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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2016 May 16;27(12):3725–3737. doi: 10.1681/ASN.2014111124

Partitioning-Defective 1a/b Depletion Impairs Glomerular and Proximal Tubule Development

Oleh Akchurin *, Zhongfang Du *, Nadira Ramkellawan *, Vidhi Dalal *, Seung Hyeok Han †, James Pullman ‡, Anne Müsch §, Katalin Susztak †, Kimberly J Reidy *,†,✉
PMCID: PMC5118470  PMID: 27185860

Abstract

The kidney is a highly polarized epithelial organ that develops from undifferentiated mesenchyme, although the mechanisms that regulate the development of renal epithelial polarity are incompletely understood. Partitioning-defective 1 (Par1) proteins have been implicated in cell polarity and epithelial morphogenesis; however, the role of these proteins in the developing kidney has not been established. Therefore, we studied the contribution of Par1a/b to renal epithelial development. We examined the renal phenotype of newborn compound mutant mice carrying only one allele of Par1a or Par1b. Loss of three out of four Par1a/b alleles resulted in severe renal hypoplasia, associated with impaired ureteric bud branching. Compared with kidneys of newborn control littermates, kidneys of newborn mutant mice exhibited dilated proximal tubules and immature glomeruli, and the renal proximal tubular epithelia lacked proper localization of adhesion complexes. Furthermore, Par1a/b mutants expressed low levels of renal Notch ligand Jag1, activated Notch2, and Notch effecter Hes1. Together, these data demonstrate that Par1a/b has a key role in glomerular and proximal tubule development, likely via modulation of Notch signaling.

Keywords: kidney development, proximal tubule, cell adhesion, renal epithelial cell, ureteric bud, podocyte

Renal epithelial cells exhibit apico-basal polarity with polarized expression of proteins and cell-cell adhesion complexes that facilitates directional solute transport. Loss of polarity and decreased cell-cell adhesion contribute to renal dysfunction in AKI.1,2 Renal epithelial cells originate from nonpolarized metanephric mesenchymal cells, and how these epithelial cells develop polarity and establish cell-cell adhesion is not fully understood.

Kidney development begins with the outgrowth of the ureteric bud from the Wolffian duct. Wnt signals from the tips of the branching ureteric bud induce the surrounding metanephric mesenchyme to undergo a mesenchymal to epithelial transition, forming a sphere of epithelial cells known as the renal vesicle.3 The renal vesicle becomes a comma- and then an S-shape body. The midportion and tip of the S-shape body become the proximal tubular epithelial cells and podocytes, respectively.4 Notch ligands Jag1 and Dll1 are expressed in the midportion, whereas Notch1 and Notch2 receptors are expressed in both the midportion and future podocytes of the S-shape body.5 Genetic defects in Jag1 and Notch2 cause Alagille syndrome, which is associated with renal aplasia, dysplasia, cystic kidney disease, and bile duct aplasia.6 Genetic modification of Notch signaling during kidney development leads to time and cell-type specific effects.7 Complete deletion of Notch2 in the kidney mesenchyme results in failure of glomerular and proximal tubular development.4 Jag1 is the critical ligand for Notch2-mediated effects on the proximal tubule, and Jag1 mutants phenocopy the effects of Notch deletion.8 Conditional deletion of Notch in the cap mesenchyme results in proximal tubular cysts,9 while conditional deletion in stromal mesenchyme results in defects in vascularization and glomerulogenesis.10,11

Apico-basal polarity proteins have cell-specific effects in the developing kidney.12–15 Partitioning-defective (Par) proteins are a family of apico-basal polarity proteins initially identified for their role in establishing anterior-posterior polarity.16,17 A complex of Pard3/Par6/aPKC proteins localizes to the apical aspect of epithelial cells in the renal vesicle12 and contributes to podocyte differentiation.13,18–20 In contrast, the basolateral protein, Scribble, is dispensable for podocyte formation.15 Knockout of Dlg5, another basolateral polarity protein, results in collecting duct renal cysts, indicating Dlg5 is required for collecting duct but not proximal tubular development.14

Par1 family members are serine threonine kinases that regulate a variety of fundamental cellular processes, including cell shape, morphogenesis, and cell-cell and cell-extracellular matrix adhesion.21–26 Four homologs of Par1 have been identified in mammalian systems: Par1a (MARK3/CTAK), Par1b (MARK2/EMK), Par1c (MARK1), and Par1d (MARK4). All four Par1 proteins have significant sequence homology and are functionally redundant on kinase assays.27–31 Knockout of individual Par1 homologs (a, b, and d) did not result in a renal developmental phenotype, likely due to compensation for one another in vivo.28–30,32,33

Interactions between Par1 and Notch signaling pathways have been described in Drosophila and Xenopus,34–36 but not in mammalian development. Pard3/Par6/aPKC can restrict Par1 localization and activity and may act upstream of Par1 interactions with Notch.35,37 Par1 localizes to the lateral aspects of MDCK cells. Block of Par1 function with a dominant negative construct in MDCK cell culture resulted in mislocalization of E-cadherin, loss of cell adhesion, and apoptosis.38

Here, we examined the role of Par1a/b in renal epithelial development in vivo. Our data demonstrate that Par1a/b deletion impairs ureteric bud branching, renal proximal tubular and glomerular epithelial development, and Notch signaling in mice.

Results

Par1a/b Are Expressed in Developing Nephrons

Par mRNA and protein expression was examined in developing mouse (Figure 1, A, B, and D, Supplemental Figures 1, 2D, and 3, C–E) and rat kidneys (Figure 1C, Supplemental Figures 2, A–C, and 3A). Par mRNA was highly expressed in embryonic rat kidneys, as compared with adult levels (Figure 1C, Supplemental Figure 3A), consistent with an earlier report.27 Peak levels of Par1a/b mRNA expression at rat embryonic day (E) 16.5 coincided with expression of Notch2, and preceded peak expression of terminal nephron differentiation markers, such as Nphs1 and Nphs2 (Supplemental Figure 2, A–C). Together, these data indicate that the temporal expression of Par1a/b mRNA in developing rat kidneys correlates with nephron segmentation. Western blot for Par1a and 1b in developing rat kidneys was congruent with mRNA expression, and demonstrated markedly decreased expression of Par1a/b in adult rat kidneys (Supplemental Figure 3A).

Figure 1.

Figure 1.

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Par proteins are upregulated during murine nephrogenesis. (A) Immunostaining demonstrating membrane Par1a (gray) expression in the developing mouse nephron (schema of developing nephron stages depicted to left). Merged images demonstrate colocalization with Six2 (induced mesenchyme, green; upper row) and Pax2 (S-shape body, green; middle row). Lower panel: Costaining with apical proximal tubular marker (LTL, green) demonstrates basal expression in newborn proximal tubules. (B and D) Immunostaining demonstrating membrane Par1b expression in developing mouse glomeruli (schema indicates early stage developing glomerulus). (B) Par1b (red) colocalized with podocalyxin (green) in developing podocytes. (D) Par1b in early and later stage developing glomeruli. Lateral podocyte membrane staining is visualized in later stage developing glomeruli. (C) Real-time PCR demonstrating increased mRNA expression of Par1 homologs Par1a (MARK3) and Par1b (MARK2) in embryonic and newborn (NB, day of life 1) rat kidneys, as compared with adult rat kidneys (8–10 weeks). A.U., arbitrary units; UB, ureteric bud.

To determine the localization of Par1a/b in the developing mouse nephron, immunostaining with antibodies specific to Par1a and Par1b was performed (Supplemental Figure 1). Developing nephron structures were colabeled using markers of induced metanephric mesenchyme (Six2), S-shape bodies (Pax2), proximal tubules (Lotus Tetragonolobus Lectin; LTL), and developing podocytes (podocalyxin) (Figure 1, A and B, Supplemental Figure 3). In newborn wild-type mouse kidneys, strong Par1a expression was identified in the nephrogenic cortex, with less expression in deeper, more mature nephrons (Figure 1A, Supplemental Figure 1A). Membrane expression of Par1a was identified in induced mesenchyme and S-shape bodies, and at the basal aspect of newborn proximal tubules (Figure 1A). Par1b expression was identified in the S-shape bodies of nephrogenic cortexes and in the renal papilla of newborn mouse kidneys (Supplemental Figures 1B and 3, C–E). This was consistent with microarray data from microdissected embryonic mouse kidneys (obtained from the GenitoUrinary Development Molecular Anatomy Project [GUDMAP]), with elevated Par1a and 1b expression in the S-shape body that correlated with Jag1 and Notch2 expression (Supplemental Figure 2D).39,40 Overlap of Par1a and 1b was present, but strong Par1b membrane expression was uniquely present at lateral aspects of developing podocytes (Figure 1B). Congruent with this, robust Par1b protein expression was identified in cultured podocytes (Supplemental Figure 3B).

Par1a/b HK and KH Kidneys Are Hypoplastic with Decreased Nephron Number

In order to examine the functional role of Par1a/b during kidney development in vivo, we next examined the renal phenotype of newborn progeny of dual heterozygous Par1a+/−:Par1b+/− (Par1a/b HH) mice. Consistent with an earlier report,29 mice with loss of three Par1a/b alleles (Par1a/b KH or HK) were born at less than predicted Mendelian ratios and no double knockout mice were obtained. High perinatal mortality was observed in Par1a/b KH and HK mice, with 25% and 75% of newborn pups dead on the first day of life, respectively. Par1a/b KH and HK pups were smaller than control littermates, with Par1a/b HK pups affected the most severely (Figure 2A).

Figure 2.

Figure 2.

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Par1a/b mutant kidneys are hypoplastic with decreased nephron number. (A) Decreased body size in Par1a/b HK newborn pup compared with the littermate control. (B) Par1a/b mutant HK and KH kidneys appeared grossly hypoplastic. (C) Decreased kidney-to-body weight ratios in Par1a/b HK and KH newborn mice. (D) Decreased glomerular number per midsagittal kidney section in Par1a/b KH and HK kidneys compared with the wild type (WW). (E–G) Hematoxylin and eosin Par1a/b control (CTR), HK, and KH newborn kidneys. (E) Low magnification demonstrating hypoplastic appearance of the Par1a/b mutant HK and KH mutant kidneys. (F) Par1a/b HK and KH kidneys have fewer nephrons. (G) Higher magnification demonstrating immature capillary loop glomeruli in Par1a/b HK kidneys, as compared with CTR and kidneys. * indicates Par1a/b KH versus WW; ** indicates Par1a/b HK versus WW; P<0.001.

Par1a/b KH and HK kidneys appeared hypoplastic compared with littermate controls (Figure 2B). Kidney-to-body weight ratio in Par1a/b KH, and particularly, HK mice was decreased, suggesting a specific effect of Par1a/b on kidney development beyond that expected with low birth weight (Figure 2C). Individual knockout of Par1a or 1b alone (Par1a/b KW, Par1a/b WK, or Par1a/b HH) did not cause a change in kidney-to-body weight ratio or morphology that was apparent upon histologic examination, indicating that two copies of either Par1a or 1b are sufficient for normal renal development (data not shown).

On histologic examination, developing nephrons were identified in the cortical nephrogenic mesenchyme of wild-type kidneys, while the deepest nephrons appeared mature (Figure 2, E–G). In contrast, Par1a/b KH and HK mutant kidneys had fewer nephrons (Figure 2, E–G). Quantification of glomeruli per midsagittal cross-section confirmed that Par1a/b KH and HK kidneys had decreased nephron number (Figure 2D).

Par1a/b Are Required for Glomerulogenesis

On light microscopy, Par1a/b KH glomeruli appeared similar to control glomeruli. In contrast, Par1a/b HK glomeruli appeared immature, and even the deepest glomeruli exhibited simple capillary loops, suggestive of a glomerulogenesis defect (Figure 2G). Other dilated blood vessels were observed, suggestive of a vascularization defect in Par1a/b HK kidneys (Figure 2F). To examine the glomerular ultrastructure, transmission electron microscopy (TEM) was performed on glomeruli from E18.5. In control littermates, well formed podocyte foot processes (fp) were identified (Figure 3A). Some well formed podocyte fp could also be identified in Par1a/b KH glomeruli (Figure 3B). In contrast, podocytes appeared cuboidal and lacked fp formation in Par1a/b HK glomeruli, indicative of immature glomeruli (Figure 3C). Fewer Par1a/b mutants produced urine and urine albumin-to-creatinine ratios were higher than in controls (Supplemental Figure 4). Together, these data indicate that Par1a/b contribute to podocyte differentiation and glomerular development.

Figure 3.

Figure 3.

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Glomerulogenesis is defective in Par1a/b HK mutants. TEM demonstrating glomeruli of (B) E18.5 Par1a/b KH and (C) HK mice and (A) their control littermates (CTR). Panels in the upper two rows show low magnification views of open glomerular capillaries (Cap). Podocyte cell bodies (P) extend fp in (A) Par1a/b control and (B) Par1a/b KH glomeruli, whereas Par1a/b HK podocytes have cuboidal cell bodies (C). Bottom row panels show higher magnification view demonstrating well formed fp in (A) Par1a/b control and (B) Par1a/b KH glomeruli, and (C) lack of fp formation in Par1a/b HK glomeruli.

Ureteric Bud Branching Is Impaired by Par1a/b Deletion

To determine the mechanism by which nephron number decreased in Par1a/b mutant kidneys, ureteric bud branching was examined in kidneys from E12.5 (Figure 4). A 30%–40% decrease in ureteric bud branches was observed in mutant kidneys, consistent with the observed changes in nephron number and kidney-to-body weight ratios. Immunostaining with Six2 revealed that cap metanephric mesenchyme was maintained in Par1a/b mutants (Supplemental Figure 5A). Thus, impaired ureteric bud branching, rather than depletion of nephron progenitors, likely accounts for the decreased nephron number in Par1a/b mutants.

Figure 4.

Figure 4.

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Par1a/b deletion impairs ureteric bud branching. (A) Brightfield images demonstrating decreased kidney size of Par1a/b HK E12.5 kidneys versus littermate control. (B) Whole-mount embryonic kidney immunostaining for cytokeratin 8 demonstrating representative Par1a/b mutant (KH and HK) and control (CTR) littermate E12.5 kidneys. (C) Quantification of primary ureteric bud branches indicating decreased branching in Par1a/b mutants versus littermate controls; P<0.05.

Par1a/b Loss Impairs Proximal Tubular Development

In addition to renal hypoplasia, dilated tubules were identified in Par1a/b KH and HK mice (Figures 2 and 5). Quantification demonstrated a shift toward dilated tubules in Par1a/b mutants as compared with controls, with 5% cysts and 16%–23% microcysts (MC) (Figure 5D). The dilated tubules were LTL- and megalin-positive, and cytokeratin-8–negative, indicative of proximal tubular origin (Figure 5, A–C). LTL labeling in Par1a/b HK kidneys revealed a more severe phenotype, with decreased proximal tubules (Supplemental Figure 6A). This was associated with decreased expression of proximal tubule genes Vil1 and Irx1 (Supplemental Figure 6B).41

Figure 5.

Figure 5.

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Par1a/b mutant kidneys have dilated and cystic proximal tubules. Proximal tubules were immunostained with (A–C) LTL and (A, B) Megalin, and distal tubules with (C) cytokeratin 8. (A, B) Decreased number of proximal tubules in Par1a/b KH newborn kidneys compared with wild type (WW), low magnification. (B) LTL and Megalin positivity confirms origination of cystic dilatations in Par1a/b KH kidneys from the proximal tubule. (C) Cystic dilatations in Par1a/b KH newborn kidneys are LTL positive but cytokeratin 8 negative, confirming that they do not originate from the collecting duct. Note cytokeratin-8–positive distal tubules adjacent to unstained cystic dilated tubule. (D) Quantification of percentage of tubular lumens classified as dilated (TD II>TD I), microcyst (MC), or cysts. Par1a/b mutants have a shift toward a larger percentage of tubules that are dilated or microcystic versus controls. Five percent of tubules are cystic in Par1a/b KH mutants.

Due to the high perinatal mortality, we were unable to assess if Par1a/b KH mutants would progress to develop grossly cystic kidneys. Alterations in proliferation and defects in primary cilia may contribute to cystogenesis.42–44 Dilated tubules in Par1a/b KH kidneys lacked cilia, and cilia in nondilated tubules appeared shorter than in control littermates, based on the results of immunofluorescence staining for acetylated tubulin (Supplemental Figure 7, A and B). Proliferation detected by Ki-67 was identified in cystic tubules (Supplemental Figure 7C). Thus, ciliary and proliferation defects may have contributed to cyst formation.

Early Differentiation into Renal Vesicles Is Intact in Par1a/b Mutant Kidneys

As mesenchymal to epithelial transition is Wnt4 dependent,3 we examined Wnt4 expression in the Par1a/b mutant kidneys by real-time PCR and in situ hybridization (ISH). No difference in Wnt4 expression level or localization was identified in Par1a/b mutants versus controls (Supplemental Figure 5, B and D). Transcription of another marker for renal vesicle differentiation, Bmp2,45 was also not altered (Supplemental Figure 5D). To further assess if Wnt signaling was intact, we examined expression of a Wnt target, Lhx1 (Supplemental Figure 5C).46 Lhx1 immunostaining revealed similar expression and localization in Par1a/b mutant renal vesicles as compared with controls (Supplemental Figure 5C).

Pard3 Polarity Protein Localization Is Altered in Par1a/b HK Podocytes

To test if Par1a/b loss affected Pard3/Par6/aPKC during renal development, we examined localization of the scaffolding protein, Pard3, in renal vesicles (Supplemental Figure 8, A and B) and in the developing podocyte (Supplemental Figure 8, C and D). Pard3 localized to apical membranes in the renal vesicle and the developing ureteric bud in both mutants and control littermates, suggesting early polarization was not affected. Immunostaining for aPKC had a similar apical pattern (not shown). However, in control podocytes from E15.5, Pard3 localized predominately to the glomerular basement membrane (Supplemental Figure 8, C and D), as previously reported.18 In Par1a/b HK podocytes, Pard3 localized over the entire podocyte cell membrane (Supplemental Figure 8, C and D). Defects in other podocyte protein localization were also detected in Par1a/b HK podocytes. In controls, podocalyxin segregated from the slit diaphragm marker ZO-1, while colocalization was observed in Par1a/b HK podocytes (Supplemental Figure 8E). Slit diaphragm proteins podocin and nephrin strongly colocalized with the glomerular basement membrane in controls, whereas more nephrin, and to a lesser degree, podocin, could be detected on apical membranes in Par1a/b HK podocytes. These data are consistent with the TEM findings, and indicate defects in differentiation and polarization in Par1a/b HK podocytes.

Par1a/b Loss Disrupts Renal Proximal Tubular Adhesion Complexes In Vivo

We next examined the effect of Par1a/b on renal epithelial cell-cell adhesion. Cell-cell adhesion regulates renal tubular diameter and contributes to cystogenesis.47 Control proximal tubules demonstrated basolateral expression of E-cadherin and β-catenin, as expected. In Par1a/b mutant proximal tubules, both E-cadherin and β-catenin subcellular distribution was severely altered, with predominantly cytoplasmic localization (Figure 6). To determine whether Par1a/b also affects adhesion complex formation in distal nephron segments, E-cadherin expression in cytokeratin-8–positive tubules was examined (Figure 6B). The majority of distal tubules and collecting ducts had intact E-cadherin localization to basolateral membranes (Figure 6B, Supplemental Figure 9). Thus, Par1a/b mutants exhibited proximal tubular specific defects in cell-cell adhesion.

Figure 6.

Figure 6.

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Sub-cellular localization of adhesion molecules is altered in Par1a/b KH proximal tubules. (A) Immunostaining for basolateral markers β-catenin (left, green) and E-cadherin (right, green), and apical proximal tubule marker Megalin (red) demonstrating appropriate expression of Megalin in wild type kidneys (WW; upper row) near the lumen, and basolateral expression of E-cadherin and β-catenin. Par1a/b KH (middle row) and HK (lower row) with unpolarized (cytoplasmic) expression of E-cadherin and β-catenin. (B) Immunostaining for collecting duct marker cytokeratin 8 and E-cadherin demonstrates preserved basolateral expression of E-cadherin in both Par1a/b KH and HK mutants and wild type, indicating that Par1a/b deletion does not affect localization of cell-cell adhesion proteins in collecting duct of the nephron.

Expression of Notch Signaling Components Is Reduced in S-Shape Bodies in Par1a/b KH and HK

Notch signaling is a key pathway in glomerular and proximal tubular development, and the Par1a/b mutant phenotype was similar to mutants with impaired Notch signaling.9,48,49 Interestingly, impaired bile duct formation similar to an Alagille phenotype was observed in Par1a/b mutant livers, associated with decreased Jag1 expression (Supplemental Figure 10).50 Par1 has been linked to localization of Notch ligands and activation of Notch signaling in Drosophila.34 Therefore, we next examined whether Par1 could affect Notch signaling pathways. Immunostaining for Par1a and Jag1 demonstrated that Par1a and Jag1 were coexpressed in S-shape bodies (Figure 7A), and Jag1 was decreased in Par1a/b KH and HK Pax2-positive S-shape bodies (Figure 7B). Quantitative PCR and immunoblotting confirmed an overall decrease in Jag1 mRNA and protein expression (Figure 7, D and E). Immunoblotting identified decreased Jag1 expression as early as E13, which correlated with decreased activated cleaved Notch2 (N2ICD) and Notch effector Hes1 (Figure 7, E and F). Jag1 ISH in embryos from E18.5 demonstrated overall fewer Jag1-positive S-shape bodies in Par1a/b mutants (Supplemental Figure 11). Low levels of Dll1 expression were identified in S-shape bodies in controls, and slightly less in Par1a/b mutants (Supplemental Figure 12). To define if Notch activity was affected within S-shape bodies, immunostaining using an antibody to the cytoplasmic tail of Notch was performed. There was a decreased level of nuclear-activated Notch2 in Par1a/b mutants (Supplemental Figure 12). Notch activation leads to Hes1 transcription, and ISH revealed decreased Hes1 mRNA in Par1a/b mutant S-shape bodies (Figure 7, C and G). Together, our data indicate that Par1a/b deletion impairs Notch signaling in the developing S-shape body.

Figure 7.

Figure 7.

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Notch activation is decreased in Par1a/b mutant kidneys. (A) Immunostaining demonstrating coexpression of Par1a (green) and Jag1 (red). (B) Immunostaining for Jag1 (red) and Pax2 (green) demonstrating membrane expression in wild-type S-shape body that is decreased in Par1a/b KH and absent in Par1a/b HK kidneys. (C, G) ISH for Hes1 demonstrating brown labeled Hes1 in representative control S-shape body. Inset shows Hes1 puncta at higher magnification. Negative control probe on same S-shape depicted to the right. Scant brown Hes1 mRNA is present in Par1a/b mutant (KH and HK) S-shape bodies. (G) Quantification of ISH as number of Hes1 puncta in S-shape bodies, demonstrating decreased Hes1 mRNA in Par1a/b mutants; *P<0.05, Par1a/b KH versus control; **P<0.05, Par1a/b HK versus control. (D) Jag1 mRNA expression is decreased in Par1a/b KH and HK kidneys (fold change±SEM depicted). (E) Western blot depicting time course of Jag1 downregulation in Par1a/b mutants (M) versus control littermates (C). Decreased Jag1 expression in Par1a/b mutants is present in E13 kidneys and persists throughout renal development. (F) Western blots demonstrating decreased activated cleaved Notch2 (N2ICD) and Notch effecter Hes1 in Par1a/b mutants (M) versus controls (C). CTR, control; WW, wild type.

Discussion

These data describe, for the first time, an essential role for Par1a/b in ureteric bud branching and proximal nephron development in vivo (Figure 8). Par1a/b loss did not affect early differentiation into the renal vesicle, but rather impaired Notch activation in the S-shape body.

Figure 8.

Figure 8.

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Par1a/b regulate ureteric bud, proximal tubule and glomerular development. Working model of Par1a/b function in the developing kidney. Upper: Par1a/b is required for ureteric bud (UB) branching and establishes nephron number. Lower: In the presence of Par1a/b (+), Jag1 is properly localized to membranes in the S-shape body. This leads to proximal tubules with proper cell-cell adhesion, with basolateral E-cadherin and β-catenin expression. In addition, glomerulogenesis occurs and podocytes extend fps. With decreased Par1a/b gene dosage (–), membrane Jag1 expression is lost from the S-shape body and Notch activation is impaired, leading to proximal tubular defects and defective glomerulogenesis.

Par1a/b deletion decreased ureteric bud branching, leading to hypoplasia and low nephron number. Ureteric bud branching requires reciprocal signals between the metanephric mesenchyme and ureteric bud.51 Our model was limited in that we were unable to determine cell-specific effects of Par1a/b deletion. However, GUDMAP data indicates that Par1a/b are expressed in the ureteric bud and in bud tips (Supplemental Figure 2), and thus may have cell-autonomous roles in ureteric bud branching. It has been proposed that Notch activation in ureteric bud tips is required to maintain their identity.52 In line with our findings, McCright et al. demonstrated that homozygous Notch2 hypomorph and combined heterozygous Jag1+/−:Notch2 hypomorph kidneys also were hypoplastic, although ureteric bud branching was not assessed.49

Par1a/b deletion had a striking effect on localization of cell-cell adhesion proteins, E-cadherin, and β-catenin. This effect was observed in proximal but not distal tubules or collecting ducts. The phenotype was similar to that observed with conditional Notch deletion in induced mesenchyme,9 where focal areas of mislocalized E-cadherin and disruption of Megalin were detected in dilated cystic tubules.9 Decreased Jag1 expression in the S-shape body may have led to proximal-tubule–specific defects in cell-cell adhesion. An alternative explanation for the lack of collecting duct phenotype in Par1a/b mutants is that other apico-basal polarity proteins, such as Dlg5, are able to compensate for loss of Par1a/b in the distal nephron.

In the kidney, E-cadherin is a major transmembrane structural component of the adherens junction (AJ). The AJ is a key contributor to the proximal tubule epithelial barrier, and its disruption leads to solute back-leak in the setting of ischemic kidney injury.53 E-cadherin also contributes to cell-cell tight junction formation and cell polarization by stimulation of protein kinase C signaling.54,55 E-cadherin/β-catenin/α-catenin link the AJ to the cortical actin cytoskeleton and microtubules, enabling the complex to modulate cell shape changes during morphogenesis.56 p120 catenin binds the cytoplasmic domain of E-cadherin and is required for its membrane localization.57 p120 catenin deletion in the metanephric mesenchyme leads to loss of proximal tubular expression of E-cadherin and β-catenin, and proximal tubular cysts.58 Thus, the observed alterations in E-cadherin and β-catenin subcellular localization likely contributed to the abnormal proximal tubule morphology in Par1a/b mutants.

Finally, these studies demonstrate that Par1a/b are required for Jag1 expression and Notch activation at a critical location, the S-shape body. The S-shape body is the peak site of Notch activation in the developing nephron.8 Notch1 and Notch2 have distinct and sometimes opposing functions in specific cell types, which may be influenced by availability and competition between Notch ligands and presence of inhibitors.59 Conditional deletion of Notch2, but not Notch1, in the metanephric mesenchyme leads to failure of podocytes and proximal tubules to form, demonstrating the dominance of Notch2 in this process.4 While Notch1 and Notch2 expression overlaps, elegant studies by Liu et al. demonstrate that the ligand binding to the Notch2 versus the Notch1 extracellular domain leads to greater Notch activation,8 explaining this dominance. Of the two ligands, Jag1 is dominant, and conditional deletion of Jag1 in the induced mesenchyme leads to marked decrease in nephron number and proximal tubules.8 Although contributions of other pathways cannot be excluded, the proximal tubular and glomerular defects in Par1a/b mutant kidneys are similar to those of Notch hypomorphs and conditional mutants,4,10,11,49 suggesting that the impaired Notch activation mediates this phenotype.

Factors that regulate Jag1 expression are relevant not only in developing organisms, but also in cancer, neural stem cells, kidney disease, and genetic diseases such as Alagille syndrome.6,60–63 In Xenopus, Ossipova et al. demonstrated that Par1 regulates Dll1 localization and Notch signaling in the ectoderm and neurogenesis.35,36 Interestingly, in Drosophila, Par1 silencing affected Notch signaling pathways by inhibiting the localization of Dll to lateral membranes.34 Similarly, failure to deliver Jag1 to lateral membranes is one potential mechanism that could contribute to decreased Jag1 expression and Notch activation observed in Par1a/b mutant S-shape bodies. Jag1 transcription is stimulated by Notch activation,64 and thus loss of Notch activation may contribute to the decreased Jag1 mRNA expression observed in newborn kidneys. Par1 is reported to affect Notch signaling via phosphorylation of mind bomb.36 Using available antibodies we were able to identify expected tubular expression65 but not S-shape body localization (data not shown). Future studies with conditional deletion of Par1a/b will be helpful to understand cell-specific effects and downstream targets.

Concise Methods

Animal Studies

All animal studies were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and Albert Einstein College of Medicine’s Institute for Animal Studies guidelines. Timed-pregnant Sprague–Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). Newborn day of life 1 and adult Sprague–Dawley rats (8–10 weeks of age) were euthanized and kidneys were removed for analysis.

Par1a/b Mutant Mice

Par1a+/- mice29 were purchased from The Jackson Laboratory (Bar Harbor, ME; B6.129×1-Mark3tm1Hpw/J, stock number 009366), Par1b+/− mice28 were a generous gift from Dr. Helena Piwnica-Worms. Par1a+/− and Par1b+/− mice were bred to generate Par1a+/−:Par1b+/− double heterozygous (Par1a/b HH) mice. Double heterozygous mice were bred and newborn pups were euthanized on the first day of life. Timed matings of Par1a/b HH mice were performed to obtain embyros.

Embryonic kidneys

The first day post overnight mating was defined as E0.5. Pregnant dams were euthanized and uteri with embryos were removed. Embryonic kidneys were obtained using a dissecting microscope.

Real-Time PCR

cDNA was synthesized from 400 (mouse) or 750 (rat) ngs of RNA using the Applied Biosystems (Foster City, CA) High Capacity cDNA Reverse Transcription Kit with RNase inhibitor, as per manufacturer’s instructions. Real-time PCR was performed using an Applied Biosystems 7900HT Fast Real-Time PCR System with Power SYBR Green PCR Master Mix. See Supplemental Material and Supplemental Table 1 for details.

Western Blotting

Par1a/b embryonic kidneys and livers were used. Proteins were extracted using RIPA or SDS buffer, and protein was quantified using the Bradford BSA kit according to manufacturer’s instructions. Equal micrograms of protein were loaded in 8%–10% acrylamide gels. Proteins were transferred to nitrocellulose/PVDF membranes, blocked with 4% milk or 5% BSA in TBS-T, and primary antibodies all diluted (see Supplemental Material) in TBST with 3% milk or 3% BSA were applied overnight at 4°C. After washing, appropriate HRP-conjugated secondary antibodies were applied and autoradiographs were obtained.

Histologic Analysis

Formalin fixed, paraffin-embedded kidney sections were stained with hematoxylin and eosin. Slides were examined and pictures were taken with a Zeiss Axioskop II and Zeiss Axiocam MRc camera. TEM was performed by the Einstein Analytic Imaging Facility (see Supplemental Material).

Immunofluorescence

Paraffin-embedded formalin-fixed sections were deparaffinized and hydrated. After antigen retrieval with sodium citrate (pH 6.0), sections were blocked with 2%–5% donkey serum in PBS and primary antibodies were applied overnight at 4°C (see Supplemental Material). After washing in PBS, appropriate secondary antibodies were applied (Jackson ImmunoResearch Laboratories, West Grove, PA). Confocal microscopy was performed with a Leica SP2 microscope. Images were analyzed with ImageJ software (NIH).

Quantification and Statistical Analyses

See Supplemental Material for details on quantification of nephron number, ureteric bud branching, tubular dilation, and cilia length. The Fisher t test was used for analyzing differences between means. All P values were two-sided and statistical significance was set at P<0.05.

Disclosures

None.

Supplementary Material

Supplemental Data
supp_27_12_3725__index.html (830B, html)

Acknowledgments

We would like to thank Dr. Helen Piwnica-Worms for providing Par1b+/− mice. We would like to thank Dr. Jonathan Barasch and his laboratory for training in embryonic dissection. We would like to thank Dr. Rani Sellers for review of liver histology. We would like to thank Leslie Gunther of the Einstein Analytic Imaging Facility. The Troma I monoclonal antibody, developed by Brulet, P. and Kemler, R., and the Lhx1 antibody, developed by Jessell, T.M. and Brenner-Morton, S., were obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development of the National Institutes of Health (NIH) and maintained at the Department of Biology, The University of Iowa, Iowa City, IA. This study used data from the GenitoUrinary Development Molecular Anatomy Project database (http://www.gudmap.org [obtained in January of 2016]), including microarray (provided by Potter and McMahon as part of GUDMAP) data.

This research was supported by a Young Investigator Award (Children’s Hospital of Montefiore/Albert Einstein College of Medicine) (to K.J.R.) and NIH NIDDK grant K08 DK091507 (to K.J.R.). Research in the Susztak lab is supported by NIH grant R01 DK076077. Research in the Müsch lab is supported by NIH grant RO1 DK064842.

Data from this manuscript was presented in abstract form at the American Society of Nephrology annual meeting held November 5–10, 2013 in Atlanta, GA and Pediatric Academic Societies annual meeting held May 3–6, 2014 in Vancouver, Canada.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

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