A fetal sheep model for studying compensatory mechanisms in the healthy contralateral kidney after unilateral ureteral obstruction

Journal of Pediatric Urology (2015) 11, 352.e1e352.e7

A fetal sheep model for studying compensatory mechanisms in the healthy contralateral kidney after unilateral ureteral obstruction a

Department of Pediatric Surgery, Medical University of Vienna, Austria

b Department of Pediatrics, Medical University of Vienna, Austria

c

Division of Biomedical Research, Medical University of Vienna, Austria d Section Ruminants, Education and Research Farm, University of Veterinary Medicine Vienna, Austria

e emergentec biodevelopment GmbH, Vienna, Austria

f

Department of Pathology, Leeds Teaching Hospitals NHS Trust, UK g

Department of Paediatric Urology, Leeds Teaching Hospitals NHS Trust, UK Correspondence to: C. Aufricht, Department of Pediatric Nephrology, Medical University of Vienna, Waehringer Guertel 18e20, 1090 Vienna, Austria, Tel.: þ43 140 400 2908; fax: þ43 140 400 6812 christoph.aufricht@ meduniwien.ac.at (C. Aufricht) Keywords Fetal uropathy; Sheep model; Signaling; Renoprotection; Stress response

Alexander Springer a, Klaus Kratochwill b, Helga Bergmeister c, Dagmar Csaicsich b, Johann Huber d, Bernd Mayer e, Irmgard Mu ¨hlberger e, Jens Stahlschmidt f, Ramnath Subramaniam g, Christoph Aufricht b Summary Introduction Fetal unilateral ureteral obstruction (UUO) triggers complex pathophysiology involving not only the affected organ but also the contralateral kidney, which undergoes evident compensatory changes. Objective We hypothesized that it would be possible to characterize a transcriptomic fingerprint and selected molecular mechanisms for compensatory growth of contralateral kidneys in UUO, specifically focusing on mediators, carriers, membrane transport, and organ crosstalk in an ovine fetal UUO model. Study design A fetal ovine model of complete UUO was created on the 60th day of gestation. For transcriptomics profiling, total RNA was extracted from vital renal biopsies of contralateral (non-obstructed) kidneys harvested on the 80th day of gestation, and kidneys of untreated fetuses served as controls. Statistical analysis provided the set of differentially regulated genes further forwarded to bioinformatics analysis for identification of eventual compensatory molecular mechanisms. Histological analysis was performed with hematoxylin and eosin and periodic acideSchiff stains. Results Contralateral kidneys showed compensatory hypertrophic renal growth, represented on the molecular side by 324 protein coding genes differentially regulated compared with the control kidney samples. Bioinformatics analysis identified an

interactome (Figure) consisting of 102 genes with 108 interactions mainly involving transporters (protein transport and protein localization as well as in protein degradation), signaling molecules, DNA/ nucleotide/RNA processing, and components of catabolism and cell cycle regulation. Within the interactome, nine receptors were identified as differentially regulated on the contralateral kidney, involving potential renoprotective ligands of the prostaglandin and the bradykinin receptor, arginine vasopressin receptor 1B, and integrin beta 4. Interestingly, a broad range of molecules found differentially expressed, has been previously described in stress response, renoprotection and repair (e.g., MAPK3, MCP1, DICER1, and others). Discussion The compensatory renal growth interactome provides a network of transcripts significantly altered in the contralateral kidney, potentially allowing novel insights into mechanisms, interactions, and signaling pathways associated with compensatory growth, and renal protection and repair. Interestingly, the finding of an embedded gene signature reflecting signaling and communication suggests a key role of these processes in CRG either by crosstalk, soluble substances, carriers, or membrane signaling. Conclusions Using a transcriptomics approach, it was possible to identify a gene expression fingerprint of contralateral renal growth in a fetal UUO model. Further studies are warranted to validate those processes and to allow incorporation of this knowledge in new fetal diagnostic or even therapeutic strategies.

Received 29 October 2014 Accepted 2 April 2015 Available online 3 July 2015

http://dx.doi.org/10.1016/j.jpurol.2015.04.041 1477-5131/ª 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Molecular mechanisms in compensatory fetal renal growth

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Figure

Introduction Compensatory renal growth (CRG) after unilateral renal damage is a clinically well-known phenomenon. Sacerdotti in 1896 first attempted to explain CRG in an experimental setting in adult dogs [1]. Since then, there has been extensive experimental work investigating CRG after unilateral nephrectomy or obstruction in adult, neonatal, and fetal animal models. However, the underlying molecular mechanisms of CRG remain only partially understood. CRG control is thought to be determined by various factors including vasoactive, growth, and sexual hormones, the kallikreinekinin system, renin, the angiotensin system, bradykinin, aldosterone, natriuretic hormone, endothelins, cytokines, extracellular matrix proteins, insulin-like growth factors, and a range of other signaling molecules [2e5]. There have been several studies using gene expression (GE) profiling techniques to portray molecular alterations in renal growth and obstructive kidney disease. In 2004, Seseke et al. [6] were the first to use cDNA microarrays to characterize GE in an adult unilateral ureteral obstruction (UUO) mouse model. Becknell et al. [7] evaluated global renal transcription with graded hydronephrosis in a megabladder mouse model identifying three primary pathways

associated with kidney remodeling/repair. Hauser et al. [8] explored the early transcriptional response of the contralateral kidney to UUO or unilateral nephrectomy in the adult rat model. Fetal renal physiology, however, is known to fundamentally differ from postnatal life in regards of renal blood flow, glomerular filtration rate, tubular function, urine production, and concentrating ability [9]. Wu et al. [10] recently described comprehensive GE changes and biological pathway analysis associated with mouse postnatal kidney development, showing widespread changes in GE over time. Over the years, several different fetal animal models have been developed showing hyperplastic and hypertrophic CRG changes depending on severity and duration of UUO [11]. One particularly well-established system is the fetal ovine model. UUO in the sheep fetus leads to hypertrophic compensatory growth in the opposite kidney with significantly larger size and normal histology, but no increase in total glomerular number [12]. In this work we sought to gain more insights into CRG specifically regarding molecular signaling mechanisms and renal crosstalk by using for the first time an exploratory, genome-wide GE approach in a fetal sheep model. Characterizing and understanding the consequences of such crosstalk, be it damaging or renoprotective for the contralateral kidney, might educate on novel

352.e3 therapeutic strategies for protecting renal function also beyond UUO.

A. Springer et al. differentially GE when comparing contralateral kidneys with healthy control kidneys a t test with p < 0.05 was performed.

Material and methods Animal model Experimental procedures were performed in accordance with the Animal Ethics Committee of the Medical University of Vienna and the Austrian Federal Ministry of Science and Research (BMWF-66.009/0235-C/GT/2007). In a procedure described previously [13], complete UUO was triggered in three female fetuses on the 60th day of gestation; three healthy twins served as control. In brief, pregnant sheep ewes underwent anesthesia at 60 days of gestation (normal gestation is approximately 130 days; intervention at 60 days refers to earlyemid trimester). After hysterotomy, ureteral ligation was performed. At 80 days of gestation, a bilateral full thickness renal biopsy was taken and immediately put into liquid nitrogen. The fetus and the ewes were euthanized. The obstructed and contralateral kidneys as well as the kidneys of healthy twin fetuses were harvested and further processed. Histological analysis was performed on 2-mm sections of formalin (4%) neutral buffered fixed, paraffin-embedded kidneys after staining with hematoxylin and eosin and periodic acideSchiff.

Sample preparation, microarray hybridization, scanning, data processing In a procedure described previously [13], biopsies were homogenized then processed. Measurements of RNA yields and quality estimates were performed and total RNA was checked for integrity. Preparation of cDNA, hybridization to the Bovine Genome Array (Affymetrix, Santa Clara, CA, USA), and scanning of the arrays were carried out according to the manufacturer’s protocols. Microarray data preprocessing was done using the Affymetrix Microarray Suite 5.0. Steps included background correction, normalization, perfect match correction, and computation of expression values from probe intensities. In order to identify

Bioinformatics analysis Affymetrix Bovine Probe IDs showing significant differential regulation were mapped to Probe IDs on the Affymetrix Human Genome U133 Plus 2.0 Array using the NetAffx tool. Respective Entrez gene IDs and gene symbols were retrieved from the Ensembl BioMart. Proteineprotein interaction information was extracted from three publicly available repositories, namely IntAct, Reactome, and BioGrid, providing us with in total 233,794 interactions. From the list of significantly differentially expressed genes separating contralateral and healthy kidney a significant fraction was represented in the interaction data, allowing us to derive an induced subgraph with features differentially regulated and interaction with another identified feature according to the reference interaction databases. Assignment to biological processes was based on the category GOTERM_BP_FAT provided by DAVID (Database for Annotation, Visualization, and Integrated Discovery) representing a subset of specific terms out of the full GO (Gene Ontology) Biological Process term ontology. A specific subset of the genes of the interactome was annotated to the term “receptor activity”, again using the DAVID Functional Annotation tool. For such identified receptors we screened for corresponding ligands by searching the HPMR (Human Plasma Membrane Receptome) database and scientific literature in PubMed. To identify publications associated with protection, stress response, or repair and regeneration, a literature search in PubMed (status as of January 2014) was performed using keywords and MeSH terms renoprotection [Title/Abstract] OR “renal protection” [Title/Abstract] OR (protection [Title/Abstract] AND kidney [MeSH Term]), stress [Title/Abstract] AND response [Title/Abstract] AND kidney [MeSH Terms], and (repair [Title/Abstract] OR regeneration [Title/Abstract]) AND kidney [MeSH Term]. Protein coding genes linked to identified PubMed IDs were

Figure 1 (A) Kidneys after renal biopsies (arrow: ureteral ligation). (B) Contralateral kidney showing regular nephrogenic zone with ureteric buds (asterisk), comma and S-shaped bodies and maturing glomerulus (H&E, original magnification, 200).

Molecular mechanisms in compensatory fetal renal growth

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obtained via gene2pubmed including all mammalian genes, being mapped to their human homologs using the HomoloGene database) where necessary.

ducts. The lining urothelium showed an adequate maturation pattern. They showed a well-demarcated nephrogenic zone containing typical immature developmental constituents including S-shaped and comma-shaped bodies, nephrogenic mesenchyme, and ureteric buds (Fig. 1B). A total of 589 bovine probes linking to 767 human probes representing 324 human protein coding genes were identified as significantly different in abundance in renal biopsies from contralateral kidneys in sheep with UUO versus control fetal sheep kidneys. Mapping the 324 differentially regulated genes on consolidated proteineprotein interaction information resulted in a network holding 102 genes with 108 interactions (Fig. 2). For specifically addressing signaling mechanisms that lead to CRG in the contralateral kidney, we screened for receptoreligand interactions. Of the 102 genes of the interactome, nine features resemble receptors, including

Results After UUO (Fig. 1A) opposite kidneys were enlarged compared to kidneys of controls with a mean length of 2.3 cm and mean width of 1.2 cm versus a mean length of 1.6 cm and a width of 1.0 cm. However, in vivo renal biopsies precluded reliable post-mortem weighing of kidneys. There were no gross lesions identifiable. Histopathological features of the UUO and the contralateral kidney were basically similar and showed good corticomedullary distinction and similar numbers of maturing glomerular rows, proximal and distal tubules, and collecting ducts. The medulla showed well-developed papillae and collecting

Figure 2 Interactome of transcripts (nodes representing protein coding genes, edges are derived from protein interaction database consolidation) significantly altered in contralateral kidneys. Polygons identify nodes being reported in publications associated with kidney compensatory growth, protection, stress response, or repair. Nodes with receptor functionality are given in red, together with ligands according to Table 1. Nodes assigned to specific functional categories are grouped into respective clusters.

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A. Springer et al.

Table 1 Receptors (gene symbols and names) identified as differentially expressed, direction of regulation in the contralateral kidney, and associated ligands (gene symbols). Gene symbol, receptor

Regulation up/down

Gene name, receptor

Ligands

AVPR1B BDKRB2 ITGB4

Up Up Down

Arginine vasopressin receptor 1B Bradykinin receptor B2 Integrin, beta 4

PTGFR

Up

Prostaglandin F receptor (FP)

AVP KNG1 LAMA1, 2, 3; LAMB1, 3; DUSP18 PTGS2

the prostaglandin F receptor (PTGFR) and the bradykinin receptor B2 (BDKRB2). For four receptors, we could retrieve information on specific ligands (Table 1). Finally, we identified a subset of differentially regulated genes being reported in the context of renoprotection, stress response, repair, and regeneration (Table 2). Finally, 12 assignments to such processes could be identified with members of the fetal CRG interactome, supporting our hypothesis of potentially protective mechanisms taking place in UUO.

Discussion We present the first data on molecular signaling mechanisms and renal crosstalk in early CRG in the fetal sheep UUO model. As expected, UUO in earlyemid trimester caused clear evidence of fetal CRG contralateral to hydronephrosis and fetal renal dysplasia [13]. At that time of gestation, control mechanisms of CRG are thought to be mediated by renal crosstalk, hemodynamics, fluid and electrolyte balance, as well as acidebase homeostasis, counterbalance, and unknown regulatory placental influences [14]. Using an exploratory, genome-wide GE approach, we could identify a core protein interaction network that might provide a new basis for improved understanding of CRG. This CRG interactome is primarily characterized by gene signatures that reflect enhanced functional and/or structural capacity as compensation for contralateral nephron loss [15]. Several genes that are involved in protein transport and protein localization as well as in protein degradation via the ubiquitineproteasome system (shown in our findings as proteasomal ubiquitin-dependent protein catabolic process) reflect activated pathways and enhanced protein turnover as expected in renal remodeling and CRG. Such increase in

Table 2

cell mass is not only demonstrated by stimulation of protein synthesis but also likely to involve altered regulation of mRNA translation [16]. In addition, CRG is known to be closely linked to cell cycle events [3]. Correspondingly, ribonucleoprotein complex assembly and biogenesis, ncRNA processing and RNA splicing reflect high turnover and growth in kidneys undergoing CRG. In the interactome, a key component in miRNA processing was identified as differentially regulated, namely DICER1, an RNase III class enzyme. It has been shown that DICER1 action plays an important role for maintaining the viability of the critical self-renewing progenitor pool of miRNA and, consequently, development of a normal nephron development [17]. Taken together, the definition of the CRG interactome provides a network of transcripts significantly altered in contralateral kidneys, potentially allowing novel insights into mechanisms, interactions, and signaling pathways associated with compensatory growth, renal protection, and repair. Interestingly, the finding of an embedded gene signature reflecting signaling and communication suggests a key role of these processes in CRG either by crosstalk, soluble substances, carriers, or membrane signaling. During fetal life, how does a kidney perceive contralateral damage? Altered renal blood flow, changes in glomerular hemodynamics, different demands of solute excretion, different solute composition, increased work regarding solute transport, hyperfiltration, and neuroendocrine crosstalk between kidneys are mechanisms thought to activate a sequence of cellular processes leading to renal growth. Therefore, the second part of our analysis specifically focused on differentially expressed genes coding for proteins with receptors function together with their ligands as identified in the interactome (PTGFR, ITGB4, AVPR1B, and BDKRB2).

Results of literature search on key words in the context of renal repair, stress response, and protection.

Query, kidney

PubMed ID

Genes

DEGs

Interactome members

Renoprotection

4990

694

BDKRB2, CCL2, MCP1, PTGFR

Stress response

1526

519

BDKRB2, CCL2, MCP-1, PTGFR, ABCB1, CHRNA7, FAT1, HSD3B1, HSD3B2, SLC9A3, ST6GAL1 AVPR1B, BDKRB2, CDH1, CSPG4, DICER1, EP400, HYOU1, MAPK3, MKI67, BMPR2, PAK1, SLC9A3

Repair, regeneration

3336

567

CDH1, EP400, MAPK3, NOLC1, ABCB1

AVPR1B, BDKRB2, CDH1, CSPG4, DICER1, EP400, HYOU1, MAPK3, MKI67 CDH1, EP400, MAPK3, NOLC1

Note. The query context, number of publications identified, total number of genes assigned, and gene symbols are given, also identified as differentially regulated in unilateral ureteral obstruction (UUO) (DEGs), and additionally embedded in the UUO interactome (Fig. 2).

Molecular mechanisms in compensatory fetal renal growth

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PTGFR and its ligand COX-2 are among the bestdescribed players in respect to fetal nephrology and UUO. COX-2 expression is subject to regulation by several conditions affected by UUO, including salt and water turnover, medullary tonicity, growth factors, cytokines, and adrenal steroids [18]. It has previously been shown that UUO enhances eicosanoid production in cortical and medullary tubules of rat kidneys [19]. It seems that oxidative stress as a consequence of UUO stimulates COX-2 expression through the activation of multiple MAPKs and that the induction of COX-2 may exert a cytoprotective function in renal medullary interstitial cells [20,21]. Upregulation of integrin beta 4 (ITGB4) was recently reported to be associated with tubulointerstitial fibrosis in human obstructive uropathy [22]. Integrins are transmembrane receptors that mediate connection between cells and the underlying extracellular matrix (ECM) [23]. ITGB4-dependent signaling is involved in the regulation of cell survival, migration, and differentiation, as well as bidirectional signal transduction across the cell membrane [22]. Ligands associated with ITGB4, such as LAMA1, play a critical role in kidney function and kidney aging [24]. Bradykinin, originating from the kallikrein system, is one of the most important vasoactive hormones acting in a paracrine manner to regulate nephrovascular growth, differentiation, and physiological functions: it binds BDKRB2, which is a G-protein coupled receptor, well-known in regulating renal blood flow and salt and water excretion during nephrogenesis intimately coordinating with the terminal differentiation of the distal nephron [25]. Potential beneficial effects of bradykinin have been shown in different forms of kidney injury with previous reports on reduced bradykinin levels in obstructed kidneys and reduced renal fibrosis upon vivo BDKRB2 activation, supporting renoprotective contralateral renal effects [26]. In the final part of our analysis, we aimed to systematically integrate our findings with evidence on protective mechanisms potentially relevant in the contralateral kidney in UUO. We hypothesized that molecular players in CRG detected in our study have been previously reported to drive stress responses and trigger renoprotection, regeneration, and cellular repair mechanisms. Our literature research indeed identified 12 such genes as involved in the CRG interactome (not discussed in detail here). Taken together, our results support the profound role in particular of well-defined ligandereceptor interactions with regards to remodeling and function of mammalian nephrons also in CRG. These signaling pathways are implicated in a wide variety of biological processes in the developing kidney and it seems obvious that our findings may open new diagnostic and/or therapeutic options in the context of CRG as these processes are amenable to known interventional approaches. Although molecular mechanisms associated with fetal CRG may signal and counteract effects of contralateral UUO on renal homeostasis, likely providing a benefit to the fetus in the short term, it is increasingly recognized that CRG implicates increased susceptibility to renal failure, kidney disease, and hypertension later in life [27]. In the long run, selected molecular features linked to CRG may thus be linked to potentially damaging processes culminating in apoptosis, inflammation, and fibrosis. For example,

whereas epithelial mesenchymal transition (EMT) may be taken as an early renoprotective mechanism in CRG preventing fibrosis, EMT also plays a crucial role in the progression of renal interstitial fibrosis. Fetal hypertrophic and hyperplastic changes may predispose to progressive increase in arterial pressure and a loss of renal function with age [28]. Clinically, the KIMONO study, a large, retrospective cohort study, clearly showed that unilateral damage due to obstruction with a solitary functioning kidney (following CRG) is a risk factor for later chronic renal disease. Children with a single functioning kidney are at a higher risk for chronic kidney disease in adulthood [29]. Functionally, the long-term renal outcome in CRG may thus be troubled with hyperfiltration and arterial hypertension mediated by specific ligands, altogether becoming clinically relevant. Knowledge of potential renoprotective mechanisms in the CRG that are amenable to therapeutic interventions will very likely allow new therapeutic approaches to be established as has already been started with COX-2 inhibitors [30]. Given the complexity of the full spectrum of obstructive uropathy, it will be necessary to develop parallel markers with high sensitivity and specificity reflecting the amount of renal damage [31]. Therefore, a further step ahead will be to correlate transcriptomic findings with those of other omics, especially proteomics. For the moment, there are not many proteomic data available on renal repair mechanisms. However, studies of urine proteomics in UUO may in fact already present the fingerprint of compensatory contralateral mechanisms. Given this fact, it is not surprising that well-known players of cellular stress response, cell cycle, and signaling are routinely reported in proteomic studies of obstructive uropathy [32]. In conclusion, this study used a transcriptomics approach to identify a GE fingerprint of contralateral renal growth in a fetal UUO model. We identified an interactome of CRG with components triggering cell communication together with a range of receptoreligand pairs potentially involved in sensing contralateral kidney status. Interestingly, a range of molecules has been previously described in stress response, renoprotection, and repair. Understanding these potentially damaging or protective mechanisms might in the future help in implementation of preventive and therapeutic strategies. Further studies are warranted to validate those processes and to allow incorporation of this knowledge in new fetal therapeutic strategies.

Conflict of interest None.

Funding None.

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