 6)

Md Ehsanul Haque; Donald Sens; Scott Garrett

doi:10.20944/preprints202504.0011.v1

Submitted:

01 April 2025

Posted:

02 April 2025

You are already at the latest version

Abstract

Studies on populations exposed to inorganic arsenic (iAs) have shown an association with the development of chronic kidney disease (CKD) and renal cell carcinoma (RCC). However, there are few studies addressing how acute exposure of the human kidney to iAs might lead to the long-term alterations that might lead to CKD or RCC. This laboratory’s hypothesis is that renal exposure to iAs might alter the renal cells responsible for the repair and regeneration of nephrons damaged by iAs exposure or other renal toxicants. The kidney possesses a minority epithelial cell population that co-express PROM1 and CD24 that are believed to be involved in renal epithelial cell repair. The purpose of this work is to understand the pathogenesis of CKD in renal cortical epithelial cells. Our model consists with acute and chronic exposure of i-As(III) to “Human Renal Tubular Precursor TERT” (HRTPT). The microarray and gene validation study demonstrated a sudden induction of microfibril associated protein 5 (MFAP5) and carcinoembryonic antigen related cell adhesion molecule 5 & 6 (CEACAM 5& 6) in chronic i-As(III) exposed cells. Chronically exposed cells also exhibited an induction of pAKT/AKT pathway, and SOX9 transcription factor. The targeting of MFAP5 & CEACAM5/6 could therefore provide a potential therapeutic approach to CKD.

Keywords:

arsenite (As III)

;

HRTPT

;

renal fibrosis

;

MFAP5

;

CEACAM 5 & 6

Subject:

Biology and Life Sciences - Toxicology

1. Introduction

Anthropogenic activities throughout the world have resulted in substantial contamination of heavy metals in the earth’s air, soil, and water. As a class, these elements present as a serious health concern [1,2,3]. Arsenic is one of the most abundant of these elements and its toxicity is one of the highest of all the heavy metal toxicants. Epidemiological studies have shown that it is carcinogenic to the skin, lungs, bladder, liver, and kidneys[4,5,6,7,8,9]. In the United States alone, over 2.1 million people are believed to be drinking arsenic-contaminated domestic well water and from other sources both known and unknown[10]. In the U.S., arsenic levels in drinking water cannot exceed 10 µg/L as recommended by the World Health Organization and enforced by the US Environmental Protection Agency[11,12,13]. Research has shown that arsenic metabolism occurs primarily in the liver, recent studies have shown that arsenic is also metabolized and eliminated by the human kidney’s proximal tubules (PT) [14,15,16]. These tubules serve as the locations for the energy-intensive reabsorption of nutrients, sodium chloride, sodium bicarbonate, and water (60–70%)[17]. Thus, the proximal tubule of the human kidney can sustain a number of injuries during the reabsorption and metabolism of xenobiotics from drugs and environmental toxins, including heavy metals. Arsenic accumulation in the proximal tubules can cause acute kidney injury (AKI) which progresses to chronic kidney disease (CKD)[18,19,20,21]. Moreover, according to epidemiological studies CKD and kidney cancer are both associated with arsenic in drinking water[22,23,24,25,26]. Clear cell carcinoma (ccRCC) accounts for most renal cancers and is thought to arise from a cell within the PT[27]. Renal progenitor cells involved in the repair of renal damage are thought to arise from dedifferentiated PTs or from scattered cells within the kidney with characteristics of PTs[28,29,30,31]. Regardless of the origin of progenitor cells, only a few studies have examined the response of these cells to iAS[32]. In this previous study, a global gene expression analysis was performed between control and iAs exposed HRTPT cells having co-expression of PROM1 and CD24 with progenitor cell properties[32,33]. An examination of the global gene expression profile showed the overexpression of carcinoembryonic antigen related cell adhesion molecules (CEACAM 5). The goal of the present study was to validate this finding and to begin determining the mechanism underlying the overexpression of the CEACAM5 genes.

2. Materials and Methods

2.1. Cell Culture and Reagents

Parental RPTEC-TERT cells were obtained from American Type Culture Collection (ATCC) and the HRTPT cell line has been established previously described[34,35]. The culture medium consisted of a 1:1 mixture of DMEM: F12 serum-free media supplemented with selenium (5 ng/mL), insulin (5 μg/mL), transferrin (5 μg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Confluent cultures of HRTPT cells were exposed to 4.5 µM i-As (III) (III)(sodium arsenite) for 24 hrs and then sub-cultured at a 1:3 ratio in the continued presence of i-As (III) until confluent. Following confluence, the cells were serially sub-cultured again in the presence of i-As (III) until confluent. This was repeated for 19 serial passages. Every passage was examined microscopically, and harvested cells were preserved for RT-qPCR and protein analysis.

2.2. RNA Extraction and RT-qPCR

The mRNA and protein expression of individual genes was determined using RT qPCR, and western blotting as described previously [34,35,36]. Confluent cell cultures were harvested to make RNA cell pellets, which were then flash-frozen under liquid nitrogen. After lysing the cell pellets with 350 µL RLT^® buffer (Qiagen, Hilden, Germany), they were dissociated using QIA shredder tubes (Qiagen) for 2 minutes at 12,500 rpm. Isolation of RNA was carried out using QIAGEN’s RNeasy Mini Plus Kit (#74034) and QIAcube instrument (Hilden, Germany), according to manufacturer’s protocols. NanoDrop spectrophotometer was used to quantify RNA (Thermo Fisher Scientific, Waltham, MA, USA). We synthesized cDNA from the total RNA using a LunaScript^® RT SuperMix Kit (New England Biolabs #E3010L, Ipswich, MA, USA) as recommended by the manufacturer. A final concentration of 20 ng/µL was obtained by diluting cDNA with nuclease-free water. Two micro liters of cDNA (20ng) were used for qPCR, and the results were analyzed using the BioRad CFX96 Touch Real-Time PCR Detection System (Hercules, CA, USA) and the Luna^® Universal qPCR Master Mix (New England Biolabs #M3003E). The parameters for the qPCR cycle were one 2-minute cycle at 95°C, forty 5-second cycles at 95°C, and thirty seconds at 60°C annealing temperature. Using 18S as the reference control gene, expression levels were calculated using threshold cycle (Ct) values using the 2-∆∆Ct method.

2.3. Western Blot

Protein expression was determined by Western blot analysis using protocols that have been previously published by this laboratory[34,35,36,37]. The HRTPT cell pellets were lysed in an ice-cold RIPA buffer containing equal volumes of protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and sodium orthovanadate (Santa Cruz) and incubated 15 mins on ice by shaking. The extracts were seared using a sonicator for about 15 Seconds two times, keeping them on ice and centrifuged for 13000xg for 10 mins at 4^oC. The supernatants were transferred to fresh, cold microfuge tubes and protein quantification was performed using BCA assay (ThermoScientific). After quantifying the samples, if required, each sample was diluted to 50 μg with RIPA; mixed with Laemmli buffer (Bio-Rad) and boiled for 5 mins at 95^oC. The samples are quickly centrifuged for 10 secs and loaded on a TGX Any Kd^TM SDS polyacrylamide gel (Bio-Rad Laboratories) and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). The blots were blocked in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% [wt/vol] nonfat dry milk for 1 hour at room temperature on the same day after being rinsed for 5 minutes in TBST. Following blocking, the membranes underwent three 15-minute TBS-T washes before being probed with the corresponding primary antibody for a whole night at 4°C in a shaker. The primary antibodies were made using 3% BSA or 5% non-fat milk in TBS-T, and they were diluted using the proper dilution factor.

3. Results

3.1. Light Microscopic Evidence Shows Fibroblast Like Growth in Human Renal Progenitor Cells at P3 and P8

After exposure to 4.5 μM of As (III), HRTPT cell lines displayed morphological changes, including spindle-shaped and irregularly shaped cells. Cells undergo this transformation after several sequential passages with As (III), becoming irregularly shaped domes or lacking domes, and exhibiting characteristics both similar to fibroblasts and more similar to mesenchymal cells (Figure 1). The domes form regularly in cells that have not been exposed to arsenite for prolonged periods. Overall, microscopic evidence indicates that renal progenitor cells exhibit fibroblast-like characteristics after exposed to As (III).

3.2. Arsenite Exposure Increased the Expression of MFAP5, CEACAM 5, and CEACAM 6.

MFAP5 mRNA levels and protein levels increased significantly with 4.5 µM As (III) exposure at P₃ and P₈ in HRTPT cells (Figure 2 A, B & C). A similar result has been observed in cells exposed to As (III) that exclusively overexpress CEACAM 5 & 6. (Figure 3 A & B). The Human Protein Atlas database shows that CEACAM5 to be expressed on plasma membrane of cell (https://www.proteinatlas.org/ENSG00000105388-CEACAM5/subcellular) but the location of CEACAM 6 is unknown (Appendix II, Figure 3C). The CEACAM is a cell adhesion molecule related to carcinoembryonic antigen that functions as a cell surface receptor. Upregulation of MFAP5 and CEACAM5 and 6 suggests a possible link to pro-fibrotic phenotype. A microarray study conducted previously in our laboratory revealed a 2.4K-fold increase in MFAP5 and a 0.4K-fold increase in CEACAMs compared with control at P3 (Appendix A). This present study further validates gene expression of both of them. In addition, we examined mRNA levels of fibrosis related genes such as fibronectin (FN1, 750-fold upregulation over control), and collagen (COL1A1, 9-fold increase over control) (Appendix B, Figure A1).

3.3. As (III) Exposure Increased the Expression of PI3K and MMP1

To gain a better understanding of the molecular cellular signaling pathways involved in cell growth, proliferation, invasion and metastasis, the mRNA level of PI3K and matrix metalloproteinase-1 (MMP1) was assessed in i-As(III) exposed cells. A higher magnitude of PI3K and MMP expression was observed in cells exposed to arsenite. Western blot anylysis shows elevated phopho-Akt in i-As (III) exposed HRTPT cells compared to unexposed control (Figure 5).

Figure 4. RT-qPCR analysis of PI3K and MMP1 (A)PI3K, and (B) MMP1; The expression of the mRNA level was normalized to the 18S housekeep-ing gene. The measurements were performed in triplicate for gene and protein data. The reported values are mean ± SEM. A one-way anova was performed, and ****; ***; **; * indicates significant differences in gene expression level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

3.4. Determine How i-As (III) Increased the Expression of p-SAPK/JNK

To gain a better understanding of the molecular cellular signaling pathways involved in renal fibrosis, the protein level of SAPK and p-SAPK was assessed in i-As(III) exposed cells. A higher magnitude of p-SAPK expression was observed in cells exposed to arsenite. Western blot anylysis shows elevated phopho-SAPK in i-As (III) exposed HRTPT cells compared to unexposed control (Figure 6).

3.5. Expression of Transcription Factors (TFs) in Cancer Associated Fibroblast Activation

In order to determine transcription factors in cancer associated fibroblast activation, which is a process that plays a role in cell proliferation and cancer progression. We evaluated the expression of several transcription factors including SOX9, POU5F1, ZEB1, JUN and FOS in HRTPT cell line exposed to arsenite. Chronically exposed cells also exhibited consistent induction of TFs, these markers commonly activated in EMT pathways involved in CKD and or in RCC.

Figure 6. mRNA level of SOX9, POU5F1, ZEB1, JUN and FOS in HRTPT cell line exposed to arsenite. (A) mRNA SOX9, (B) mRNA POU5F1, (C) mRNA ZEB1 (D) mRNA JUN, (E) mRNA FOS; ****; ***; **; * indicates significant differences in mRNA level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

4. Discussion

According to American Cancer Society, 9 out of 10 kidney cancers are renal cell carcinomas, while about 7 out of 10 people with RCC have clear cell renal cell carcinoma (ccRCC) [27]. Thus, RCC has become one of the greatest public health problems in the world, and American health care systems are forced to spend billions of dollars treating it. The recurrence of ccRCC occurs locally or distantly within five years after nephrectomy for 20-30% of patients. Cancer recurrence following surgery is one of the major factors negatively affecting patient survival. Using biomarkers, doctors and researchers can predict treatment outcomes, make informed treatment decisions, monitor cancer, and calculate the risk of recurrence[38,39,40,41]. It is therefore imperative to gain a better understanding of the pathogenesis and search for new biomarkers or therapeutic targets.

The present study is focused on exploring how exposure to nephrotoxins might alter the renal epithelial cells charged with the repair and regeneration of the epithelial cells comprising a damaged nephron. For this study, the laboratory employed an immortalized human cell culture model, HRTPT, that co-expresses PROM1 and CD24 and displayed the expected properties of a renal epithelial cell with capabilities for renal repair and regeneration[33]. In a previous study, the laboratory employed these cells to determine their response to i-As exposure[32]. It was shown that the HRTPT cells responded to i-As exposure by undergoing an epithelial to mesenchymal transition (EMT) which was mostly, but not fully reversed, upon removal of exposure of the cells to iAs[32]. As part of this study, global gene expression was performed on the HRTPT cells in the presence and absence of exposure to i-As. While the most attention was focused on the EMT and MET response of the HRTPT cells, it was also noted that the expressions of MFAP5 and CEACAM5 were highly expressed due to i-As exposure. These genes and their regulation were chosen for validation and further study since two recent studies suggest they might have a role in renal fibrosis and RCC[42,43,44,45]. One study showed that CEACAMs are prognostic markers for some malignancies, including ccRCC[43], while another study shown MFAP5 to be involved in cancer-associated fibroblasts activation in pancreatic cancer[42]. The validation of these two markers showed a concurrent upregulation, which suggests a potential link to development of renal fibrosis and cell transformation. This potential linkage was further explored through the analysis of several additional pathways likely to be involved in i-As-induced alterations in HRTPT cells. The i-As exposed HRTPT cells showed high levels of mRNA for PI3K and p-AKT/AKT which is interesting since PI3K activates signaling cascades that promote cell growth and division. The PI3K/AKT signaling pathway itself serves a major role in regulating cell physiology and pathology, including cell proliferation, survival and invasion[46,47,48]. Other studies found that EMT and metastasis was promoted by overactive PI3K/AKT pathways because of their potent effects on cell migration[49,50,51]. The present study demonstrates that significant PI3K/AKT upregulation correlates with the many alterations in the microarray analysis associated with EMT and those associated with renal fibrosis.

The stress-activated protein kinase/Jun-amino-terminal kinase SAPK/JNK was also shown to be activated which is interesting since it is activated by a variety of environmental stresses and inflammatory cytokines[52]. The activation of these pathways also correlates to the overexpression of matric components such as FN1, COL1A1, ACTA2 and TAGLN in i-As exposed HRTPT cells (Appendix A & B). The expression of SOX9 was also examined since it is a transcription factor known for its role in promoting EMT and can bind to specific DNA sequences that regulate genes involved in cell phenotypic changes, migration, and invasion[53,54]. The HRTPT cells exposed to i-As upregulated factors such as POU5F1, Zeb1, JUN and FOS at defined points within the time course of exposure to i-As.

Overall, these studies suggest a number of changes in the gene expression of progenitor-like cells when exposed to i-As. The present study is limited in scope due to several related factors. The HRTPT cells are immortalized which presents advantages and disadvantages. An advantage is that renal fibrosis and renal cancer require long-term exposure to nephrotoxic agents. A disadvantage is the fact that they are immortalized, and this process itself may influence data interpretation. An additional advantage is that the HRTPT cells offer a longer term of exposure compared to primary cultures of renal cells that have a very limited lifespan along with difficulties in tissue acquisition. Thus, the present study largely provides insight into potential alterations in gene expression that might guide precise genomic analysis of intact human renal tissue by single cell technology and spatial analysis. While the current results are descriptive, the data provides insight on further in vitro studies to identify alterations elicited by i-As on cells co-expression both PROM1 and CD24. Gene knockdowns and knock-ins will be needed in future studies to begin to define cause and effect among the genes identified through global gene analysis.

Author Contributions

D. Sens, S. Garrett, and Md Ehsanul Haque Conceptualization, Md Ehsanul Haque and S. Garrett designed and performed experiments, analyzed data and manuscript preparation. Md Ehsanul Haque performed experiments, curated and analyzed data. Md Ehsanul Haque analyzed data and revised and helped prepare the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The research described was supported by funds provided by the Department of Pathology and the School of Medicine and Health Sciences, University of North Dakota. Undergraduate research, graduate student mentoring, core facilities for bioinformatics, microscopy, statistics, and gene expression were supported by the ND INBRE IDeA program P20 GM103442 from the National Institute of General Medical Sciences, NIH. University of North Dakota School of Medicine and Health Sciences pilot grant.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Microarray data shows fold change of top 30 upregulated genes in As (III) exposed HRTPT cells at P3 over control.

Appendix B

Figure A1. mRNA level of FN1 & COL1A1 in HRTPT cell lines exposed with 4.5 µM of i-As up to P8 passages. RT-qPCR analysis of mRNA_ ****; ***; **; * indicates significant differences in gene expression level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

Figure A2. mRNA level of ACTA2 & TAGLN in HRTPT cell lines exposed with 4.5 µM of i-As (III). RT-qPCR analysis of mRNA_ ****; ***; **; * indicates significant differences in gene expression level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

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Figure 1. Light microscopic images of HRTPT cells exposed to 4.5 μM of arsenite shows fibroblast-like growth at passage at P₃ (B), & P₈ (C). A. Control- without arsenite; B-C- exposed to arsenite passaged up to P₈ and grown in 1:1 DMEM/F12 media.

Figure 2. Expression of MFAP5 marker in HRTPT cell lines exposed with 4.5 µM of iAs up to P₈ passages. RT-qPCR and Wester blot analysis of (A) mRNA_MFPA5 and (B) protein_MFPA5 (C) western blot. The expression of the MFAP5 gene and protein was normalized to the 18S housekeep-ing gene and β-actin, respectively. The measurements were performed in triplicate for gene and protein data. The reported val-ues are mean ± SEM. A one-way anova was performed, and ****; ***; **; * indicates significant differences in gene/protein expression level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.****; ***; **; * indicates significant differences in gene and protein expression level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

Figure 3. RT-qPCR analysis of CECAM 5 and 6 (A)CEACAM5, and (B) CEACAM6 ****; ***; **; * indicates significant differences in mRNA level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively. And (C) Expression of CEACAM 5 on the surface plasma mebrane (light green color) but location of CEACAM 6 is unknown. The expression of the CEACAMs was normalized to the 18S housekeep-ing gene. The measurements were performed in triplicate for gene data. The reported values are mean ± SEM. A one-way anova was performed, and ****; ***; **; * indicates significant differences in gene expression level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

Figure 5. Western blot of Akt, phospho-Akt and p-Akt/Akt in HRTPT cell line exposed to arsenite. (A) Akt, (B) p-Akt; (C) p-Akt/Akt (D) western blot; ****; ***; **; * indicates significant differences in protein level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

Figure 6. Western blot of SAPK, phospho-SAPK and p-SAPK/SAPK in HRTPT cell line exposed to arsenite. (A) Akt, (B) p-Akt; (C) p-Akt/Akt (D) western blot; ****; ***; **; * indicates significant differences in protein level compared to the control 0.0 µM arsenite concentration at p-value of ≤ 0.0001; ≤ 0.001; ≤ 0.01; ≤ 0.05 respectively.

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