Neonatal Hyperoxia Downregulates Claudin-4, Occludin, and ZO-1 Expression in Rat Kidney Accompanied by Impaired Proximal Tubular Development.

Hyperoxia is essential to manage in preterm infants but causes injury to immature kidney. Previous study indicates that hyperoxia causes oxidative damage to neonatal kidney and impairs renal development. However, the underlying mechanisms by which neonatal hyperoxia effects on immature kidney still need to be elucidated. Tight junction, among which the representative proteins are claudin-4, occludin, and ZO-1, plays a crucial role in nephrogenesis and maintaining renal function. Inflammatory cytokines are involved in the pleiotropic regulation of tight junction proteins. Here, we investigated how neonatal hyperoxia affected the expression of key tight junction proteins and inflammatory factors (IL-6 and TNF-α) in the developing rat kidneys and elucidated their correlation with renal injury. We found claudin-4, occludin, and zonula occludens-1 (ZO-1) expression in proximal tubules was significantly downregulated after neonatal hyperoxia. The expression of these tight junction proteins was positively correlated with that of IL-6 and TNF-α, while claudin-4 expression was positively correlated with injury score of proximal tubules in mature kidneys. These findings indicated that impaired expression of tight junction proteins in kidney might be a potential mechanism of hyperoxia-induced nephrogenic disorders. It provides new insights to further study oxidative renal injury and development disorders and will be helpful for seeking potential therapeutics for hyperoxia-induced renal injury in the future.

Matrigel-Free Laminin-Entactin Matrix to Induce Human Renal Proximal Tubule Structure Formation In Vitro.

A successful in vitro tissue model must recapitulate the native tissue features while also being reproducible. Currently, Matrigel is the principal biomaterial used to induce the formation of proximal convoluted tubules (PCTs) in vitro, because of its similar composition and structure with the kidney tubular basement membrane and the presence of critical growth factors. However, Matrigel is not well-defined, and batch-to-batch variability is a significant issue. Here, we define a Matrigel-free method, using a laminin-entactin (L-E) matrix to support the formation of proximal tubular-like structures in vitro using immortalized human renal epithelial cells (RPTEC/TERT1) cocultured with murine fibroblast stromal cells (FOXD1lacZ+). The matrix supports the presence of specific components of the tubular basement membrane (laminin, entactin/nidogen, and heparan sulfate proteoglycan) in addition to fibroblast growth factor 8a (FGF-8a). The matrix also induces tubulogenesis, leading to the formation of PCTs based on several key markers, including E-cadherin, aquaporin-1, and Na+/K+ ATPase. Moreover, these PCT structures displayed cell polarity and a well-defined lumen after 18 days in culture. This laminin-entactin (L-E) matrix constitutes a defined and consistent biomaterial that can be used in kidney tissue engineering for understanding in vitro proximal tubule development and for nephrotoxicity studies.

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.

Hnf4a deletion in the mouse kidney phenocopies Fanconi renotubular syndrome.

Different nephron tubule segments perform distinct physiological functions, collectively acting as a blood filtration unit. Dysfunction of the proximal tubule segment can lead to Fanconi renotubular syndrome (FRTS), with major symptoms such as excess excretion of water, glucose, and phosphate in the urine. It has been shown that a mutation in HNF4A is associated with FRTS in humans and that Hnf4a is expressed specifically in proximal tubules in adult rat nephrons. However, little is known about the role of Hnf4a in nephrogenesis. Here, we found that Hnf4a is expressed in both presumptive and differentiated proximal tubules in the developing mouse kidney. We show that Hnf4a is required for the formation of differentiated proximal tubules but is dispensable for the formation of presumptive proximal tubules. Furthermore, we show that loss of Hnf4a decreased the expression of proximal tubule-specific genes. Adult Hnf4a mutant mice presented with FRTS-like symptoms, including polyuria, polydipsia, glycosuria, and phosphaturia. Analysis of the adult Hnf4a mutant kidney also showed proximal tubule dysgenesis and nephrocalcinosis. Our results demonstrate the critical role of Hnf4a in proximal tubule development and provide mechanistic insight into the etiology of FRTS.

The von Hippel-Lindau Gene Is Required to Maintain Renal Proximal Tubule and Glomerulus Integrity in Zebrafish Larvae.

BACKGROUND: von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene. METHODS: We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays. RESULTS: Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL. CONCLUSIONS: Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.

Endocytic receptor LRP2/megalin-of holoprosencephaly and renal Fanconi syndrome.

Megalin (or LRP2) is an endocytic receptor that plays a central role in embryonic development and adult tissue homeostasis. Loss of this receptor in congenital or acquired diseases results in multiple organ dysfunctions, including forebrain malformation (holoprosencephaly) and renal reabsorption defects (renal Fanconi syndrome). Here, we describe current concepts of the mode of receptor action that include co-receptors and a repertoire of different ligands, and we discuss how these interactions govern functional integrity of the kidney and the brain, and cause disease when defective.

Expression of Cyt-c-Mediated Mitochondrial Apoptosis-Related Proteins in Rat Renal Proximal Tubules during Development.

BACKGROUND: Apoptosis regulates embryogenesis, organ metamorphosis and tissue homeostasis. Mitochondrial signaling is an apoptotic pathway, in which Cyt-c and Apaf-1 are transformed into an apoptosome, which activates procaspase-9 and triggers apoptosis. This study evaluated Cyt-c, Apaf-1 and caspase-9 expression during renal development. METHODS: Kidneys from embryonic (E) 16-, 18-, and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups were obtained. Immunohistochemical analysis, dual-labeled immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique assay and Western blot were performed in addition to histological analysis. RESULTS: Immunohistochemistry showed that Cyt-c was strongly expressed in proximal and distal tubules (DTs) at all time points. Caspase-9 and Apaf-1 were strongly expressed in proximal tubules (PTs) but only weakly expressed in DTs. Dual-labeled immunofluorescence showed that most tubules expressed both Cyt-c and Apaf-1, except for some tubules that only expressed Cyt-c. The TUNEL assay showed a greater percentage of apoptotic cells in PTs compared to DTs. Apaf-1 and cleaved caspase-9 protein expression gradually increased during the embryonic period and peaked during the early postnatal period but apparently declined from P7. Cyt-c protein expression was weak during the embryonic period but obviously increased after P1. CONCLUSION: This study showed that PTs are more sensitive to apoptosis than DTs during rat renal development, even though both tubule segments contain a large number of mitochondria. Furthermore, Cyt-c-mediated mitochondrial apoptosis-related proteins play an important role in PTs during the early postnatal kidney development.

Absence of renal enlargement in fructose-fed proximal-tubule-select insulin receptor (IR), insulin-like-growth factor receptor (IGF1R) double knockout mice.

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated AktT308 and IRY1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-ß (TGF-ß) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet.

Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development.

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.

Apobec-1 Complementation Factor (A1CF) Inhibits Epithelial-Mesenchymal Transition and Migration of Normal Rat Kidney Proximal Tubular Epithelial Cells.

Apobec-1 complementation factor (A1CF) is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, which participates in site-specific posttranscriptional RNA editing of apolipoprotein B (apoB) transcript. The posttranscriptional editing of apoB mRNA by A1CF in the small intestine is required for lipid absorption. Apart from the intestine, A1CF mRNA is also reported to be highly expressed in the kidneys. However, it is remained unknown about the functions of A1CF in the kidneys. The aim of this paper is to explore the potential functions of A1CF in the kidneys. Our results demonstrated that in C57BL/6 mice A1CF was weakly expressed in embryonic kidneys from E15.5dpc while strongly expressed in mature kidneys after birth, and it mainly existed in the tubules of inner cortex. More importantly, we identified A1CF negatively regulated the process of epithelial-mesenchymal transition (EMT) in kidney tubular epithelial cells. Our results found ectopic expression of A1CF up-regulated the epithelial markers E-cadherin, and down-regulated the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in NRK52e cells. In addition, knockdown of A1CF enhanced EMT contrary to the overexpression effect. Notably, the two A1CF variants led to the similar trend in the EMT process. Taken together, these data suggest that A1CF may be an antagonistic factor to the EMT process of kidney tubular epithelial cells.

Hepatocyte nuclear factors 4α and 1α regulate kidney developmental expression of drug-metabolizing enzymes and drug transporters.

The transcriptional regulation of drug-metabolizing enzymes and transporters (here collectively referred to as DMEs) in the developing proximal tubule (PT) is not well understood. As in the liver, DME regulation in the PT may be mediated through nuclear receptors, which are thought to "sense" deviations from homeostasis by being activated by ligands, some of which are handled by DMEs, including drug transporters. Systems analysis of transcriptomic data during kidney development predicted a set of upstream transcription factors, including hepatocyte nuclear factor 4α (Hnf4a) and Hnf1a, as well as Nr3c1 (Gr), Nfe2l2 (Nrf2), peroxisome proliferator-activated receptor α (Pparα), and Tp53. Motif analysis of cis-regulatory enhancers further suggested that Hnf4a and Hnf1a are the main transcriptional regulators of DMEs in the PT. Available expression data from tissue-specific Hnf4a knockout tissues revealed that distinct subsets of DMEs were regulated by Hnf4a in a tissue-specific manner. Chromatin immunoprecipitation combined with massively parallel DNA sequencing was performed to characterize the PT-specific binding sites of Hnf4a in rat kidneys at three developmental stages (prenatal, immature, adult), which further supported a major role for Hnf4a in regulating PT gene expression, including DMEs. In ex vivo kidney organ culture, an antagonist of Hnf4a (but not a similar inactive compound) led to predicted changes in DME expression, including among others Fmo1, Cyp2d2, Cyp2d4, Nqo2, as well as organic cation transporters and organic anion transporters Slc22a1 (Oct1), Slc22a2 (Oct2), Slc22a6 (Oat1), Slc22a8 (Oat3), and Slc47a1 (Mate1). Conversely, overexpression of Hnf1a and Hnf4a in primary mouse embryonic fibroblasts, sometimes considered a surrogate for mesenchymal stem cells, induced expression of several of these proximal tubule DMEs, as well as epithelial markers and a PT-enriched brush border marker Ggt1. These cells had organic anion transporter function. Taken together, the data strongly supports a critical role for HNF4a and Hnf1a in the tissue-specific regulation of drug handling and differentiation toward a PT-like cellular identity. We discuss our data in the context of the "remote sensing and signaling hypothesis" (Ahn and Nigam, 2009; Wu et al., 2011).

Stem cells derived from neonatal mouse kidney generate functional proximal tubule-like cells and integrate into developing nephrons in vitro.

We have recently shown that kidney-derived stem cells (KSCs) isolated from the mouse newborn kidney differentiate into a range of kidney-specific cell types. However, the functionality and integration capacity of these mouse KSCs remain unknown. Therefore, the main objectives of this study were (1) to determine if proximal tubule-like cells, generated in vitro from KSCs, displayed absorptive function typical of proximal tubule cells in vivo, and (2) to establish whether the ability of KSCs to integrate into developing nephrons was comparable with that of metanephric mesenchyme (MM), a transient population of progenitor cells that gives rise to the nephrons during kidney organogenesis. We found that proximal tubule-like cells generated in vitro from mouse KSCs displayed megalin-dependent absorptive function. Subsequently, we used a chimeric kidney rudiment culture system to show that the KSCs could generate proximal tubule cells and podocytes that were appropriately located within the developing nephrons. Finally, we compared the ability of KSCs to integrate into developing kidneys ex vivo with that of metanephric mesenchyme cells. We found that KSCs integrated into nascent nephrons to a similar extent as metanephric mesenchyme cells while both were excluded from ureteric bud branches. Our analysis of the behavior of the two cell types shows that some, but not all KSC characteristics are similar to those of the MM.

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development.

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.

MDM-2 antagonists induce p53-dependent cell cycle arrest but not cell death in renal cancer cell lines.

Renal cell cancers (RCC) are notoriously resistant to chemotherapy and radiotherapy. While mutations of the p53 tumor suppressor gene frequently contribute to therapy resistance in other epithelial cancers, p53 mutations are relatively rare in RCC. To date, there is conflicting evidence as to whether p53 signaling and function are otherwise proficient or defective in tumors with wild-type p53. In this study, we assayed p53 function in a series of RCC cell lines and normal proximal epithelial tubule cells using two different MDM-2 antagonists, Nutlin-3a and MI-219. Most cell lines with wild-type p53 responded to MDM-2 antagonists as evidenced by induction of p53 and its target gene p21. RCC cell lines treated with MDM-2 antagonists consistently accumulated in the G2/M phase of the cell cycle and this event was associated with inhibition of proliferation in RCC cell lines but not in normal proximal epithelial tubule cells. MDM-2 antagonists did not induce significant cell death in RCC cell lines, even with induction of p53-dependent pro-apoptotic genes. In contrast, MDM-2 antagonists caused significant cell death in LNCaP prostate adenocarcinoma cells. RCC cell lines with reduced p53, either by mutation or through ectopic expression of p53 shRNA, demonstrated enhanced sensitivity to cell death following sequential treatment with DNA damage and G2/M checkpoint abrogation. Our results suggest that wild-type p53 RCC cell lines are proficient in p53-dependent cell cycle arrest but defective in p53-dependent cell death.

Expression of OAT1 and OAT3 in differentiating proximal tubules of the mouse kidney.

Organic anion transporter 1 (OAT1) and OAT3 in the proximal tubules (PT) of the kidney play important roles in the elimination of harmful endogenous compounds and xenobiotics from the body. We investigated the temporal and spatial expression of OAT1 and OAT3 in the differentiating PT in mouse kidney. Ontogenic expression of OAT1 and OAT3 was investigated by immunohistochemical analysis. The S1, S2, and S3 segments of the PT were identified using antibodies to aquaporin 1 (AQP1), Na+-HCO3- cotransporter 1 (kNBC1), and AQP4. OAT1 immunoreactivity was first detected at PT in the inner cortex of 15-day-old fetuses (F15) and in the outer cortex of 7-day old pups. OAT3 was first observed in the distal tubule of F14 and in S2 segment of the PT of F16 and in S1 and S3 segments around the time of birth; expression increased through postpartum day 21. The ontogenic pattern of expression of OAT1 and OAT3 in the differentiating PT suggests that both transporters may function in the S2 segment in the fetus, but not until after birth in S1 and S3 segments.

Cellular effect evaluation of micropollutants using transporter functions of renal proximal tubule cells.

Issues pertaining to the effects of micropollutants in reclaimed water are arising in terms of their effect on human health. However, current cellular methodologies face some difficulties to detect subtle effects of waterborne micropollutants at environmental concentrations (ngL(-1)-microgL(-1)) on human and animal cells. In this study, an appropriate cellular model capable of detecting the subtle effects of aquatic micropollutants at environmental concentrations using the functions of primary cultured rabbit renal proximal tubule cells (PTCs) is proposed. Tris-(2-chloroethyl)-phosphate (TCEP) was chosen as the representative micropollutant from eight typical micropollutants via lactate dehydrogenase assay. TCEP significantly decreased not only ion (sodium, calcium, and phosphate) uptake from 10(-2) mg L(-1) (64.8-82.5%, 60.4-68.8%, and 91.9-93.8% of the control, respectively), but also the expression of ion transporters (NHE-3 and L-type Ca channel) from 10(-2) mg L(-1) (53.9-87.4% and 38.6-63.6% of the control, respectively). Moreover, TCEP significantly decreased both the non-ion (glucose, fructose, and l-arginine) uptake and the expression of non-ion transporters (SGLT 1, GLUT 5, and rBAT) from 10(-2) mg L(-1). Therefore, the results demonstrated that the function of PTCs as a cellular model can be used to determine subtle effects of environmental micropollutants at low concentrations.

Placental oxidative stress in malnourished rats and changes in kidney proximal tubule sodium ATPases in offspring.

1. Intrauterine malnutrition has been linked to the development of adult cardiovascular and renal diseases, which are related to altered Na(+) balance. Here we investigated whether maternal malnutrition increases placental oxidative stress with subsequent impact on renal ATP-dependent Na(+) transporters in the offspring. 2. Maternal malnutrition was induced in rats during pregnancy by using a basic regional diet available in north-eastern Brazil. Placental oxidative stress was evaluated by measuring thiobarbituric acid-reactive substances, which were 35-40% higher in malnourished dams (MalN). Na(+) pumps were evaluated in control and prenatally malnourished rats (at 25 and 90 days of age). 3. Identical Na(+)/K(+)-ATPase activity was found in both groups at 25 days (approximately 150 nmol P(i)/mg per min). However, although Na(+)/K(+)-ATPase increased by 40% with growth in control rats, it remained constant in pups from MalN. 4. In juvenile rats, the activity of the ouabain-insensitive Na(+)-ATPase was higher in MalN than in controls (70 vs 25 nmol P(i)/mg per min). Nevertheless, activity did not increase with kidney and body growth: at 90 days, it was 50% lower in MalN than in controls. The maximal stimulation of the Na(+)-ATPase by angiotensin (Ang) II was 35% lower in MalN than in control rats and was attained only with a much higher concentration of the peptide (10(-10) mol/L) than in controls (10(-14) mol/L). 5. Protein kinase C activity, which mediates the effects of AngII on Na(+)-ATPase was only one-third of normal values in the MalN group. 6. These results indicate that placental oxidative stress may contribute to fetal undernutrition, which leads to later disturbances in Na(+) pumps from proximal tubule cells.

Podocyte-derived BMP7 is critical for nephron development.

Individuals with congenital renal hypoplasia display a defect in the growth of nephrons during development. Many genes that affect the initial induction of nephrons have been identified, but little is known about the regulation of postinductive stages of kidney development. In the absence of the growth factor bone morphogenic protein 7 (BMP7), kidney development arrests after induction of a small number of nephrons. The role of BMP7 after induction, however, has not been fully investigated. Here, we generated a podocyte-specific conditional knockout of BMP7 (Bmp7(flox/flox);Nphs2-Cre(+) [BMP7 CKO]) to study the role of podocyte-derived BMP7 in nephron maturation. By postnatal day 4, 65% of BMP7 CKO mice had hypoplastic kidneys, but glomeruli demonstrated normal patterns of laminin and collagen IV subunit expression. Developing proximal tubules, however, were reduced in number and demonstrated impaired cellular proliferation. We examined signaling pathways downstream of BMP7; the level of cortical phosphorylated Smad1, 5, and 8 was unchanged in BMP CKO kidneys, but phosphorylated p38 mitogen-activated protein kinase was significantly decreased. In addition, beta-catenin was reduced in BMP7 CKO kidneys, and its localization to intracellular vesicles suggested that it had been targeted for degradation. In summary, these results define a BMP7-mediated regulatory axis between glomeruli and proximal tubules during kidney development.

Developmental changes in proximal tubule NaCl transport.

The proximal tubule reabsorbs two thirds of the filtered NaCl in an isoosmotic fashion. In the adult proximal tubule, active NaCl transport is mediated by the parallel operation of Na(+)/H(+) and Cl(-)/base exchangers, and a substantive amount of chloride transport occurs passively across the paracellular pathway. Studies in the neonatal proximal tubule have resulted in unexpected results. The isoform of the Na(+)/H(+) exchanger mediating proximal tubule sodium absorption, NHE3, is virtually absent in the neonatal rat kidney. NHE8, an isoform of the Na(+)/H(+) exchange, in low abundance on the apical membrane of the adult proximal tubule, is present in high abundance in the neonatal segment. Whereas chloride permeability is high in the adult, favoring passive paracellular chloride flux, the neonatal proximal tubule is virtually impermeable to chloride ions. This is again due to a developmental change in isoforms of proteins forming the tight junction. The permeability properties of epithelia are due to a family of tight junction proteins called claudins. Claudins 6 and 9 are expressed in the neonatal proximal tubule at a time when chloride permeability is low, but these claudin isoforms are virtually absent in the adult segment. The causes for these postnatal changes in proximal tubular transport and developmental isoform changes are also discussed in this review.

Interaction of membrane skeletal protein, protein 4.1B and p55, and sodium bicarbonate cotransporter1 in mouse renal S1-S2 proximal tubules.

Our recent studies demonstrated the localization of protein 4.1B, a member of the 4.1 skeletal membrane proteins, to the basolateral membranes of the S1-S2 renal proximal tubules. In the present studies, we investigated the presence of binding partners that could form a molecular complex with the 4.1B protein. Immunohistochemistry revealed the localization of p55, a membrane-associated guanylate kinase, and the sodium bicarbonate cotransporter1 (NBC1), to the basolateral membrane domain of S1-S2 in mouse renal proximal tubules. Using immunoprecipitation of kidney lysates with anti-p55 antibody, a positive band was blotted with anti-4.1B antibody. GST fusion proteins including the NBC1 and 4.1B regions were confirmed to bind with each other by electrophoresis after mixing. Both NBC1- and 4.1B-specific bands were detected in renal protein mixtures immunoprecipated by either anti-4.1B- or NBC1-specific antibodies. It is likely that NBC1, 4.1B, and p55 form a molecular complex in the basolateral membrane of the kidney S1-S2 proximal tubules. We propose that the 4.1B-containing membrane skeleton may play a role in regulating the Na(+) and HCO(3)(-) reabsorption in S1-S2 proximal tubules.