Basic Research

GRHL2 Is Required for Collecting Duct Epithelial Barrier Function and Renal Osmoregulation

Christian Hinze, Janett Ruffert, Katharina Walentin, Nina Himmerkus, Elham Nikpey, Olav Tenstad, Helge Wiig, Kerim Mutig, Zeliha Yesim Yurtdas, Janet D. Klein, Jeff M. Sands, Federica Branchi, Michael Schumann, Sebastian Bachmann, Markus Bleich and Kai M. Schmidt-Ott
JASN March 2018, 29 (3) 857-868; DOI: https://doi.org/10.1681/ASN.2017030353

Abstract

Collecting ducts make up the distal-most tubular segments of the kidney, extending from the cortex, where they connect to the nephron proper, into the medulla, where they release urine into the renal pelvis. During water deprivation, body water preservation is ensured by the selective transepithelial reabsorption of water into the hypertonic medullary interstitium mediated by collecting ducts. The collecting duct epithelium forms tight junctions composed of barrier-enforcing claudins and exhibits a higher transepithelial resistance than other segments of the renal tubule exhibit. However, the functional relevance of this strong collecting duct epithelial barrier is unresolved. Here, we report that collecting duct–specific deletion of an epithelial transcription factor, grainyhead-like 2 (GRHL2), in mice led to reduced expression of tight junction–associated barrier components, reduced collecting duct transepithelial resistance, and defective renal medullary accumulation of sodium and other osmolytes. In vitro, Grhl2-deficient collecting duct cells displayed increased paracellular flux of sodium, chloride, and urea. Consistent with these effects, Grhl2-deficient mice had diabetes insipidus, produced dilute urine, and failed to adequately concentrate their urine after water restriction, resulting in susceptibility to prerenal azotemia. These data indicate a direct functional link between collecting duct epithelial barrier characteristics, which appear to prevent leakage of interstitial osmolytes into urine, and body water homeostasis.

Kidneys facilitate osmoregulation by excreting or reabsorbing water. Mechanisms of water reabsorption include passive paracellular diffusion and transcellular passage mediated by aquaporin (AQP) water channels.1–5 Although about 85% of water reabsorption occurs in the proximal tubules and the thin descending limbs, fine tuning occurs in the collecting ducts (CDs). CD water reabsorption occurs passively and depends on AQP-mediated transcellular passage of free water toward a hypertonic interstitium. Medullary hypertonicity is achieved by interstitial sodium chloride accumulation, which is accomplished via active transport in the thick ascending limbs of Henle loop. Interstitial sodium facilitates urinary concentration toward the papillary tip, resulting in a urea gradient between the urine and the interstitium. In case of water restriction, specialized urea transporters are inserted into the cell membrane,6–8 and urea follows its gradient into the medulla.9

Compared with other segments of the nephron, the CD epithelium forms particularly tight barriers between the urinary space and the interstitial environment. A steep increase of transepithelial resistance (TER) is observed from proximal tubule to CD.10–12 Because cell membranes generally feature very high electrical resistances, the increase in TER is assumed to predominantly reflect a decreasing permeability of the paracellular barrier.13 Paracellular permeability of epithelial cell layers is mainly regulated by tight junctions.13,14 Claudin (CLDN) proteins are central components of tight junctions and show a nephron segment–specific expression pattern.15–18 The tight epithelia of the distal nephron and CDs express predominantly barrier-enforcing CLDNs, including CLDN3, CLDN4, and CLDN8.18 The functional relevance of this increasing barrier function toward distal segments of the renal tubule is unknown.

Grainyhead family transcription factors are implicated in the maintenance of epithelial barriers in different tissues across several species.19–24 Three grainyhead-like (GRHL) factors, GRHL1, GRHL2, and GRHL3, constitute mammalian homologs of a common ancestor called grainyhead.25 In mice and humans, Grhl2 is typical of epithelia of the distal nephrons and CDs.26,27 In addition to the adult kidney, Grhl2 is expressed in embryonic epithelia, such as the surface ectoderm, gut tube, nephric ducts, and ureteric buds of the developing kidneys25–28; adult lung epithelia29,30; and placental trophoblast epithelia.31 The widespread expression of Grhl2 in barrier-forming epithelia and its function as a transcriptional activator of barrier-associated signature genes24,26,27,29–37 make Grhl2 one key candidate regulator of epithelial barrier function.

We previously reported that Grhl2 is important for adequate transepithelial barrier function in monolayers of a mouse cell line of kidney epithelial cells with characteristics of the renal CD (inner medullary collecting duct 3 [IMCD3] cells).26 However, analyses of Grhl2 functions in the adult renal CD in vivo were previously precluded by the midembryonic lethality of globally Grhl2-deleted embryos.27 We now generated mice with a conditional CD-specific Grhl2 inactivation. Phenotypical characterization of the structural, molecular, and functional defects in these mice together with analyses of isolated CDs and cultured cells identify a novel functional role of the CD epithelial barrier in renal osmoregulation.

Results

Mice Lacking Grhl2 in CDs Have Normal Nephron Numbers and Occasional Unilateral Renal Agenesis

In embryonic day 18 kidneys, nuclear GRHL2 protein was strongly expressed in the CD, connecting tubule, distal convoluted tubule, and the urothelium of both renal pelvis and ureter (Figure 1, Supplemental Figure 1A). GRHL2 expression was maintained at high levels in principal and intercalated cells of postnatal day 30 (P30) kidneys (Supplemental Figure 1B). This was consistent with previously published GRHL2 expression domains in murine and human tissues.26–28

Figure 1.
Figure 1.

Grhl2CD−/− mice exhibit collecting duct-specific deletion of the transcription factor GRHL2. (A) In wildtype mice, GRHL2 is expressed in AQP2-labeled renal CD, connecting tubule (CNT), and distal convoluted tubule (DCT). (B) Grhl2CD−/− mice exhibit a knockout of Grhl2 within the majority of CD cells but not in CNT or DCT. E18, embryonic day 18.

To delineate Grhl2 in vivo function, we generated mice with a homozygous CD-specific inactivation of Grhl2 (Grhl2CD−/− mice). CD specificity in Grhl2CD−/− mice was obtained via Hoxb7Cre-transgene–mediated recombination of a conditional Grhl2 allele that we had previously generated (Grhl2flox).31 Consistent with previous reports of Hoxb7Cre activity,38–42 we observed deletion of GRHL2 protein in most cells of the CD but not in connecting tubule or distal convoluted tubule (Figure 1B, Supplemental Figure 1, B and C).

Grhl2CD−/− mice were viable, were fertile, and lived to an age of at least 1 year old. We found no significant deviations from the expected Mendelian genotype distribution (22% Grhl2CD−/− mice of a total of 218 littermates; P=0.30, chi-squared test). Interestingly, we observed unilateral renal agenesis in 8.8% of Grhl2CD−/− mice (n=114) compared with only 0.9% of control littermates (n=214; P<0.001) (Figure 2A). In the remaining mice, Grhl2CD−/− kidneys were macroscopically normal, with a slightly reduced kidney weight (Figure 2B) and slightly smaller pole-to-pole kidney diameter (reduced by 10% in embryonic day 18 mice, n=4, P=0.02 and 5% in P30 mice, n=5, P=0.08); however, nephron quantification revealed similar nephron numbers (Figure 2C), and the density of CDs in the region between inner medulla and outer medulla was not different (Supplemental Figure 1D). Grhl2CD−/− mice had normal kidney function (Tables 1–3) and showed no signs of kidney fibrosis according to Masson Trichrome staining (Supplemental Figure 1E). A detailed analysis of CD tubular morphology revealed that Grhl2CD−/− CDs displayed a more simplified epithelium with smaller rounded and more densely packed cells, resulting in a reduced CD lumen diameter (Supplemental Figure 2). However, number and distribution of principal and intercalated cells were unchanged in Grhl2CD−/− mice (Supplemental Figure 3), and CD epithelia displayed normal distribution of apicobasal polarity markers and primary cilia (Supplemental Figure 4). Transmission electron microscopy of CDs showed no obvious structural alterations of the tight junction in Grhl2CD−/− kidneys with preserved apicobasal tight junction depths (Supplemental Figure 5). Hematoxylin and eosin–stained kidney sections from P30 and adult Grhl2CD−/− mice revealed no apparent age-dependent differences in renal histology (Supplemental Figure 6).

Figure 2.
Figure 2.

Grhl2CD−/− mice are viable and display occasional unilateral renal agenesis. (A) Representative images of urinary systems in Grhl2CD−/− and control mice at P30. Grhl2CD−/− mice exhibit a higher incidence of unilateral renal agenesis in comparison with control littermates (8.8% in Grhl2CD−/− versus 1.1% in controls; P<0.001). a, adrenal; b, bladder; k, kidney; u, ureter. (B) Kidney weight and (C) nephron numbers at P30 in Grhl2CD−/− and control mice. ***P<0.001.

View this table:
Table 1.

Physiologic parameters in Grhl2CD−/− mice versus respective control littermates (1–3 months old)

View this table:
Table 2.

Physiologic parameters in Grhl2CD−/− mice versus respective control littermates (1–3 months old)

View this table:
Table 3.

Physiologic parameters in Grhl2CD−/− mice versus respective control littermates (1–3 months old)

Grhl2 Controls a Molecular Network Governing Epithelial Barrier Function in CDs

To assess the molecular program controlled by GRHL2, we performed microarray gene expression analyses comparing Grhl2CD−/− with control kidneys (P30, n=6) and integrated these data with Grhl2 chromatin immunoprecipitation sequencing data from wild-type embryonic and adult kidneys.26 Microarray data analysis yielded 774 genes that were downregulated and 672 genes that were upregulated in Grhl2-deficient kidneys (Figure 3A, Supplemental Table 1). We overlapped differentially expressed genes with published databases from microdissected rodent nephron segments (rat RNA-seq43 and mouse SAGE44), indicating that most of these differentially expressed genes were expressed in CDs (87.3% and 74.7%, respectively). GRHL2 chromatin immunoprecipitation peaks associated more often with genes downregulated in Grhl2CD−/− mice versus control mice (Figure 3A), supporting the previously suggested function of GRHL2 as a transcriptional activator.26,31

Figure 3.
Figure 3.

GRHL2 target genes encode for barrier-enforcing tight junction components. (A, left panel) Heat map of all differentially expressed genes sorted by fold change between kidneys of control and Grhl2CD−/− mice (P30; fold change in average controls divided by average Grhl2CD−/−). Genes are maximum normalized on a per gene basis. (A, right panel) Densities plots displaying GRHL2 chromatin immunoprecipitation (ChIP) peak occurrence in the vicinity of genes shown on the heat map. For these plots, a peak was considered associated with a gene if it occurred within 2 kb from the gene. Density plots were generated by applying a sliding average with a window size of 100 genes. (B) Validation of discovered tight junction–associated GRHL2 targets by quantitative RT-PCR in microdissected medullary CDs (n=3, male mice, P50–P60). *P<0.05. (C) GRHL2-dependent expression and GRHL2 binding near tight junction–associated GRHL2 target genes. (D) Putative scheme of GRHL2-mediated control of barrier-enforcing tight junction components in CD epithelia. DE, differentially expressed; qPCR, quantitative PCR; TJ, tight junction; TJP, tight junction protein; TSS, transcription start site.

Two hundred ninety-two differentially expressed genes from our microarray data also displayed a GRHL2 chromatin immunoprecipitation peak in adult kidney <2 kb away from the gene, suggesting that these genes represented direct targets of GRHL2 (Supplemental Table 2). Among these genes were several known GRHL2 target genes (Figure 3B).26,30 Ontology analysis of these genes revealed “Tight junction” as the only significantly enriched Kyoto Encyclopedia of Genes and Genomes pathway.45 Tight junction–associated GRHL2 target genes included our previously published26 as well as new targets (Figure 3, B and C). We microdissected medullary CDs and performed RT-PCR, confirming downregulation of these genes in CDs from Grhl2CD−/− versus control kidneys (Figure 3, B and C, Supplemental Figure 7). These data indicated that GRHL2 directly controls expression levels of genes that encode tight junction–associated proteins in the CD (Figure 3D shows a schematic diagram).

GRHL2 Controls CD Barrier Function and Maintains Medullary Interstitial Osmolality

To assess the role of GRHL2 in paracellular epithelial barrier function in vivo, we analyzed TER in freshly isolated CDs from Grhl2CD−/− and control mice (P30). We performed tubular perfusion using a double-barreled perfusion system as described previously (Figure 4A).46,47 CDs of the inner stripe of the outer medulla were perfused under conditions mimicking the conditions in this part of the CD.6 The TER was markedly reduced by about 50% in CDs of Grhl2CD−/− mice (Figure 4B). Transepithelial voltage (Vte) measurements revealed no relevant electrogenic net transport in both control and Grhl2CD−/− CDs (Supplemental Figure 8).

Figure 4.
Figure 4.

Grhl2CD−/− mice display reduced TER of the CD epithelium and fail to establish adequate corticomedullary osmolality gradients. (A) Representative micrographs of CDs isolated from the inner stripe of the outer medulla (ISOM) of control and Grhl2CD−/− mice. (B) ISOM CDs of Grhl2CD−/− mice (P30) exhibit a significantly reduced TER (mean±SEM; nanimals=5, nCDs=9 [control] versus 14 [Grhl2CD−/−]). ***P<0.001 (t test). (C) Tissue osmolality measurements in the cortex (C), the ISOM, and the inner medulla (IM) in control and Grhl2CD−/− mice (mean±SEM; n=5). OSOM, outer stripe of the outer medulla. *P<0.05; **P<0.01. (D) Measurements of sodium concentrations in indicated regions of the kidney in 24-hour water-restricted Grhl2CD−/− and control mice (median ± quartiles, n=5, Mann-Whitney U test [MWU]). *P<0.05; **P<0.01. (E) Paracellular osmolyte flux across layers of wild-type (WT IMCD3) and Grhl2-KO-IMCD3) cells (median ± quartiles, n=5, MWU). *P<0.05. (F) Schematic diagram of leaky tight junctions in the CDs of Grhl2CD−/− mice leading to a presumed paracellular osmolyte leakage into the urine and a flattened corticomedullary osmolality gradient. DT, distal tubule; LOH, loop of Henle; PT, proximal tubule.

To further characterize ion and osmolyte transport across Grhl2-deficient epithelia, we used inner medullary collecting duct 3 cells with a CRISPR/Cas9-induced knockout of grainyhead-like 2 (Grhl2-KO-IMCD3) (Q Ming et al., unpublished data) (Supplemental Material). Consistent with in vivo data, confluent Transwell layers of Grhl2-KO-IMCD3 cells displayed a reduced TER compared with wild-type IMCD3 cells (Supplemental Figure 9). We then applied gradients of sodium, chloride, and urea in Transwells to measure paracellular transport rates across cell layers. This analysis revealed markedly increased paracellular flux rates for sodium, chloride, and urea across Grhl2-KO-IMCD3 cell layers versus wild-type IMCD3 layers (Figure 4E). Because IMCD3 cells did not express urea transporters (data not shown) and experiments were carried out in the presence of amiloride to block transcellular sodium transport, we concluded that sodium, chloride, and urea fluxes occurred primarily via the paracellular route.

We hypothesized that increased paracellular flux of osmolytes via the Grhl2-deficient CD epithelium would lead to reduced ability to accumulate interstitial osmolytes (primarily sodium and urea) in the renal medulla. We, therefore, measured tissue osmolality in the cortex, the inner stripe of the outer medulla, and the inner medulla of control and Grhl2CD−/− kidneys using previously described methods.48 Tissue osmolality was significantly reduced in Grhl2CD−/− mice in the inner stripe of the outer medulla and the inner medulla, with no difference in renal cortex osmolality (Figure 4C). Because the reduced TER suggested an increased loss of sodium, we measured medullary sodium levels. Sodium concentration was significantly decreased in medullas and papillas of Grhl2CD−/− mice compared with control mice (Figure 4D). Our data were consistent with a continuous paracellular leakage of osmolytes across the Grhl2-deficient CD epithelium, leading to a decreased corticomedullary osmolality gradient (a schematic diagram is in Figure 4F).

Grhl2CD−/− Mice Show Urine-Concentrating Disability and Are Prone to Prerenal Azotemia

We analyzed urinary excretion over 24 hours in P30 and adult Grhl2CD−/− mice (Figure 5A, Supplemental Figure 10, Tables 1–3). Grhl2CD−/− mice showed significantly increased urinary output as well as a significant decrease in urine osmolality compared with control mice. Grhl2CD−/− mice had an increased daily water intake and tended to be hypernatremic (Figure 5A, Tables 1–3). Acid-base homeostasis was normal in Grhl2CD−/− mice (Supplemental Table 2).

Figure 5.
Figure 5.

Grhl2CD−/− mice display diabetes insipidus and fail to adequately concentrate urine during water restriction. (A) Baseline daily urinary output, urine osmolality, and drinking volume were determined in 1- to 3-month-old Grhl2CD−/− mice and respective control littermates (controls n=13 and Grhl2CD−/− mice n=10). (B) Cumulative urine volumes (upper panel) and urine osmolality (lower panel) during 24 hours of water depletion in Grhl2CD−/− (n=11) and control mice (n=16) at P30. Note that the osmolality measurements at 24 hours were limited by the fact that 56.3% (nine of 16) of the control mice and 18.2% (two of 11) of the Grhl2CD−/− mice produced insufficient amounts of urine during the last 6 hours of the water restriction experiment (hours 19–24). (C) Plasma creatinine (left panel) and urea (right panel) concentrations at baseline and after 28 and 60 hours of water depletion in P30 mice (baseline: controls n=12, Grhl2CD−/− mice n=4; 28 hours: controls n=4, Grhl2CD−/− mice n=7; 60 hours: controls n=15, Grhl2CD−/− mice n=12). bw, Body weight. *P<0.05 (Mann-Whitney U test [MWU]); **P<0.01 (MWU); ***P<0.001 (MWU).

Next, we determined urine concentration ability within 24 hours of water restriction. Urine output and urine osmolality were measured every 6 hours while harboring the mice in metabolic cages. Grhl2CD−/− mice produced significantly higher urine volumes than control animals within the first 24 hours and produced 45.9% more urine (Figure 5B). We found a significantly lower urine osmolality in Grhl2CD−/− mice after 6 hours of water depletion (Figure 5B) as well as a lower peak osmolality after 24 hours (Figure 5B).

Grhl2CD−/− mice had normal creatinine and slightly increased urea levels compared with control mice after 24 hours of water deprivation (Figure 5C). After prolonged water restriction (60 hours), Grhl2CD−/− mice (P30) showed a significantly higher rate of prerenal azotemia with markedly higher plasma creatinine and plasma urea levels (Figure 5C). Quantitative RT-PCR analysis revealed a significant upregulation of Grhl2 mRNA expression in kidneys after 60 hours of water deprivation compared with baseline conditions (Supplemental Figure 11).

Grhl2CD−/− Mice Display Normal CD Water Transport, Vasopressin (Arginine Vasopressin) Signaling, and Urea Transport

Microarray analyses and quantitative PCR indicated mild downregulation of Aqp2 and Aqp4 in Grhl2CD−/− kidneys (Supplemental Figure 12A). AQP2 and AQP4 proteins were appropriately localized in CD cells of Grhl2CD−/− kidneys according to immunofluorescence analyses (Supplemental Figure 12B). Western blots indicated decreased AQP2 protein levels in medulla but similar levels in the cortex of Grhl2CD−/− kidneys. Western blots for AQP3 and AQP4 on cortex and medulla revealed no differences in protein abundance between Grhl2CD−/− kidneys and control kidneys (Supplemental Figure 13). These findings were consistent with the reduced tissue osmolality in Grhl2CD−/− kidneys and the known responsiveness of Aqp2 transcription to interstitial tissue osmolality.49–52 To rule out that reduced AQP2 levels in Grhl2CD−/− medullas were due to a potential direct regulation of AQP2 by GRHL2, we prepared primary cultures from medullary CD cells53 of control and Grhl2CD−/− kidneys and exposed them to 600 mosmol/kg medium. After 3 days of culture, AQP2 levels were similar between Grhl2CD−/− and control cells, indicating that regulation of AQP2 in these cells was independent from GRHL2 when controlling for extracellular osmolality (Supplemental Figure 14). To functionally determine CD water transport characteristics in Grhl2CD−/− mice, we performed swelling and deswelling experiments on freshly isolated CDs, revealing no evidence of altered AQP-mediated water transport across the basolateral and apical membranes of Grhl2CD−/− CDs compared with control CDs (Supplemental Figure 14). These findings indicated that AQP-mediated transcellular water transport mechanisms were intact in Grhl2CD−/− CDs.

Plasma arginine vasopressin (AVP) levels (measured by copeptin ELISA) were similar between Grhl2CD−/− and control mice (Supplemental Figure 15). Also, AQP2 trafficked to the plasma membrane after AVP stimulation in primary cultures from medullary CD cells isolated from Grhl2CD−/− kidneys, indicating normal responsiveness of principal cells to AVP (Supplemental Figure 14). Furthermore, AQP2 displayed intact apical localization in water-deprived Grhl2CD−/− mice (Supplemental Figure 12B). Together, these data indicated unaltered AVP signaling and responsiveness in Grhl2CD−/− mice.

IMCD urea transporters contribute to the establishment of medullary hypertonicity.7 To rule out that Grhl2CD−/− kidneys exhibit a defect of urea transporter expression, we analyzed UT-A1 or UT-A3 expression in medullas of control and Grhl2CD−/− kidneys using Western blot and immunofluorescence, revealing similar expression levels and unchanged cellular localization (Supplemental Figure 16). Together, these data indicated that CD water transport systems, AVP signaling, and medullary urea transporter expression were normal in Grhl2CD−/− mice.

Discussion

Although it has been known for decades that the CD is made up of a particularly tight epithelium,54,55 the functional implications of a defect of this CD epithelial barrier were previously unclear. Here, we show that the transcription factor GRHL2 serves to activate a molecular program of tight junction barrier enforcement and enhance paracellular barrier function of the CD epithelium in vivo. We provide evidence that the primary function of the Grhl2-mediated epithelial barrier in the CD is to shield the hypertonic medullary interstitium of the kidney from hypotonic urine within the CD lumen. In CD cells, Grhl2 deficiency induces paracellular leakage of sodium, chloride, and urea, and Grhl2-deficient mice display reduced medullary osmolarity, presumably due to loss of medullary osmolytes. Consistently, Grhl2-deficient mice develop diabetes insipidus, polyuria, and a defect of urinary concentrating ability (Figure 6 shows a schematic overview). Hence, our study adds an essential new player to the renal concentrating mechanism: the epithelial barrier function of the CD.

Figure 6.
Figure 6.

Schematic outline of the novel role of GRHL2-dependent CD epithelial barrier function in renal osmoregulation. Wildtype control animals (left) form an efficient CD barrier, allowing retention of interstitial osmolytes in the renal medulla and effective urinary concentration. In contrast, Grhl2CD−/− mice (right) exhibit a leaky CD epithelium, causing defective interstitial osmolyte accumulation and a urinary concentration defect.

Previous studies had provided ample evidence of renal concentrating defects in mice that lacked essential components of the pathway that involves AVP-dependent transcellular reabsorption of free water via AQP water channels in CD principal cells.56–59 In addition, urinary concentrating defects were observed in mice with genetic defects that perturbed the accumulation of sodium chloride (via functions of the thick ascending loop of Henle)60–62 or urea (via specific urea transporters).7,63 We examined these different components of the urinary concentration machinery in Grhl2CD−/− mice. AVP levels were normal, water reabsorption was functionally unaltered as shown by swelling/deswelling experiments in isolated CDs, and AQP2 membrane translocation in response to cAMP stimulation in isolated CD cells occurred normally. Medullary AQP2 levels were reduced in Grhl2CD−/− mice, but this was likely secondary to the reduced medullary osmolarity, a known regulator of AQP2 expression.49–52 Direct regulation of AQP2 by GRHL2 is unlikely given the absence of GRHL2 binding near the Aqp2 gene and considering that AQP2 was reduced only in medulla but was not reduced in cortex of Grhl2CD−/− mice. Furthermore, freshly isolated Grhl2-deficient principal cells expressed normal AQP2 levels when placed under conditions that controlled for extracellular osmolarity. In addition, the key CD urea transporters UT-A1 and UT-A3 were expressed at normal levels and showed apparently unaltered cellular localization in Grhl2CD−/− mice.

Urea reabsorption is passive and dependent on medullary sodium accumulation.9,64 Because we found a sharp reduction of tissue sodium concentration in Grhl2CD−/− medulla, we propose that the reduced sodium content in the interstitium resulting from paracellular sodium leakage may be the primary driver of the decreased tissue osmolarity. Because we were unsuccessful in directly measuring tissue urea concentrations in Grhl2CD−/− medulla, it remains to be formally determined whether medullary urea concentration was reduced compared with control mice. We did not observe evidence of a salt-wasting phenotype in Grhl2CD−/− mice, because urinary sodium excretion rates and arterial BP were not different between Grhl2CD−/− mice and control mice.

Additional prerequisites for urinary concentration include an intact structure and function of the renal vasculature and of the ascending loop of Henle.65 Because the deletion of Grhl2 is specific to the CD in our mouse model, we consider it unlikely that defects of sodium transport in the loop of Henle and/or altered renal hemodynamics account for the observed phenotype, but these aspects were not studied in detail in this study.

Among the key target genes controlled by GRHL2 was Cldn4, a known barrier-forming CLDN involved in paracellular chloride transport.18,66–68 CLDN4 is sufficient to increase TER in Grhl2-deficient IMCD3 cells.26 Hence, decreased Cldn4 expression may explain some of the phenotypical features observed in Grhl2CD−/− mice. However, a comparison of the phenotypes described in Cldn4 knockout mice and those that we observe in Grhl2CD−/− mice shows differences. Mice with a global or CD-specific knockout of Cldn4 displayed some polyuria, but unlike Grhl2 knockout mice, they did not display evidence of diabetes insipidus or a decreased urinary concentrating ability.69,70 Instead, CD-specific Cldn4 knockout mice displayed increased urinary excretion of Na+ and Cl− and arterial hypotension, indicative of chloride wasting secondary to a lack of CLDN4 pore-dependent Cl− reabsorption. We did not observe any of these alterations in Grhl2CD−/− mice and in fact, observed increased paracellular chloride transport across Grhl2-deficient IMCD3 layers. This suggests that the global barrier defect in Grhl2CD−/− CDs supersedes the more specific defect of chloride reabsorption that relates to CLDN4 deficiency. Additional target genes of GRHL2, such as Shroom3, Myh10, and Tjp3, may participate in barrier enforcement within the CD epithelium, but their individual effects on CD barrier function or urinary concentration have not been determined. Transmission electron microscopy did not show evident differences in tight junction morphology in Grhl2CD−/− CDs, consistent with our analyses of Grhl2-deficient otic vesicle epithelia.27 These observations suggest that tight junctions are assembled in Grhl2-deficient epithelia but display defects on a molecular level that alter epithelial barrier characteristics. Others have made similar observations of apparently unaffected tight junction ultrastructure, despite profound alterations of barrier characteristics.71

In a subset of Grhl2CD−/− mice (about 8%), we observed unilateral renal agenesis. This finding is interesting in light of reports of humans carrying homozygous mutations of Grhl2 who displayed renal agenesis.72,73 The underlying developmental mechanisms remain to be determined.

Our findings link the transcriptional control of molecular components of epithelial barrier function with renal osmoregulation and the maintenance of kidney function in the setting of limited fluid supplies. This is of translational importance given that our study identifies the CD barrier as a novel potential target for therapeutic intervention.

Concise Methods

All animal experiments were approved by the Berlin Animal Review Board and conducted according to National Institutes of Health Guide for the Care and Use of Laboratory Animals standards. Laboratory techniques are outlined in detail in Supplemental Material.

Animals

Mice with a Grhl2-deficient (Grhl2+/−) and a conditional floxed Grhl2 allele (Grhl2flox/flox) were generated and genotyped by PCR as described previously.27,31 Breeding of Grhl2flox/flox mice with heterozygous Grhl2+/− carrying the Hoxb7Cre transgene (Hoxb7Cre;Grhl2+/−) enabled selective homozygous Grhl2 inactivation in the ureteric bud and the CDs (Hoxb7Cre;Grhl2−/flox).39–41 Grhl2flox/flox, Hoxb7Cre;Grhl2flox/flox, and Hoxb7Cre;Grhl2−/flox mice were on a mixed 129/C57BL/6 genetic background. Conditional CD-specific Hoxb7Cre;Grhl2flox/flox and Hoxb7Cre;Grhl2−/flox knockout mice are jointly referred to as Grhl2CD−/− mice in the manuscript. We observed occasional unilateral agenesis in Grhl2CD−/− mice. These mice were excluded from functional studies.

Urine Analyses

Mice were housed with (baseline conditions) or without drinking water (water deprivation) in metabolic cages for the indicated time periods. Urine was collected and stored at −20°C until measured. Chloride, potassium, sodium, urea, and creatinine concentrations were measured using a Roche Cobas 8000 Analyzer (Labor 28, Berlin, Germany). Osmolalities were measured using a cryo-osmometer.

Tissue Osmolality Measurements

Tissue osmolality measurements were carried out as described previously by Herrera and Garvin.48 Osmolality was calculated for each sample and related to tissue water content. Results are expressed as milliosmoles per kilogram H2O.

Tissue Electrolyte Measurements

The kidney poles of the shock-frozen kidneys were cut off perpendicular to the long axis with a scalpel while still frozen. The remaining 2- to 3-mm-thick slice containing the papilla was dissected on a plate kept at −20°C into a pyramidal-shaped tissue block with the papillary tip as the apex and the cortex as the basis. From this block, a tissue sample of about 5 mg was cut off from papilla, medulla, and cortex and transferred to a preweighed Eppendorf tube containing 300 μl Milli-Q water for equilibration at room temperature. After equilibration, 5 μl of each eluate was diluted in 60 μl Milli-Q water, and the concentrations of sodium were measured by ion chromatography (Dionex ICS-4000 Ion Chromatography System with RFIC-EG, conductivity, and charge detection; Dionex CCES 300 Cation Capillary Electrolytic Suppressor; and a Dionex capillary methanesulfonic acid eluent generator cartridge). The analyzed cations were baseline separated in a 4–12 mM MSA gradient by a Dionex IonPac CS19 Capillary column (0.4×250 mm) at a flow rate of 12 μl/min. For dry weights, tissue samples were vacuum dried and weighed.

Renal Tubule Perfusion and TER Measurement

Perfusion and TER measurements in freshly isolated mouse inner stripe of the outer medulla CDs were performed similar to the perfusion of the thick ascending limb of the loop of Henle in the work by Plain et al.47 CDs were isolated freshly using fine forceps under a dissection microscope from kidneys of both control and Grhl2CD−/− mice housed under standard conditions. Tubules were transferred into the bath on a heated microscope stage, perfused using a concentric glass pipette system with a double-barreled perfusion pipette,46 and perfused and superperfused (bath exchange 8–10 ml/min) with a control solution (600 mosmol/kg, NaCl 245 mM, KH2PO4 0.4 mM, K2HPO4 1.6 mM, MgCl2 1 mM, glucose 5 mM, Ca-gluconate 1.3 mM, and urea 100 mM, pH 7.4). One barrel was used for voltage measurement and perfusion (perfusion rate 10–20 nl/min; 6.1±0.3-μm inner diameter), whereas the other barrel was used for constant current injection (Iinj=13 nA). All perfusions and recordings were conducted at 37°C. Transepithelial voltage was recorded during the whole experiment, and length and diameter of tubules were obtained from recorded images. Measured inner stripe of the outer medulla CDs was rather short but with high TER that excluded the use of the cable equation without measurement of the transepithelial voltage unperfused end of the tubule, which however, itself could be source of potential flaws in calculation of the TER (improper sealing or generation of potentials between different agar bridges). We, therefore, used a simplified method to calculate an estimated TER Rte′ by taking a simple schematic circuit diagram as basis with two resistors in parallel, resistance of the tubule lumen Rlu, and Rte′, resulting in Rtotal. Rtotal was calculated according to the Ohm law from the voltage deflections ΔVte elicited by Iinj. Rlu was calculated Rlu=ρ×l/A (ρ, resistivity of the solution; l, tubular length; A, circular area of the tubule). Rte′ was calculated according to the Kirchhoff law and normalized to the calculated tubular surface.

Swelling and Deswelling Experiments in Isolated CDs

In swelling experiments, isolated CDs were initially kept in a 600 mosmol/kg basolateral solution, which was then acutely lowered to 300 mosmol/kg, whereas the luminal solution remained unchanged at 600 mosmol/kg. In the presence of basolateral AQPs, this leads to water influx and cell swelling. After a fixed time, 1 μM forskolin was added to the 300 mosmol/kg basolateral solution to activate cAMP signaling, leading to apical shuttling of AQP2. This causes deswelling by introducing a shortcut for water. Imaging was used to assess the dynamics of swelling and deswelling.

Additional methods can be found in Supplemental Material.

Disclosures

None.

Acknowledgments

We thank Friedrich C. Luft for insightful comments on the manuscript. We thank Tatjana Luganskaja, Anna Maren Maier, and Gabriel Kirchgraber for their excellent technical assistance. In addition, we also thank Ilona Kamer for providing service and expertise in telemetric BP analysis, Marcus Mildner and Duygu Elif Yilmaz for their help with electron microscopy, and Claudia Heldt for her excellent work for the Ussing chamber experiments. We thank Dr. Anje Sporbert (Max Delbrück Center, Advanced Light Microscopy Facility).

C.H. is supported by the Berlin Institute of Health Charité Clinical Scientist Program funded by Charité Universitätsmedizin and the Berlin Institute of Health. J.R. and K.M.S.-O. are generously supported by the Urological Research Foundation (Berlin). J.D.K. and J.M.S. are supported by US National Institutes of Health grant R01 DK41707. This work was supported by Western Norway Regional Health Authority grant 911888 (to H.W.), Research Council of Norway grant 262079 (to H.W.), and Deutsche Forschungsgemeinschaft grants FOR 1368 (to K.M.S.-O.), Schm 1730/2-1 (to K.M.S.-O.), and Schm1730/3-1 (to K.M.S.-O.).

Footnotes

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GRHL2 Is Required for Collecting Duct Epithelial Barrier Function and Renal Osmoregulation
Christian Hinze, Janett Ruffert, Katharina Walentin, Nina Himmerkus, Elham Nikpey, Olav Tenstad, Helge Wiig, Kerim Mutig, Zeliha Yesim Yurtdas, Janet D. Klein, Jeff M. Sands, Federica Branchi, Michael Schumann, Sebastian Bachmann, Markus Bleich, Kai M. Schmidt-Ott
JASN Mar 2018, 29 (3) 857-868; DOI: 10.1681/ASN.2017030353
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