Impact of Pals1 on Expression and Localization of Transporters Belonging to the Solute Carrier Family

Pals1 is part of the evolutionary conserved Crumbs polarity complex and plays a key role in two processes, the formation of apicobasal polarity and the establishment of cell-cell contacts. In the human kidney, up to 1.5 million nephrons control blood filtration, as well as resorption and recycling of inorganic and organic ions, sugars, amino acids, peptides, vitamins, water and further metabolites of endogenous and exogenous origin. All nephron segments consist of polarized cells and express high levels of Pals1. Mice that are functionally haploid for Pals1 develop a lethal phenotype, accompanied by heavy proteinuria and the formation of renal cysts. However, on a cellular level, it is still unclear if reduced cell polarization, incomplete cell-cell contact formation, or an altered Pals1-dependent gene expression accounts for the renal phenotype. To address this, we analyzed the transcriptomes of Pals1-haploinsufficient kidneys and the littermate controls by gene set enrichment analysis. Our data elucidated a direct correlation between TGFβ pathway activation and the downregulation of more than 100 members of the solute carrier (SLC) gene family. Surprisingly, Pals1-depleted nephrons keep the SLC’s segment-specific expression and subcellular distribution, demonstrating that the phenotype is not mainly due to dysfunctional apicobasal cell polarization of renal epithelia. Our data may provide first hints that SLCs may act as modulating factors for renal cyst formation.


SM2-SM7 Description of excel sheets
Only in case that the DEGs (from: differentially expressed genes) enrichment is above the value 10^3 terms are included of the list. Labeling of columns: GO Term: GO term number; Description: name of GO term; FDR (q-Value) is the false discovery rate; Enrichment (N, B, n, b); N -is the total number of genes; B -is the total number of genes associated with a specific GO term; n -is the number of genes in the top of the user's input list or in the target set when appropriate; b -is the number of genes in the intersection. The enrichment is defined as the ratio between (b/n) / (B/N). Highlighted in yellow are the SLC genes that can be found in the different categories.
SM2 Matched cellular components GO subsets for upregulated DEGs. This excel sheet includes gene ontologies (GO) terms of the category cellular components that were matches by differentially upregulated genes in Pals1-deficient kidneys.

SM3 Matched biological processes GO subsets for upregulated DEGs.
This excel sheet includes gene ontologies (GO) terms of the category biological processes that were matches by differentially upregulated genes in Pals1-deficient kidneys.

SM4 Matched molecular function GO subsets for upregulated DEGs.
This excel sheet includes gene ontologies (GO) terms of the category molecular function that were matches by differentially upregulated genes in Pals1-deficient kidneys.

SM5 Matched cellular components GO subsets for downregulated DEGs.
This excel sheet includes gene ontologies (GO) terms of the category cellular components that were matches by differentially upregulated genes in Pals1-deficient kidneys.

SM6 Matched biological processes GO subsets for downregulated DEGs.
This excel sheet includes gene ontologies (GO) terms of the category biological processes that were matches by differentially upregulated genes in Pals1-deficient kidneys.

SM7 Matched molecular function GO subsets for downregulated DEGs.
This excel sheet includes gene ontologies (GO) terms of the category molecular function that were matches by differentially upregulated genes in Pals1-deficient kidneys.

SM8: Data set: transcriptome analysis
Gene set enrichment analyses (GSEA) of differentially regulated genes in Pals1-deficient kidneys. (A) ReviGO images demonstrating the enrichment of GO terms of the categories molecular function for upregulated DEGs (for details: Table ST3). (B) ReviGO illustration of matched GO terms of downregulated DEGs in the category biological processes (for details: Table ST5). (C) GO terms of downregulated DEGs in the category molecular function showed a striking clustering of terms linked to transmembrane transporter activities (Zoom left site, for details ST6). The heat map indicates the p-value. The plot size indicates the number of regulated genes that match the different GO terms. The asterisks marks GO subsets including transporters of the SLC family. (D/E) Quantitative real-time RT-PCR analyses of mRNA levels derived from Pals1-deficient kidneys (∆Pals1) and their littermate controls (suppl. data set A). In Pals1deficient kidneys target genes of the TGFβ (Serpine1) and Hippo-pathways marker genes Ctgf and Cyr61 (D) as well as renal injury markers Lcn2 and Ccl2 (E) are upregulated.

SM9 Solute carrier gene family expression in Pals1-deficient kidneys
This excel sheet summarizes all members of the SLC gene superfamily that are differentially expressed in the mouse kidneys following Pals1-silencing. The first column gives the SLC subfamily number, the second column the predicted role of the subfamily, and the third column the SLC members that are found in kidney transcriptomes of Pals1-haplodeficient mice and their littermate controls (Weide et al., 2017). The fourth column gives the fold change values of Pals1-deficient (Cre+) mice versus their littermate controls (Cre-). In red are fold change values marked that are more than 1.5fold, but below 5 (> 1.5x < 5x) regulated. Dark red values indicate fold changes above 5 (>5x). Upregulated SCLs are labeled in green. (There were no values above 5x). The experimental protocols and methods in this work involving animals were approved by and conducted in accordance with all guidelines and regulations set forth by the German regional authorities (Az.: 84-02.04.2014 A405; LANUV). Animals were housed under standard specific pathogen-free conditions with free access to tap water and standard animal chow. Pals1 conditional knockout and Six2-Cre transgenic mice, their genotyping PCRs and primers (Table  ST8) have been described earlier (Kobayashi et al., 2008;Kim et al., 2010;Weide et al., 2017).
Methods used (for experiments shown in suppl. data set A, including SDS-PAGE, genotyping of mice and analyses of histologic section) have been described earlier in detail (Weide et al., 2017) SM11 Data Set: Immunohistochemical analyses using antibodies against Slc5a2, Slc22a7 and Slc22a8 The transcriptome analyses show that Pals1 gene silencing in mouse induce downregulation of various renal mRNA transporters of the SLC family including Slc5a2 (Sglt2), or Slc22a7 (Oat2), and a trend for Slc22a8 (Oat3). In Pals1-deficient kidneys (Cre+), most of the proximal convoluted tubules (PCT; S1/S2 segments) as well proximal straight tubules (PST; S3 segments) were significantly dilated in comparison to those in the littermate wildtype controls (Cre -). Also, our immunohistochemical (IHC) data indicate that cysts could possibly originate from both proximal and distal nephron segments. Although the proximal tubules are dilated, basolateral and apical membrane domain of their epithelium look morphologically preserved.
Tested downregulated SLC transporters were mainly located in the BBM (cortical S1/S2 segments for Slc5a2/Sglt2 and S3 segments in the outer stripe for Slc22a7/Oat2), indicating that Pals1 might has an impact on SLC transporters located in the apical membrane domain of proximal tubule epithelium in contrast to less sensitive SLC transporters located in the basolateral membrane domain. The Na/K-ATPaserelated staining was used to label the basolateral membrane domain of proximal and distal tubules of mouse nephrons.
These labelings confirm data shown in Fig. 2C, indicating that polarity of proximal tubules is preserved in the Pals1-haplodeficient kidneys. Furthermore, tested renal SLC membrane transporters are located on the same epithelial domain of proximal tubules in both Pals1-depleted and wildtype epithelia confirming the preservation of cell polarization in Pals1-haplodeficient nephrons. In addition to the staining given in Fig. 2C, we performed further IHC using specific antibodies against Slc5a2 (Sglt2), Slc22a7 (Oat2), and Slc22a8 (Oat3) as well as Na/K-ATPase (α1-subunit).

SM11-a:
Impact of Pals1 silencing on the Slc5a2 (Sglt2) localization in the mouse kidney SM11-b: Localization of Slc5a2 (Sglt2) in the kidney of control (wildtype) animals; rats vs. mice SM11-c: Impact of Pals1 silencing on the Slc22a7 (Oat2) localization in the mouse kidney SM11-d: Localization of Slc22a7 (Oat2) in the kidney of control (wildtype) animals; rats vs. mice SM11-e: Double labeling of Slc22a7 (Oat2) and Na/K-ATPase (α1-subunit) in the mouse kidney; wildtype vs. Pals1 deficient SM11-f: Impact of Pals1 silencing on the Slc22a8 (Oat3) localization in the mouse kidney SM11-g: Localization of Slc22a8 (Oat3) in the kidney of control (wildtype) mice SM11-h: Impact of Pals1 silencing on the Na/K-ATPAse (α1-subunit) localization in mouse kidney Slc5a2 SM11-a: Impact of Pals1 silencing on the Slc5a2 (Sglt2) localization in the mouse kidney In the kidney of Cre negative wildtype mice (littermate control), the anti Slc5a2/Sglt2 antibody strongly stained the brush border membrane (BBM) of proximal convoluted tubules (PCT, S1/S2 segments) in the cortex where the Slc5a2-related fluorescence intensity was similar in males and females. In the kidney of Pals1-deficient (Cre positive) mice, the anti Slc5a2/Sglt2 antibody stained the same tubular epithelial domain, i.e. the BBM of PCT S1/S2 segments. However, fluorescence intensity of Slc5a2 BBM staining of PCT in the kidney of Pals1-deficient (Cre positive) mice was lower in comparison to wildtype (Cre positive) mice; this phenomenon was observed in both male and female mice. In the kidney of Pals1-deficient (Cre positive) mice almost all PCT was significantly dilated in comparison to PCT of wildtype Cre negative mice. Furthermore, in the kidney of Pals1-deficient (Cre positive) mice, the presence of many cysts was detected (not shown). Bar = 20 µm. SM11-d: Localization of Slc22a7 (Oat2) in the kidney of control (wildtype) animals; rats vs. mice In the kidney of 3 months (mo) old rat, the Slc22a7/Oat2 antibody strongly stained the brush border membrane (BBM) of proximal straight tubules (PST, S3 segments) in the outer stripe. In the kidney of 3-mo/6-weeks (wk) old C57Bl/6 mice, the Slc22a7/Oat2 antibody strongly stained the BBM of PST S3 segments in the outer stripe, whereas other nephron segments were Oat2-negative. These results are in accordance to our previously reported data and were described in detail (Ljubojevic et al., 2007). The same pattern of Slc22a7/Oat2 -related immunostaining was observed in the kidney of 21 days old wildtype (Cre negative) mice. Bar = 20 µm.
SM11-e: Double labeling of Slc22a7 (Oat2) and Na/K-ATPase (α1-subunit) in the mouse kidney; wildtype vs. Pals1-deficient In the kidney of wildtype (Cre negative) mice, the Slc22a7/Oat2 antibody (red) strongly stained the brush border membrane (BBM) of proximal straight tubules (PST) (S3 segments) in the outer stripe, whereas fluorescence intensity of this immunostaining was significantly reduced in the kidney of Pals1-deficient (Cre positive) mice. In the outer stripe of wildtype (Cre negative) and Pals1-deficient (Cre positive) mice, the Na/K-ATPase-antibody (green) stained the BLM of PST as well the medullary thick ascending limb of Henle (MTALH). However, in the outer stripe of Pals1deficient (Cre positive) mice most of the PST S3 segments were dilated, and presence of many cysts was detected. Merged imaged (red + green fluorescence) indicated that Oat2-related immunostaining was almost undetectable in the PST S3 segments of Pals1-deficient (Cre positive) mice in comparison to wildtype (Cre negative) mice. Bar = 20 µm.