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.: Grhl2 CD−/− 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) Grhl2 CD−/− 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 viaHoxb7Cre-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.: Grhl2 CD−/− mice are viable and display occasional unilateral renal agenesis. (A) Representative images of urinary systems in Grhl2 CD−/− and control mice at P30. Grhl2 CD−/− mice exhibit a higher incidence of unilateral renal agenesis in comparison with control littermates (8.8% in Grhl2 CD−/− 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 Grhl2 CD−/− and control mice. ***P<0.001.
Table 1. -
Physiologic parameters in
Grhl2CD−/− mice versus respective control littermates (1–3 months old)
| General Parameters |
Control, n=13 |
Grhl2CD−/−
, n=10 |
P Value |
| Body weight, g |
21.39±2.3 |
22.5±2.8 |
0.31 |
| Urine volume, μl/24 h g body wt |
92.11±16.3 |
119.5±29.7 |
0.01 |
| Urine osmolality, mosmol/kg |
938±89 |
779±137 |
0.003 |
| Water uptake, ml/24 h g body wt |
0.043±0.035 |
0.086±0.044 |
0.02 |
| Mean arterial pressure, mm Hg |
98±3.2 |
101±3.4 |
0.12 |
Table 2. -
Physiologic parameters in
Grhl2CD−/− mice versus respective control littermates (1–3 months old)
| Urinary Excretion Rates (Normalized to Body Weight) |
Control, n=13 |
Grhl2CD−/−
, n=10 |
P Value |
| Creatinine, μg/g body wt per 24 h |
16.64±3.2 |
18.35±2.6 |
0.19 |
| Urea, μmol/g body wt per 24 h |
44.75±10.1 |
47.34±6.2 |
0.48 |
| Na+, μmol/g body wt per 24 h |
7.51±1.1 |
7.98±2.0 |
0.47 |
| K+, μmol/g body wt per 24 h |
10.86±1.9 |
11.05±1.5 |
0.81 |
| Cl−, μmol/g body wt per 24 h |
5.78±1.2 |
5.82±1.7 |
0.96 |
| Total osmolytes, μosmol/g body wt per 24 h |
86.4±17.5 |
90.0±12.7 |
0.59 |
Na+, sodium; K+, potassium; Cl−, chloride.
Table 3. -
Physiologic parameters in
Grhl2CD−/− mice versus respective control littermates (1–3 months old)
| Blood Analyses |
Control, n=5 |
Grhl2CD−/−
, n=5 |
P Value |
| Creatinine, mg/dl |
0.17±0.05 |
0.13±0.03 |
0.21 |
| Urea, mg/dl |
48.8±11.4 |
44.0±7.2 |
0.49 |
| Na+, mmol/L |
147.6±1.5 |
153.8±7.1 |
0.10 |
| K+, mmol/L |
6.1±0.6 |
5.9±0.6 |
0.72 |
| Cl−, mmol/L |
107.6±2.1 |
109±0.8 |
0.25 |
Na+, sodium; K+, potassium; Cl−, chloride.
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.: 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 Grhl2 CD−/− mice (P30; fold change in average controls divided by average Grhl2 CD−/−). 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.: Grhl2 CD−/− 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 Grhl2 CD−/− mice. (B) ISOM CDs of Grhl2 CD−/− mice (P30) exhibit a significantly reduced TER (mean±SEM; n animals=5, n CDs=9 [control] versus 14 [Grhl2 CD−/−]). ***P<0.001 (t test). (C) Tissue osmolality measurements in the cortex (C), the ISOM, and the inner medulla (IM) in control and Grhl2 CD−/− 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 Grhl2 CD−/− 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 Grhl2 CD−/− 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.: Grhl2 CD−/− 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 Grhl2 CD−/− mice and respective control littermates (controls n=13 and Grhl2 CD−/− mice n=10). (B) Cumulative urine volumes (upper panel) and urine osmolality (lower panel) during 24 hours of water depletion in Grhl2 CD−/− (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 Grhl2 CD−/− 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, Grhl2 CD−/− mice n=4; 28 hours: controls n=4, Grhl2 CD−/− mice n=7; 60 hours: controls n=15, Grhl2 CD−/− 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.: 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, Grhl2 CD−/− 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).39–41Grhl2flox/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.
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.).
References
1. Devuyst O: Physiopathology and diagnosis of nephrogenic
diabetes insipidus. Ann Endocrinol (Paris) 73: 128–129, 201222503803
2. Moeller HB, Fuglsang CH, Fenton RA: Renal aquaporins and water balance disorders. Best Pract Res Clin Endocrinol Metab 30: 277–288, 201627156764
3. Vukićević T, Schulz M, Faust D, Klussmann E: The trafficking of the water channel aquaporin-2 in renal principal cells-a potential target for pharmacological intervention in cardiovascular diseases. Front Pharmacol 7: 23, 201626903868
4. Knepper MA, Kwon T-H, Nielsen S: Molecular physiology of water balance. N Engl J Med 372: 1349–1358, 201525830425
5. Moeller HB, Fenton RA: Cell biology of vasopressin-regulated aquaporin-2 trafficking. Pflugers Arch 464: 133–144, 201222744229
6. Kurbel S, Dodig K, Radić R: The osmotic gradient in kidney medulla: A retold story. Adv Physiol Educ 26: 278–281, 200212443999
7. Jiang T, Li Y, Layton AT, Wang W, Sun Y, Li M, Zhou H, Yang B: Generation and phenotypic analysis of mice lacking all urea transporters. Kidney Int 91: 338–351, 201727914708
8. Sands JM, Layton HE: Advances in understanding the urine-concentrating mechanism. Annu Rev Physiol 76: 387–409, 201424245944
9. Klein JD, Blount MA, Sands JM: Urea transport in the kidney. Compr Physiol 1: 699–729, 201123737200
10. Claude P, Goodenough DA: Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia. J Cell Biol 58: 390–400, 19734199658
11. Gonzalez-Mariscal L, Namorado MC, Martin D, Luna J, Alarcon L, Islas S, Valencia L, Muriel P, Ponce L, Reyes JL: Tight junction proteins ZO-1, ZO-2, and occludin along isolated renal tubules. Kidney Int 57: 2386–2402, 200010844608
12. Denker BM, Sabath E: The biology of epithelial cell tight junctions in the kidney. J Am Soc Nephrol 22: 622–625, 201121415157
13. Anderson JM, Van Itallie CM: Physiology and function of the tight junction. Cold Spring Harb Perspect Biol 1: a002584, 200920066090
14. Dragsten PR, Blumenthal R, Handler JS: Membrane asymmetry in epithelia: Is the tight junction a barrier to diffusion in the plasma membrane? Nature 294: 718–722, 19817322203
15. Kiuchi-Saishin Y, Gotoh S, Furuse M, Takasuga A, Tano Y, Tsukita S: Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments. J Am Soc Nephrol 13: 875–886, 200211912246
16. Reyes JL, Lamas M, Martin D, del Carmen Namorado M, Islas S, Luna J, Tauc M, González-Mariscal L: The renal segmental distribution of claudins changes with development. Kidney Int 62: 476–487, 200212110008
17. Angelow S, Ahlstrom R, Yu AS: Biology of claudins. Am J Physiol Renal Physiol 295: F867–F876, 200818480174
18. Amasheh S, Fromm M, Günzel D: Claudins of intestine and nephron - a correlation of molecular tight junction structure and barrier function. Acta Physiol (Oxf) 201: 133–140, 201120518752
19. Bray SJ, Kafatos FC: Developmental function of Elf-1: An essential transcription factor during embryogenesis in Drosophila. Genes Dev 5: 1672–1683, 19911909284
20. Tao J, Kuliyev E, Wang X, Li X, Wilanowski T, Jane SM, Mead PE, Cunningham JM: BMP4-dependent expression of Xenopus Grainyhead-like 1 is essential for epidermal differentiation. Development 132: 1021–1034, 200515705857
21. Wilanowski T, Caddy J, Ting SB, Hislop NR, Cerruti L, Auden A, Zhao LL, Asquith S, Ellis S, Sinclair R, Cunningham JM, Jane SM: Perturbed desmosomal cadherin expression in grainy head-like 1-null mice. EMBO J 27: 886–897, 200818288204
22. Narasimha M, Uv A, Krejci A, Brown NH, Bray SJ: Grainy head promotes expression of septate junction proteins and influences epithelial morphogenesis. J Cell Sci 121: 747–752, 200818303052
23. Yu Z, Mannik J, Soto A, Lin KK, Andersen B: The epidermal differentiation-associated Grainyhead gene Get1/Grhl3 also regulates urothelial differentiation. EMBO J 28: 1890–1903, 200919494835
24. Han Y, Mu Y, Li X, Xu P, Tong J, Liu Z, Ma T, Zeng G, Yang S, Du J, Meng A: Grhl2 deficiency impairs otic development and hearing ability in a zebrafish model of the progressive dominant hearing loss DFNA28. Hum Mol Genet 20: 3213–3226, 201121610158
25. Wilanowski T, Tuckfield A, Cerruti L, O’Connell S, Saint R, Parekh V, Tao J, Cunningham JM, Jane SM: A highly conserved novel family of mammalian developmental transcription factors related to Drosophila grainyhead. Mech Dev 114: 37–50, 200212175488
26. Aue A, Hinze C, Walentin K, Ruffert J, Yurtdas Y, Werth M, Chen W, Rabien A, Kilic E, Schulzke JD, Schumann M, Schmidt-Ott KM: A grainyhead-like 2/ovo-like 2 pathway regulates renal
epithelial barrier function and lumen expansion. J Am Soc Nephrol 26: 2704–2715, 201525788534
27. Werth M, Walentin K, Aue A, Schönheit J, Wuebken A, Pode-Shakked N, Vilianovitch L, Erdmann B, Dekel B, Bader M, Barasch J, Rosenbauer F, Luft FC, Schmidt-Ott KM: The transcription factor grainyhead-like 2 regulates the molecular composition of the epithelial apical junctional complex. Development 137: 3835–3845, 201020978075
28. Auden A, Caddy J, Wilanowski T, Ting SB, Cunningham JM, Jane SM: Spatial and temporal expression of the Grainyhead-like transcription factor family during murine development. Gene Expr Patterns 6: 964–970, 200616831572
29. Varma S, Cao Y, Tagne JB, Lakshminarayanan M, Li J, Friedman TB, Morell RJ, Warburton D, Kotton DN, Ramirez MI: The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation. J Biol Chem 287: 37282–37295, 201222955271
30. Gao X, Vockley CM, Pauli F, Newberry KM, Xue Y, Randell SH, Reddy TE, Hogan BL: Evidence for multiple roles for grainyhead-like 2 in the establishment and maintenance of human mucociliary airway epithelium. [corrected]. Proc Natl Acad Sci U S A 110: 9356–9361, 201323690579
31. Walentin K, Hinze C, Werth M, Haase N, Varma S, Morell R, Aue A, Pötschke E, Warburton D, Qiu A, Barasch J, Purfürst B, Dieterich C, Popova E, Bader M, Dechend R, Staff AC, Yurtdas ZY, Kilic E, Schmidt-Ott KM: A Grhl2-dependent gene network controls trophoblast branching morphogenesis. Development 142: 1125–1136, 201525758223
32. Pyrgaki C, Liu A, Niswander L: Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion. Dev Biol 353: 38–49, 201121377456
33. Senga K, Mostov KE, Mitaka T, Miyajima A, Tanimizu N: Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among claudin3, claudin4, and Rab25. Mol Biol Cell 23: 2845–2855, 201222696678
34. Cieply B, Riley P 4th, Pifer PM, Widmeyer J, Addison JB, Ivanov AV, Denvir J, Frisch SM: Suppression of the epithelial-mesenchymal transition by Grainyhead-like-2. Cancer Res 72: 2440–2453, 201222379025
35. Tanimizu N, Mitaka T: Role of grainyhead-like 2 in the formation of functional tight junctions. Tissue Barriers 1: e23495, 201324665375
36. Gao X, Bali AS, Randell SH, Hogan BL: GRHL2 coordinates regeneration of a polarized mucociliary epithelium from basal stem cells. J Cell Biol 211: 669–682, 201526527742
37. Ray HJ, Niswander LA: Grainyhead-like 2 downstream targets act to suppress epithelial-to-mesenchymal transition during neural tube closure. Development 143: 1192–1204, 201626903501
38. Yu J, Carroll TJ, McMahon AP: Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney. Development 129: 5301–5312, 200212399320
39. Rubera I, Loffing J, Palmer LG, Frindt G, Fowler-Jaeger N, Sauter D, Carroll T, McMahon A, Hummler E, Rossier BC: Collecting duct-specific gene inactivation of alphaENaC in the mouse kidney does not impair sodium and potassium balance. J Clin Invest 112: 554–565, 200312925696
40. Zhao H, Kegg H, Grady S, Truong HT, Robinson ML, Baum M, Bates CM: Role of fibroblast growth factor receptors 1 and 2 in the ureteric bud. Dev Biol 276: 403–415, 200415581874
41. Rojek A, Füchtbauer EM, Kwon TH, Frøkiaer J, Nielsen S: Severe urinary concentrating defect in renal collecting duct-selective AQP2 conditional-knockout mice. Proc Natl Acad Sci U S A 103: 6037–6042, 200616581908
42. Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre JV, Valerius MT, McMahon AP, Duffield JS: Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 176: 85–97, 201020008127
43. Lee JW, Chou CL, Knepper MA: Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes. J Am Soc Nephrol 26: 2669–2677, 201525817355
44. Cheval L, Pierrat F, Dossat C, Genete M, Imbert-Teboul M, Duong Van Huyen JP, Poulain J, Wincker P, Weissenbach J, Piquemal D, Doucet A: Atlas of gene expression in the mouse kidney: New features of glomerular parietal cells. Physiol Genomics 43: 161–173, 201121081658
45. Kamburov A, Wierling C, Lehrach H, Herwig R: ConsensusPathDB--a database for integrating human functional interaction networks. Nucleic Acids Res 37: D623–D628, 200918940869
46. Greger R, Hampel W: A modified system for in vitro perfusion of isolated renal tubules. Pflugers Arch 389: 175–176, 19817193859
47. Plain A, Wulfmeyer VC, Milatz S, Klietz A, Hou J, Bleich M, Himmerkus N: Corticomedullary difference in the effects of dietary Ca
2+ on tight junction properties in thick ascending limbs of Henle’s loop. Pflugers Arch 468: 293–303, 201626497703
48. Herrera M, Garvin JL: A high-salt diet stimulates thick ascending limb eNOS expression by raising medullary osmolality and increasing release of endothelin-1. Am J Physiol Renal Physiol 288: F58–F64, 200515353403
49. Hasler U, Jeon US, Kim JA, Mordasini D, Kwon HM, Féraille E, Martin PY: Tonicity-responsive enhancer binding protein is an essential regulator of aquaporin-2 expression in renal collecting duct principal cells. J Am Soc Nephrol 17: 1521–1531, 200616641150
50. Storm R, Klussmann E, Geelhaar A, Rosenthal W, Maric K: Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells. Am J Physiol Renal Physiol 284: F189–F198, 200312388395
51. López-Rodríguez C, Antos CL, Shelton JM, Richardson JA, Lin F, Novobrantseva TI, Bronson RT, Igarashi P, Rao A, Olson EN: Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression. Proc Natl Acad Sci U S A 101: 2392–2397, 200414983020
52. Yi MH, Lee YS, Kang JW, Kim SJ, Oh SH, Kim YM, Lee YH, Lee SD, Kim DW: NFAT5-dependent expression of AQP4 in astrocytes. Cell Mol Neurobiol 33: 223–232, 201323180003
53. Faust D, Geelhaar A, Eisermann B, Eichhorst J, Wiesner B, Rosenthal W, Klussmann E: Culturing primary rat inner medullary collecting duct cells [published online ahead of print June 21, 2013]. J Vis Exp doi:10.3791/5036623852264
54. Muto S, Tsuruoka S, Miyata Y, Fujimura A, Kusano E: Effect of trimethoprim-sulfamethoxazole on Na and K+ transport properties in the rabbit cortical collecting duct perfused in vitro. Nephron, Physiol 102: 51–60, 200616286787
55. Lehrmann H, Thomas J, Kim SJ, Jacobi C, Leipziger J: Luminal P2Y2 receptor-mediated inhibition of Na+ absorption in isolated perfused mouse CCD. J Am Soc Nephrol 13: 10–18, 200211752016
56. Li JH, Chou CL, Li B, Gavrilova O, Eisner C, Schnermann J, Anderson SA, Deng CX, Knepper MA, Wess J: A selective EP4 PGE2 receptor agonist alleviates disease in a new mouse model of X-linked nephrogenic
diabetes insipidus. J Clin Invest 119: 3115–3126, 200919729836
57. Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS: Neonatal mortality in an aquaporin-2 knock-in mouse model of recessive nephrogenic
diabetes insipidus. J Biol Chem 276: 2775–2779, 200111035038
58. Yang B, Zhao D, Qian L, Verkman AS: Mouse model of inducible nephrogenic
diabetes insipidus produced by floxed aquaporin-2 gene deletion. Am J Physiol Renal Physiol 291: F465–F472, 200616434568
59. Ma T, Song Y, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS: Nephrogenic
diabetes insipidus in mice lacking aquaporin-3 water channels. Proc Natl Acad Sci U S A 97: 4386–4391, 200010737773
60. Takahashi N, Chernavvsky DR, Gomez RA, Igarashi P, Gitelman HJ, Smithies O: Uncompensated polyuria in a mouse model of Bartter’s syndrome. Proc Natl Acad Sci U S A 97: 5434–5439, 200010779555
61. Kemter E, Rathkolb B, Bankir L, Schrewe A, Hans W, Landbrecht C, Klaften M, Ivandic B, Fuchs H, Gailus-Durner V, Hrabé de Angelis M, Wolf E, Wanke R, Aigner B: Mutation of the Na(+)-K(+)-2Cl(-) cotransporter NKCC2 in mice is associated with severe polyuria and a urea-selective concentrating defect without hyperreninemia. Am J Physiol Renal Physiol 298: F1405–F1415, 201020219826
62. Grahammer F, Haenisch N, Steinhardt F, Sandner L, Roerden M, Arnold F, Cordts T, Wanner N, Reichardt W, Kerjaschki D, Ruegg MA, Hall MN, Moulin P, Busch H, Boerries M, Walz G, Artunc F, Huber TB: mTORC1 maintains renal tubular homeostasis and is essential in response to ischemic stress. Proc Natl Acad Sci U S A 111: E2817–E2826, 201424958889
63. Li X, Chen G, Yang B: Urea transporter physiology studied in knockout mice. Front Physiol 3: 217, 201222745630
64. Fenton RA, Knepper MA: Urea and renal function in the 21st century: Insights from knockout mice. J Am Soc Nephrol 18: 679–688, 200717251384
65. Ren H, Gu L, Andreasen A, Thomsen JS, Cao L, Christensen EI, Zhai XY: Spatial organization of the vascular bundle and the interbundle region: Three-dimensional reconstruction at the inner stripe of the outer medulla in the mouse kidney. Am J Physiol Renal Physiol 306: F321–F326, 201424305474
66. Sonoda N, Furuse M, Sasaki H, Yonemura S, Katahira J, Horiguchi Y, Tsukita S: Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier. J Cell Biol 147: 195–204, 199910508866
67. Van Itallie C, Rahner C, Anderson JM: Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 107: 1319–1327, 200111375422
68. Hou J, Renigunta A, Yang J, Waldegger S: Claudin-4 forms paracellular chloride channel in the kidney and requires claudin-8 for tight junction localization. Proc Natl Acad Sci U S A 107: 18010–18015, 201020921420
69. Fujita H, Hamazaki Y, Noda Y, Oshima M, Minato N: Claudin-4 deficiency results in urothelial hyperplasia and lethal hydronephrosis. PLoS One 7: e52272, 201223284964
70. Gong Y, Yu M, Yang J, Gonzales E, Perez R, Hou M, Tripathi P, Hering-Smith KS, Hamm LL, Hou J: The Cap1-claudin-4 regulatory pathway is important for renal chloride reabsorption and blood pressure regulation. Proc Natl Acad Sci U S A 111: E3766–E3774, 201425157135
71. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, Hashimoto N, Furuse M, Tsukita S: Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161: 653–660, 200312743111
72. Walne AJ, Collopy L, Cardoso S, Ellison A, Plagnol V, Albayrak C, Albayrak D, Kilic SS, Patıroglu T, Akar H, Godfrey K, Carter T, Marafie M, Vora A, Sundin M, Vulliamy T, Tummala H, Dokal I: Marked overlap of four genetic syndromes with dyskeratosis congenita confounds clinical diagnosis. Haematologica 101: 1180–1189, 201627612988
73. Balci S, Engiz O, Erekul A, Gozdasoglu S, Vulliamy T: An atypical form of dyskeratosis congenita with renal agenesis and no mutation in DKC1, TERC and TERT genes. J Eur Acad Dermatol Venereol 23: 607–608, 200919415813
