Jonathan A. Lindquist,Anja Bernhardt,Charlotte Reichardt,Eva Sauter,Sabine Brandt,Rajiv Rana,Maja T. Lindenmeyer,Lars Philipsen,Berend Isermann,Cheng Zhu,Peter R. Mertens
Cold Shock Domain Protein DbpA Orchestrates Tubular Cell Damage and Interstitial Fibrosis in Inflammatory Kidney Disease
kidney injury,kidney fibrosis,cold shock proteins
DNA-binding protein A (DbpA) belongs to the Y-box family of cold shock domain proteins that exert transcriptional and translational activities in the cell via their ability to bind and regulate mRNA. To investigate the role of DbpA in kidney disease, we utilized the murine unilateral ureter obstruction (UUO) model, which recapitulates many features of obstructive nephropathy seen in humans. We observed that DbpA protein expression is induced within the renal interstitium following disease induction. Compared with wild-type animals, obstructed kidneys from Ybx3-deficient mice are protected from tissue injury, with a significant reduction in the number of infiltrating immune cells as well as in extracellular matrix deposition. RNAseq data from UUO kidneys show that Ybx3 is expressed by activated fibroblasts, which reside within the renal interstitium. Our data support a role for DbpA in orchestrating renal fibrosis and suggest that strategies targeting DbpA may be a therapeutic option to slow disease progression.
Cold Shock Domain Protein DbpA Orchestrates Tubular Cell Damage and Interstitial Fibrosis in Inflammatory Kidney Disease

DNA-binding protein A (DbpA) belongs to the Y-box family of cold shock domain proteins that exert transcriptional and translational activities in the cell via their ability to bind and regulate mRNA. To investigate the role of DbpA in kidney disease, we utilized the murine unilateral ureter obstruction (UUO) model, which recapitulates many features of obstructive nephropathy seen in humans. We observed that DbpA protein expression is induced within the renal interstitium following disease induction. Compared with wild-type animals, obstructed kidneys from Ybx3-deficient mice are protected from tissue injury, with a significant reduction in the number of infiltrating immune cells as well as in extracellular matrix deposition. RNAseq data from UUO kidneys show that Ybx3 is expressed by activated fibroblasts, which reside within the renal interstitium. Our data support a role for DbpA in orchestrating renal fibrosis and suggest that strategies targeting DbpA may be a therapeutic option to slow disease progression.

Renal fibrosis resulting from excessive deposition of the extracellular matrix is a common feature of chronic kidney disease (CKD) [1,2]. Fibrotic deposits alter the tissue architecture, resulting in decreased renal function that ultimately leads to organ failure. Currently, there is limited therapy that can slow or stop disease progression [3]. Therefore, a better understanding of the underlying molecular mechanisms that contribute to fibrogenesis may identify new targets for therapeutic intervention. Y-box binding protein-1 (YB-1; gene Ybx1) and DNA-binding protein A (DbpA; gene Ybx3) belong to the Y-box family of cold shock domain proteins that bind single-stranded nucleic acids via their evolutionarily conserved cold shock domain [4,5]. Therefore, these proteins play a central role in regulating a number of cellular processes, including transcription, translation, and the cell cycle. We have shown that kidney injury induces the expression of both YB-1 and DbpA [6,7]. While YB-1 is a key regulator of fibrosis-related genes, less is known about the function(s) of DbpA [4,8,9]. DbpA was first identified as a transcription factor that binds to a responsive element within the epidermal growth factor receptor (EGFR) promoter and is known by several names, including mouse Y-box protein 3 (MSY3), mouse Y-box protein 4 (MSY4), cold shock domain A (CSDA), and zonula occludens-1 (ZO-1)-associated nucleic acid binding protein (ZONAB) [10]. There are two isoforms of DbpA that result from alternative splicing, hence the names MSY3 and MSY4 [11]. Both isoforms possess an ancestral cold shock domain, but differ by a single exon, encoding 69 amino acids. DbpA is mainly expressed in smooth muscle cells of the heart, vasculature, and skeletal muscle, whereas YB-1 is ubiquitously expressed [12,13]. Upregulated DbpA expression has been observed in various cancers [14,15,16,17,18,19]. DbpA was first described as a component of tight junctions, due to its interaction with ZO-1 (also known as tight junction protein 1). When cells lose their cell–cell contacts, DbpA is released from the plasma membrane and translocates to the nucleus, where it triggers cell proliferation by regulating target genes such as thymidine kinase, cyclin D1, and proliferating cell nuclear antigen (PCNA) [20,21,22,23,24,25]. DbpA shows a strong expression in developing embryos, but is rapidly downregulated after birth [21]. Congruently, we recently demonstrated that the DbpA protein was barely detected in healthy kidney tissue; however, its expression was strongly induced upon the induction of mesangioproliferative glomerulonephritis [7]. DbpA also plays a crucial role in the differentiation and proliferation of proximal tubular cells [21]. However, little is known about the role of DbpA in kidney disease. The aim of this study was to determine the contribution of DbpA to disease progression. Therefore, we applied a surgical model of ureteral obstruction to wild-type and Ybx3-deficient mice. This model recapitulates many features of obstructive nephropathy in humans, including subacute renal injury characterized by tubular cell damage, interstitial inflammation, and the development of fibrosis [26,27].

Animal studies were performed using genetically modified mice on a C57BL/6N background. The animals were housed and bred in the Central Animal Facility of the Medical Faculty at the Otto-von-Guericke University Magdeburg as previously described [28].

First, the tissue biopsy was lysed with 200 µL direct PCR lysis buffer and 50 µg of proteinase K. PCR was performed with primer pairs for mouse MSY4 (MSY4F3: CAGACTCAGAAAATGAAGGGCA; MSY4R3: AGATGGGCAAGATTCTCAAGTC; and PGK1: TGAGACGTGCTACTTCCATTTG) under the following conditions: MSY4 PCR, 35 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 60 s. Products were separated on 2% agarose gels.

Unilateral ureter obstruction was performed on 14 healthy C57BL/6N mice (Janvier, 20 to 30 g, 12 to 14 weeks of age) and 13 healthy MSY4-/- mice (T. Ley, Washington University School of Medicine, St. Louis, MO, USA) as previously described [28].

The kidney tissue was mechanically crushed, resuspended in digestion buffer (RPMI, 0.1% BSA, 1 mg/mL collagenase D, and 100 µg/mL DNase I), and incubated for 30 min (37 °C, 5% CO2, 100% humidity). After repeated mechanical grinding, the entire contents of the well were placed on a 70 µm cell strainer, rinsed, and then placed and rinsed on a 40 µm cell strainer. After centrifugation, erythrocytes were lysed, and the cells were washed and analyzed via flow cytometry [29].

Cells (1 × 106) were used for the staining procedure. The cells were stained with a directly labeled antibody for 30 min at 4 °C in the dark. Fixing and permeabilization of the cells were performed using the Fix/Perm buffer set (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. To prevent non-specific binding, the cells were incubated with 5% mouse serum for 10 min at 4 °C. The list of surface markers includes: CD11b clone M1/70 APC/Cy7, CD3 clone 145-2C11 APC, CD45 clone 30-F11 PE, CD73 clone TY/11.8 Alexa Fluor® 647 (all from BioLegend), and PDGFRβ clone APB5 PE (AbCam, Cambridge, UK). The flow cytometry measurements were performed using a FACSAria II instrument (BD Immunocytometry Systems). Data collection and analysis were performed using the FlowJo software version 10. Controls were used for gating analyses to distinguish positively from negatively stained cell populations (Supplementary Figure S1).

The total RNA of the kidney was extracted using an RNAeasy mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. The total RNA (1 μg) was reverse-transcribed using a SuperScript III First-Strand Synthesis System (Thermo Fischer Scientific Inc., Rockford, IL, USA). The real-time PCR reactions were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TaqMan Universal PCR Master Mix (Applied Biosystems). Collagen type I, collagen type III, and 18S rRNA mRNAs were detected using FAM-labeled specific primers purchased from Applied Biosystems. The thermal cycling conditions were as follows. After an initial hold of 2 min at 50 °C and 10 min at 95 °C, the samples were cycled 40 times at 95 °C for 15 sec and 60 °C for 1 min. Relative levels of αSMA, collagen type I, and collagen type III mRNAs in each sample were evaluated after normalization with 18S expression. Beta-Actin (Actb) (Mm00607939_s1) served as an internal control (Life Technologies GmbH, Darmstadt, Germany). The relative abundance (fold change against uninfected controls) was calculated using the delta Ct method (2−ΔΔCt).

Methacarn-fixed, paraffin-embedded tissue sections were used for immunohistochemistry and immunofluorescence staining as previously described [28].

The staining procedure was performed as previously described [28].

Kidney fibrosis was quantified using Sirius red staining as previously described [28].

Tissue protein extracts were prepared and separated via SDS-PAGE (10% Bis-Tris gels) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Non-specific binding was blocked with 5% milk in TBS-tween, before adding primary antibodies: anti-YB-1 C-terminal (aa 306-321, Eurogentec, Seraing, Belgium), anti-DbpA N-terminal (aa 51-63, Eurogentec), anti-GAPDH (14C10, Cell Signaling, Danvers, MA, USA), anti-PDGFRβ (28E1, Cell Signaling), anti-β-catenin (Cell Signaling), anti-vinculin (V284, Santa Cruz, CA, USA), anti-E-cadherin (24E10, Cell Signaling), anti-MSY2 (ab154829, Abcam). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Southern Biotech, Birmingham, AL, USA) were used for detection. Antibodies were visualized using SuperSignal chemiluminescence substrate Pierce ECL (Thermo Fischer Scientific) according to the manufacturer’s instructions.

Automated multidimension microscopy was performed as previously reported [28].

Human renal biopsy specimens were collected in an international multicenter study, the European Renal cDNA Bank-Kröner–Fresenius Biopsy Bank (ERCB-KFB) [30]. Biopsies were obtained from patients after receiving informed written consent and with approval of the local ethics committees. Biopsies were prepared as previously reported [31]. Glomerular and tubular samples were analyzed for mRNA expression levels (GSE32591, GSE35489, GSE37463, GSE47185, GSE 99340). CEL file normalization was performed with the Robust Multichip Average method using RMAExpress (Version 1.20) and the human Entrez-Gene custom CDF annotation from Brain Array version 25 (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/CDF_download.asp, accessed on 28 November 2022). The log-transformed dataset was corrected for a batch effect using ComBat from the GenePattern pipeline (http://www.broadinstitute.org/cancer/software/genepattern/, accessed on 28 November 2022). To identify differentially expressed genes, the SAM (significance analysis of microarrays) method was applied using the SAM function in Multiple Experiment Viewer (TiGR MeV, Version 4.9) [32]. A q-value below 5% was considered to be statistically significant.

The results were calculated and presented as means ± SDs. The Student t-test was applied for two-group comparisons, with * p < 0.05 considered statistically significant; ** p < 0.01, *** p < 0.001.

We first analyzed the changes in YBX3 expression in both the glomerular and tubulointerstitial compartments of microdissected patient biopsies. Our data suggest disease-specific changes in YBX3 transcript levels (Figure 1). To further investigate the role of DbpA in kidney disease progression, we examined a model of tubulointerstitial nephritis, namely unilateral ureter obstruction (UUO), in wild-type and MSY4-deficient mice, hereafter referred to as Ybx3-deficient mice. As previously reported, deletion of the Ybx3 gene has no overt effect on either the birth rate or development of knockout animals; however, male mice display decreased fertility after 4 months of age [33]. Deletion of the gene was confirmed by both PCR genotyping (Figure 2A) and Western blotting (Figure 2B). Since DbpA is expressed in two isoforms, we isolated mRNA from the testis and kidney of wild-type, heterozygous, and knockout mice to determine which isoform(s) is (are) expressed in the kidney [11]. Primers were designed corresponding to the N- and C-terminus of the DbpA coding sequence, which should yield products of 1083 and 875 bp, respectively. Analysis of the amplification products identified three bands present in both the testis and kidney of wild-type mice, which were reduced in intensity in the heterozygote, and absent in the knockout (Figure 2C). Sequence analysis confirmed the identity of the upper and lower bands as the long and short isoforms of DbpA, i.e., DbpA_a and DbpA_b, respectively. The identity of the third band (*), provided by the Proteomics Database, may be a premature termination product of the long isoform reported in humans [34]. Thus, our analysis showed that the long isoform DbpA_a was predominantly expressed in the testis, which is consistent with the Western blot results, whereas DbpA_b predominated in kidney tissue. Generation of the knockout induced truncated RNA products, which were not seen in wild-type tissue. Sequencing confirmed that the upper band, indicated as KO_a, corresponds to the expected truncated mRNA product containing exons 1, 7, 8, and 9 of the Ybx3 gene (Figure 2D) [33]. This truncated message encodes a second open reading frame that corresponds to the 113-amino-acid sequence of the cold shock domain protein A, isoform CRA_a (Supplementary Figure S2A) [35]. Further analysis showed that the lower band visible in the knockout kidney, indicated as KO_b, results from alternative splicing and lacks exon 7. This mRNA encodes a potential fusion protein consisting of the 79 N-terminal residues of DbpA fused to the last 86 residues of CRA_a (Supplementary Figure S2B). To test whether this mRNA encodes a functional protein, we cloned the PCR products into the vector pEGFP-N1. Western blot analysis of transfected cell lysates with N-terminal anti-DbpA sera (aa 51-63) and anti-GFP confirmed the existence of CRA_a as well as the fusion protein, with products visible in the 35–50 kDa range (Figure 2E). This is the first report that shows CSDA_CRA_a encodes a gene product of unknown function. The growth of the knockout mice was comparable with that of their wild-type littermates, despite a trend of reduced weight. Ybx3-deficient mice showed normal kidney development and function, and a normal distribution of blood cells, indicating that hematopoiesis is not impaired (Figure 3A–H). Consistent with published results showing that Ybx3 mRNA is downregulated during kidney development, we previously reported that the DbpA protein is not detectable in healthy kidney tissue; however, DbpA expression is induced in mesangioproliferative diseases [7]. Therefore, we first analyzed tissue lysates 5 and 14 days after UUO induction to determine to what extent DbpA was expressed (Figure 4A). Immunostaining with anti-DbpA serum showed a strong induction of a 50 kDa protein corresponding to the short isoform DbpA_b at 14 days in kidney lysates from wild-type mice, which was not observed in the knockout. The induction of platelet-derived growth factor receptor-β (PDGFR-β), E-cadherin, and β-catenin served as a control for successful disease induction, whereas glyceraldehyde-3-phosphate dehydrogenase (GAPDH) staining showed that comparable amounts of tissue lysates were loaded [36,37,38]. Activation of YB-1 was also seen upon disease induction, as previously reported [28]. E-cadherin and YB-1 expression appeared enhanced in the Ybx3 knockout. The latter finding is supported by observations that these cold shock proteins counter-regulate one another [39,40,41]. Immunohistochemistry showed that in healthy kidneys, DbpA was primarily expressed only in vascular smooth muscle cells, which is in agreement with our previous observation [7]. However, after UUO induction, a dramatic upregulation of DbpA expression was observed within the kidney cortex (Figure 4B). Closer examination showed that DbpA was primarily expressed in the interstitium, with little or no staining visible in the glomeruli or tubuli. DbpA showed a membranous staining in the vasculature of healthy kidneys, which fits the reported junction-associated activities of DbpA [11,21]. In contrast, the disease-induced DbpA showed greater nuclear staining after UUO induction. Published single-cell RNA sequencing data from kidneys of UUO mice show an induction of Ybx3 expression in proliferating proximal tubular cells and activated fibroblasts (Figure 4C) [42,43]. Surprisingly, the pattern of DbpA expression in the kidney resembles that of tissue-resident dendritic cells (Figure 4D) as well as type I collagen (Col1a1), which is upregulated in the tubulointerstitium during fibrosis (Figure 4E) [2,44,45,46]. Both collagen I staining and the number of CD11c-positive cells were reduced in obstructed Ybx3-deficient kidneys compared with wild-type kidneys.

To assess tissue damage, we performed periodic acid–Schiff (PAS) staining on contralateral, 5-day, and 14-day UUO kidney sections from both wild-type and Ybx3-deficient mice (Figure 5A). Analysis of the 5-day and 14-day UUO sections from the wild-type mice showed the expected induction of tubular damage, as evidenced by the large vacuole-like structures resulting from cell death. Ybx3-deficient mice demonstrated a significantly higher number of cells attached to the basement membranes along the nephron, starting from the proximal to distal tubular segments and the collecting duct on day 5 of UUO. These differences were no longer seen on day 14 of the disease, suggesting that the loss of DbpA protects against the immediate early damage caused by UUO. One component of tubulointerstitial nephritis is an increased recruitment of immune cells into the kidney following disease induction. In order to better quantify the number of immune cells present, the kidneys were harvested after 5 or 14 days of UUO and analyzed via flow cytometry following tissue disruption. Flow cytometry showed no difference in the number of leukocytes in the healthy contralateral kidneys of wild-type and Ybx3 knockout animals. However, following the induction of UUO, we observed an increase in the number of infiltrating CD45-positive cells in both wild-type and Ybx3 knockout animals; however, the number of infiltrating cells was reduced in Ybx3 knockout mice compared to the wild type (Figure 5B and Figure S3). Among the CD45+ subsets, we observed a similar trend of reduced numbers of infiltrating CD11b+ myeloid cells, macrophages, NK cells, neutrophils, and CD3+ T cells. Importantly, no differences were observed in the blood, indicating that the lack of cell infiltration does not result from a hematopoietic defect in these animals (Figure 3 and Figure S4).

Since maximum DbpA expression was observed late (day 14) and appeared most abundant in the cortical renal interstitium, we focused our analysis on the development of renal fibrosis, a key feature in the progression of chronic kidney disease. Tenascin-C is a major component of the fibrogenic niche, which is known to be upregulated in UUO [47]. Whereas wild-type mice showed the expected induction of tenascin-C expression upon disease induction, Ybx3 knockout mice showed a marked reduction in tenascin-C expression compared to the wild type (Figure 6A). Cortical alpha smooth muscle actin (αSMA) is considered a marker of myofibroblast activation and thus fibrosis. αSMA staining of healthy kidneys showed a predominant vascular pattern similar to that of DbpA. However, following disease induction, a marked increase in αSMA positivity was observed within the interstitium of WT mice, which was significantly reduced in the absence of DbpA (Figure 6B). To measure the changes in total collagen, tissue lysates were hydrolyzed, and the hydroxyproline content was determined. As expected, the collagen content increased upon disease induction, consistent with the development of fibrosis; however, no differences were observed between wild-type and knockout mice (Figure 7A). Sirius red staining of the tissue showed enhanced collagen deposition in wild-type mice after disease induction that was reduced in the Ybx3 knockout (Figure 7B,C). Utilizing polarized light to determine the ratio of collagen type I to type III showed that this too was altered between the wild type and knockout (Figure 7D). The determination of transcript numbers via qRT-PCR confirmed the induction of both collagen type I and type III following UUO, which is consistent with the development of fibrosis. However, PCR analysis showed that transcripts for both collagen type I and type III were reduced in the Ybx3 knockout animals compared with the wild type (Figure 7E).

Here, we show, for the first time, that Ybx3 deletion does not appear to influence kidney development or function in healthy mice. However, following ureter ligation, we observed a strong induction of the DbpA protein within the renal tubulointerstitium in wild-type mice. We observed a significant difference in tissue damage, especially tubular cell loss, on day 5 of disease, along with a significant reduction in the number of infiltrating immune cells in Ybx3 knockout animals. Since we observed no differences within the composition of cells within the blood, this suggests that DbpA does not play a role in hematopoiesis. Considering that DbpA is expressed in the vasculature of healthy kidneys and that immune cell infiltration is reduced by the loss of DbpA, we cannot exclude a potential role for DbpA in regulating either the expression or activation of adhesion molecules (e.g., selectins and integrins) that are used by immune cells for transmigration. However, given that the expression pattern of DbpA-positive cells in the tubulointerstitium is remarkably similar to that of CD11c+ tissue-resident renal dendritic cells (DCs), it may be that DbpA protein expression is induced in renal DCs upon activation in response to tissue injury. Renal DCs share a number of markers with infiltrating monocytes/macrophages (e.g., F4/80, CD11b, MHC-II), making it difficult to distinguish these cell types from one another. Renal DCs adopt a pro-inflammatory phenotype following ureter obstruction, even though they do not appear to directly contribute to fibrosis [48]. This is consistent with our previous observations that immune cell infiltration and fibrosis are distinct and separable events [29]. In this context, dendritic cells within the renal interstitium are ideally positioned for immune surveillance, as their foot processes extend into the tubules, allowing them to take up antigens [49]. Here, renal DCs play an important role in initiating immune cell infiltration by acting as sentinels and producing chemokines in response to damage-associated molecular patterns (DAMPs) released during tissue injury [50]. DCs also express receptors of the C-type lectin (CLEC) family, such as the mannose receptor, which bind collagen types I–IV via a fibronectin type II domain [51]. In the kidney, pericytes and fibroblasts are recognized as the primary sources of myofibroblasts, which are the major matrix-producing cells, including collagen type I [52]. These cells are clearly present in the tubulointerstitium, consistent with our data. Further investigation of the key markers of renal fibrosis showed a reduced induction of both tenascin-C and αSMA in Ybx3 knockouts, as well as collagen I and III. The reduction in collagen expression may result from an increase in the YB-1 protein, as YB-1 is a known negative regulator of collagen expression [53]. Overall, the data clearly indicate a role for DbpA in supporting tissue fibrosis. The hypothesis that Ybx3 expression is induced in activated fibroblasts is supported by single-cell RNA sequencing data from the kidneys of UUO mice (Figure 4C) [42,43]. This dataset also reports DbpA expression in proliferating proximal tubules, which we did not observe. However, since DbpA is released from the cell membranes when tight junctions are disrupted, and translocates to the nucleus to drive proximal tubule cell proliferation, one cannot rule out the possibility that an experimental artifact was introduced into the dataset during the preparation of the tissue for single-cell analysis [21]. There are several possible mechanisms by which DbpA may regulate fibrosis. First, as a member of the cold shock domain protein family, DbpA is similar to YB-1 in both structure and function. Both proteins share an evolutionarily conserved cold shock domain that binds single-stranded RNA/DNA [4]. Recently, it was demonstrated that both proteins share an overlapping affinity for substrates, which is not surprising, as both proteins are found within the ribonucleoprotein complexes that regulate RNA stability and splicing [41]. Therefore, one possible mechanism is that DbpA translationally regulates the mRNA of tenascin-C and αSMA. However, since fibrosis is determined by the rate of extracellular matrix production as well as the rate of degradation, the latter being regulated by the levels of matrix metalloproteases (MMPs) and their inhibitors (TIMPs), it is foreseeable that DbpA could exert its influence by regulating the expression of these proteins as well. To further complicate matters, both microRNAs and lncRNAs are known to exert regulatory effects on mRNA expression and are themselves regulated by cold shock proteins [54,55]. Since the cold shock domain is conserved between Ybx1 and Ybx3, it is possible that some of the beneficial effects observed upon targeting YB-1 are indeed mitigated by DbpA. It is interesting to speculate that manipulating factors that are responsible for inducing DbpA expression may be a means to treat renal fibrosis.

Here, we report disease-specific upregulation of YBX3 transcripts within different compartments of the kidney in patient biopsies, which agrees with our previous immune histochemical findings [7]. Since growth and kidney function do not appear to be influenced by the deletion of Ybx3, we can conclude that DbpA is not as essential for development as Ybx1 has been shown to be [56,57]. Following disease onset, we showed a strong induction of the short (50 kDa) isoform of the Ybx3 protein (DbpA_b) in the kidney interstitium, where the DbpA-positive cells reside. How the two isoforms differ in function remains to be shown. However, the reduction in immune cell infiltration and the reduced expression of fibrotic markers in knockout mice lead us to conclude that DbpA upregulation contributes to the development of fibrosis in our disease model. Whether this conclusion holds up in other disease models and other organs has yet to be demonstrated. However, it is interesting to speculate that targeting DbpA may be a means to slow the development of fibrosis and thereby prolong kidney function.