Primary and Immortalized Cultures of Human Proximal Tubule Cells Possess Both Progenitor and Non-Progenitor Cells That Can Impact Experimental Results

Abstract

Studies have reported the presence of renal proximal tubule specific progenitor cells which co-express PROM1 and CD24 markers on the cell surface. The RPTEC/TERT cell line is a telomerase-immortalized proximal tubule cell line that expresses two populations of cells, one co-expressing PROM1 and CD24 and another expressing only CD24, identical to primary cultures of human proximal tubule cells (HPT). The RPTEC/TERT cell line was used by the authors to generate two new cell lines, HRTPT co-expressing PROM1 and CD24 and HREC24T expressing only CD24. The HRTPT cell line has been shown to express properties expected of renal progenitor cells while HREC24T expresses none of these properties. The HPT cells were used in a previous study to determine the effects of elevated glucose concentrations on global gene expression. This study showed the alteration of expression of lysosomal and mTOR associated genes. In the present study, this gene set was used to determine if pure populations of cells expressing both PROM1 and CD24 had different patterns of expression than those expressing only CD24 when exposed to elevated glucose concentrations. In addition, experiments were performed to determine whether cross-talk might occur between the two cell lines based on their expression of PROM1 and CD24. It was shown that the expression of the mTOR and lysosomal genes was altered in expression between the HRTPT and HREC24T cell lines based on their PROM1 and CD24 expression. Using metallothionein (MT) expression as a marker demonstrated that both cell lines produced condition media that could alter the expression of the MT genes. It was also determined that PROM1 and CD24 co-expression was limited in renal cell carcinoma (RCC) cell lines.

Keywords: CD24; PROM1; RCC; cancer stem cell; conditioned medium; glucose; kidney progenitor cells.

Figure 1

Expression of lysosomal and mTOR genes in RPTEC/TERT1 cells treated with 5.5 mM, 7.5 mM, 11 mM and 16 mM glucose for 3 passages. RT-qPCR analysis of (A) FKBP2; (B) FKBP11; (C) LGMN; (D) LIPA; (E) MLST8; (F) NPC2; (G) RHEB; (H) RRAGC; (I) RRAGD; (J) SQSTM1; (K) TSC1; (L) RIC; (M) RAP; (N) NEU1; (O) LDLR; (P) LAMP1; (Q) IGF2R; (R) CLCN7; (S) eIF4EBP1; (T) DEPTOR; (U) CTSA; (V) mTOR; (W) MCOLN1. ****; ***; **; * indicate significant differences in gene expression level compared to the 1.0 control 5.5 mM glucose concentration at p-value of ≤0.0001; ≤0.001; ≤0.01; ≤0.05, respectively.

Figure 2

Expression of lysosomal and mTOR genes in HRTPT cells treated with 5.5 mM, 7.5 mM, 11 mM and 16 mM glucose for 10 passages. RT-qPCR analysis of (A) CLCN7; (B) IGF2R; (C) LAMP1; (D) HEXB; (E) LGMN; (F) LIPA; (G) NPC2; (H) NEU1; (I) MCOLN1; (J) RRAGC; (K) SQTM1; (L) elF4EBP1; (M) RAPTOR; (N) RICTOR; (O) CTSA; (P) FKBP2; (Q) FKBP11; (R) LDLR; (S) MLST8; (T) RHEB; (U) RRAGD; (V) mTOR. ****; ***; **; * indicate significant differences in gene expression level compared to the 1.0 control 5.5 mM glucose concentration at p-value of ≤0.0001; ≤0.001; ≤0.01; ≤0.05, respectively.

Figure 3

Expression of lysosomal and mTOR genes in HREC24T cells treated with 5.5 mM, 7.5 mM, 11 mM and 16 mM glucose for 7 passages. RT-qPCR analysis of (A) CLCN7; (B) HEXB; (C) IGF2R; (D) LAMP1; (E) FNBP1; (F) LDLR; (G) NEU1; (H) NPC2; (I) LIPA; (J) PSAP; (K) RRAGC; (L) RRAGD (M) MCOLN1; (N) LGMN; (O) SQTM1; (P) elF4EBP1; (Q) RAPTOR; (R) RICTOR; (S) CTSA; (T) FKBP2; (U) FKBP11; (V) RHEB; (W) MLST8; (X) mTOR. ****; ***; **; * indicate significant differences in gene expression level compared to the 1.0 control 5.5 mM glucose concentration at p-value of ≤0.0001; ≤0.001; ≤0.01; ≤0.05, respectively.

Figure 4

Expression of metallothionein genes in HRTPT and HREC24T cells exposed to conditioned media. RT-qPCR analysis of (A) MT1A, (B) MT1E, (C) MT1F, (D) MT1X, (E) MT2A in RPTEC/TERT1, HRTPT and HREC24T cells; (F) MT1A, (G) MT1E, (H) MT1F, (I) MT1X, (J) MT2A, in HRTPT control, HRTPT exposed to conditioned media from HRTPT, and HREC24T cells exposed to conditioned media from HREC24T; (K) MT1A, (L) MT1E, (M) MT1F, (N) MT1X, (O) MT2A in HREC24T control, HRTPT exposed to conditioned media from HREC24T and HREC24T cells exposed to conditioned media from HREC24T. ****; ***; **; * indicate significant differences in copy number and gene expression level compared to the control either RPTEC/TERT1 or HRTPT or HREC24T at p-value of ≤0.0001; ≤0.001; ≤0.01; ≤0.05, respectively.

Figure 5

Expression of MT2A associated genes in HRTPT and HREC24T cells exposed to conditioned media. (A) MT2A showed numerous protein–protein associations, and the top 10 genes with highest string-db score were OAS2, OASL, HLA-A, SP100, B2M, CD44, GBP1, VCAM1, ICAM1, and IRF1. RT-qPCR analysis of (B) B2M; (C) GBP1; (D) HLA-A; (E) ICAM1; (F) IRF1; (G) OAS2; (H) OASL; (I) SP100 in HRTPT control, HRTPT exposed to conditioned media from HRTPT and HREC24T cells exposed to conditioned media from HREC24T; (J) B2M; (K) GBP1; (L) HLA-A; (M) ICAM1; (N) IRF1; (O) OAS2; (P) OASL; (Q) SP100 in HREC24T control, HRTPT exposed to conditioned media from HREC24T and HREC24T cells exposed to conditioned media from HREC24T. ****; ***; **; * indicate significant differences in gene expression level compared to the control either HRTPT or HREC24T at p-value of ≤0.0001; ≤0.001; ≤0.01; ≤0.05, respectively.