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Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzyme deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria, promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome. It has been shown to inhibit neuronal lactate dehydrogenase 5 enzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, stiripentol decreased the synthesis of oxalate by hepatocytes in a dose-dependent manner. In vivo, oral administration of stiripentol significantly reduced urine oxalate excretion in rats. Stiripentol protected the kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, stiripentol significantly improved renal function. Patients affected by Dravet syndrome and treated with stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received stiripentol for several weeks, and urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.

Acute kidney injury (AKI) can lead to chronic kidney disease (CKD) if injury is severe and/or repair is incomplete. However, the pathogenesis of CKD following renal ischemic injury is not fully understood. Capillary rarefaction and tubular hypoxia are common findings during the AKI to CKD transition. We investigated the tubular stress response to hypoxia and demonstrated that a stress responsive transcription factor, FoxO3, was regulated by prolyl hydroxylase. Hypoxia inhibited FoxO3 prolyl hydroxylation and FoxO3 degradation, thus leading to FoxO3 accumulation and activation in tubular cells. Hypoxia-activated Hif-1α contributed to FoxO3 activation and functioned to protect kidneys, as tubular deletion of Hif-1α decreased hypoxia-induced FoxO3 activation, and resulted in more severe tubular injury and interstitial fibrosis following ischemic injury. Strikingly, tubular deletion of FoxO3 during the AKI to CKD transition aggravated renal structural and functional damage leading to a more profound CKD phenotype. We showed that tubular deletion of FoxO3 resulted in decreased autophagic response and increased oxidative injury, which may explain renal protection by FoxO3. Our study indicates that in the hypoxic kidney, stress responsive transcription factors can be activated for adaptions to counteract hypoxic insults, thus attenuating CKD development.

Summary To determine whether adult kidney papillary label-retaining cells (pLRCs) are specialized precursors, we analyzed their transcription profile. Among genes overexpressed in pLRCs, we selected candidate genes to perform qPCR and immunodetection of their encoded proteins. We found that Zfyve27, which encodes protrudin, identified a subpopulation of pLRCs. With Zfyve27-CreERT2 transgenic and reporter mice we generated bitransgenic animals and performed cell-lineage analysis. Post tamoxifen, Zfyve27-CreERT2 marked cells preferentially located in the upper part of the papilla. These cells were low cycling and did not generate progeny even after long-term observation, thus they did not appear to contribute to kidney homeostasis. However, after kidney injury, but only if severe, they activated a program of proliferation, migration, and morphogenesis generating multiple and long tubular segments. Remarkably these regenerated tubules were located preferentially in the kidney medulla, indicating that repair of injury in the kidney is regionally specified. These results suggest that different parts of the kidney have different progenitor cell pools., Graphical Abstract, Highlights • Zfyve27 (protrudin) identified a subpopulation of kidney papilla label-retaining cells • Zfyve27-CreERT2 marked cells do not contribute to kidney cellular homeostasis • Following severe renal injury, Zfyve27-marked cells generate new medullary tubules • Thus, different regions of the kidney may have distinct precursor cell populations, In this article, Al-Awqati, Oliver, and colleagues report that Zfyve27, which encodes PROTRUDIN, identified a subpopulation of adult kidney papillary label-retaining cells. In Zfyve27-CreERT2;Rosa26-tdTomato mice, marked cells preferentially located in the upper papilla and seldom cycled in homeostasis, but after severe kidney injury the cells generated multiple tubular segments but mostly located in the kidney medulla.

The nephron cortical collecting duct (CCD) is composed of principal cells, which mediate Na, K, and water transport, and intercalated cells (ICs), which are specialized for acid-base transport. There are two canonical IC forms: acid-secreting α-ICs and HCO3-secreting β-ICs. Chronic acidosis increases α-ICs at the expense of β-ICs, thereby increasing net acid secretion by the CCD. We found by growth factor quantitative PCR array that acidosis increases expression of mRNA encoding SDF1 (or CXCL12) in kidney cortex and isolated CCDs from mouse and rabbit kidney cortex. Exogenous SDF1 or pH 6.8 media increased H+ secretion and decreased HCO3 secretion in isolated perfused rabbit CCDs. Acid-dependent changes in H+ and HCO3 secretion were largely blunted by AMD3100, which selectively blocks the SDF1 receptor CXCR4. In mice, diet-induced chronic acidosis increased α-ICs and decreased β-ICs. Additionally, IC-specific Cxcr4 deletion prevented IC subtype alterations and magnified metabolic acidosis. SDF1 was transcriptionally regulated and a target of the hypoxia-sensing transcription factor HIF1α. IC-specific deletion of Hif1a produced no effect on mice fed an acid diet, as α-ICs increased and β-ICs decreased similarly to that observed in WT littermates. However, Hif1a deletion in all CCD cells prevented acidosis-induced IC subtype distribution, resulting in more severe acidosis. Cultured principal cells exhibited an HIF1α-dependent increase of Sdf1 transcription in response to media acidification. Thus, our results indicate that principal cells respond to acid by producing SDF1, which then acts on adjacent ICs.

The kidney, like other organs, grows in constant proportion to the rest of the body. When one kidney is removed, the remaining one hypertrophies. In a comprehensive series of studies, Chen et al. show that growth during maturation is mediated by the mTORC1 signaling pathway, which is induced by EGF-like peptides, and requires PI3K, PDK, AKT, mTORC2, and activation of mTORC1 through the combined effects of TSC and RHEB as part of a multiprotein complex localized on lysosomes. However, compensatory growth is mediated by amino acids, which act on mTORC1 independently of the previous pathway, and requires a class III PI3K (VPS34) that is known to be involved in vesicle trafficking to the lysosomes.

Although most nephron segments contain one type of epithelial cell, the collecting ducts consists of at least two: intercalated (IC) and principal (PC) cells, which regulate acid-base and salt-water homeostasis, respectively. In adult kidneys, these cells are organized in rosettes suggesting functional interactions. Genetic studies in mouse revealed that transcription factor Tfcp2l1 coordinates IC and PC development. Tfcp2l1 induces the expression of IC specific genes, including specific H+-ATPase subunits and Jag1. Jag1 in turn, initiates Notch signaling in PCs but inhibits Notch signaling in ICs. Tfcp2l1 inactivation deletes ICs, whereas Jag1 inactivation results in the forfeiture of discrete IC and PC identities. Thus, Tfcp2l1 is a critical regulator of IC-PC patterning, acting cell-autonomously in ICs, and non-cell-autonomously in PCs. As a result, Tfcp2l1 regulates the diversification of cell types which is the central characteristic of 'salt and pepper' epithelia and distinguishes the collecting duct from all other nephron segments.

S5D-SRCRB is a novel mouse secretory glycoprotein belonging to the ancient and highly conserved scavenger receptor cysteine-rich superfamily of protein receptors. Available evidence indicates that S5D-SRCRB interacts with conserved microbial cell wall components, as well as with some endogenous proteins, and presents a restricted tissue expression pattern. This study further analyzes the expression of S5D-SRCRB along the mouse urogenital tract. Immunohistochemical staining for S5D-SRCRB was observed in spermatocytes from seminiferous tubules and in the epithelial surface from urethra and bladder, as well as in kidney tubules, mainly from medulla and papilla. Double stainings showed that S5D-SRCRB is expressed in both principal (P) and intercalated (IC) cells from renal collecting ducts (CD). By using an in vitro cell model of IC cell differentiation, preferential expression of S5D-SRCRB was observed in the apical border of terminally differentiated IC. Colocalization of S5D-SRCRB with galectin-3 (Gal-3) was also observed in kidney and bladder, but not in testis, supporting concurrent biochemical studies demonstrating the carbohydrate-dependent interaction of Gal-3 and S5D-SRCRB. Furthermore, upregulation of S5D-SRCRB expression was observed in in vitro and in vivo models of bacterial aggression, reinforcing the emerging view that CD, and specially IC, are important players in innate defense of the urinary tract against infection. Taken together, the results indicate that S5D-SRCRB is an integral component of the urogenital tract involved in innate immune functions.

Although most nephron segments contain one type of epithelial cell, the collecting ducts consists of at least two: intercalated (IC) and principal (PC) cells, which regulate acid-base and salt-water homeostasis, respectively. In adult kidneys, these cells are organized in rosettes suggesting functional interactions. Genetic studies in mouse revealed that transcription factor Tfcp2l1 coordinates IC and PC development. Tfcp2l1 induces the expression of IC specific genes, including specific H+-ATPase subunits and Jag1. Jag1 in turn, initiates Notch signaling in PCs but inhibits Notch signaling in ICs. Tfcp2l1 inactivation deletes ICs, whereas Jag1 inactivation results in the forfeiture of discrete IC and PC identities. Thus, Tfcp2l1 is a critical regulator of IC-PC patterning, acting cell-autonomously in ICs, and non-cell-autonomously in PCs. As a result, Tfcp2l1 regulates the diversification of cell types which is the central characteristic of 'salt and pepper' epithelia and distinguishes the collecting duct from all other nephron segments. DOI: http://dx.doi.org/10.7554/eLife.24265.001

Initial studies on stem cells in many adult organs found them to be slow-cycling. When exposed to a DNA label (e.g., bromodeoxyuridine or iododeoxyuridine) during a period of cycling, all cells will incorporate it, but cells that continue cycling (in the absence of the label) will lose it whereas only the slow-cycling cells will retain it. Thus, these label-retaining cells (LRCs) have been found to be progenitor cells in many adult organs. The kidney contains LRCs, largely in the papilla, and these cells can proliferate in response to kidney injury in a specific zone in the upper papilla. The cells can then migrate in a manner depending on the SDF1/CXCR4 pathway and replace damaged cells in the cortex. Isolation of these cells has allowed a gene expression study that should facilitate lineage tracing of the fate of their progeny. Recent studies in other organs have shown that each organ contains many pools of progenitor cells, each with a specific function. Several of these pools, including LRCs, interconvert among themselves, which suggests a previously unappreciated complexity in the biology of these cell populations in response to normal homeostasis or repair.

The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795–804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair.

Epithelia, the most abundant cell type, differentiate to protoepithelia from stem cells by developing apical and basolateral membrane domains and form sheets of cells connected by junctions. Following this differentiation step, the cells undergo a second step (terminal differentiation), during which they acquire a mature phenotype, which unlike the protoepithelial one is tissue and organ specific. An extracellular matrix (ECM) protein termed hensin (DMBT1) mediates this differentiation step in the kidney intercalated cells. Although hensin is secreted as a soluble monomer, it requires polymerization and deposition in the ECM to become active. The polymerization step is mediated by the activation of inside-out signaling by integrins and by the secretion of two proteins: cypA (a cis-trans prolyl isomerase) and galectin 3.

The kidney papilla contains a population of cells with several characteristics of adult stem cells, including the retention of proliferation markers during long chase periods (i.e., they are label-retaining cells [LRCs]). To determine whether the papillary LRCs generate new cells in the normal adult kidney, we examined cell proliferation throughout the kidney and found that the upper papilla is a site of enhanced cell cycling. Using genetically modified mice that conditionally expressed green fluorescence protein fused to histone 2B, we observed that the LRCs of the papilla proliferated only in its upper part, where they associate with “chains” of cycling cells. The papillary LRCs decreased in number with age, suggesting that the cells migrated to the upper papilla before entering the cell cycle. To test this directly, we marked papillary cells with vital dyes in vivo and found that some cells in the kidney papilla, including LRCs, migrated toward other parts of the kidney. Acute kidney injury enhanced both cell migration and proliferation. These results suggest that during normal homeostasis, LRCs of the kidney papilla (or their immediate progeny) migrate to the upper papilla and form a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury.

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

The kidney developmental program encodes the intricate branching and organization of approximately 1 million functional units (nephrons). Branching regulation is poorly understood, as is the source of a 10-fold variation in nephron number. Notably, low nephron count increases the risk for developing hypertension and renal failure. To better understand the source of this variation, we analyzed the complete gestational trajectory of mouse kidney development. We constructed a computerized architectural map of the branching process throughout fetal life and found that organogenesis is composed of two distinct developmental phases, each with stage-specific rate and morphologic parameters. The early phase is characterized by a rapid acceleration in branching rate and by branching divisions that repeat with relatively reproducible morphology. The latter phase, however, is notable for a significantly decreased yet constant branching rate and the presence of nonstereotyped branching events that generate progressive variability in tree morphology until birth. Our map identifies and quantitates the contribution of four developmental mechanisms that guide organogenesis: growth, patterning, branching rate, and nephron induction. When applied to organs that developed under conditions of malnutrition or in the setting of growth factor mutation, our normative map provided an essential link between kidney architecture and the fundamental morphogenetic mechanisms that guide development. This morphogenetic map is expected to find widespread applications and help identify modifiable targets to prevent developmental programming of common diseases.

{alpha}–Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC–dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.

Cyclosporin A (CsA), a widely used immunosuppressant, causes distal renal tubular acidosis (dRTA). It exerts its immunosuppressive effect by a calcineurin-inhibitory complex with its cytosolic receptor, cyclophilin A. However, CsA also inhibits the peptidyl prolyl cis-trans isomerase (PPIase) activity of cyclophilin A. We studied HCO3− transport and changes in β-intercalated cell pH on luminal Cl− removal in isolated, perfused rabbit cortical collecting tubules (CCDs) before and after exposure to media pH 6.8 for 3 h. Acid incubation causes adaptive changes in β-intercalated cells by extracellular deposition of hensin ( J Clin Invest 109: 89, 2002). Here, CsA prevented this adaptation. The unidirectional HCO3− secretory flux, estimated as the difference between net flux and that after Cl− removal from the lumen, was −6.7 ± 0.2 pmol·min−1·mm−1 and decreased to −1.3 ± 0.2 after acid incubation. CsA in the bath prevented the adaptive decreases in HCO3− secretion and apical Cl−:HCO3− exchange. To determine the mechanism, we incubated CCDs with FK-506, which inhibits calcineurin activity independently of the host cell cyclophilin. FK-506 did not prevent the acid-induced adaptive decrease in unidirectional HCO3− secretion. However, [AD-Ser]8 CsA, a CsA derivative, which does not inhibit calcineurin but inhibits PPIase activity of cyclophilin A, completely blocked the effect of acid incubation on apical Cl−:HCO3− exchange. Acid incubation resulted in prominent “clumpy” staining of extracellular hensin and diminished apical surface of β-intercalated cells [smaller peanut agglutinin (PNA) caps]. CsA and [AD-Ser]8 CsA prevented most hensin staining and the reduction of apical surface; PNA caps were more prominent. We suggest that hensin polymerization around adapting β-intercalated cells requires the PPIase activity of cyclophilins. Thus CsA is able to prevent this adaptation by inhibition of a peptidyl prolyl cis-trans isomerase activity. Such inhibition may cause dRTA during acid loading.

Many epithelial organs, such as the kidney, gastrointestinal tract, skin, lung, and brain, are segmented such that each region has its characteristic cell type. Close examination of these segments reveals that they are homogenous and contain only one epithelial cell type. With a bit of training, it

Many adult organs contain stem cells, which are pluripotent and are involved in organ maintenance and repair after injury. In situ, these cells often have a low cycling rate and locate in specialized regions (niches). To detect such cells in the kidney, we administered a pulse of the nucleotide bromodeoxyuridine (BrdU) to rat and mouse pups and, after a long (more than 2-month) chase, examined whether the kidney contained a population of low-cycling cells. We found that in the adult kidney, BrdU-retaining cells were very sparse except in the renal papilla, where they were numerous. During the repair phase of transient renal ischemia, these cells entered the cell cycle and the BrdU signal quickly disappeared from the papilla, despite the absence of apoptosis in this part of the kidney. In vitro isolation of renal papillary cells showed them to have a plastic phenotype that could be modulated by oxygen tension and that when injected into the renal cortex, they incorporated into the renal parenchyma. In addition, like other stem cells, papillary cells spontaneously formed spheres. Single-cell clones of these cells coexpressed mesenchymal and epithelial proteins and gave rise to myofibroblasts, cells expressing neuronal markers, and cells of uncharacterized phenotype. These data indicate that the renal papilla is a niche for adult kidney stem cells.

Single-layered epithelia are the first differentiated cell types to develop in the embryo, with columnar and squamous types appearing immediately after blastocyst implantation. Here, we show that mouse embryonic stem cells seeded on hensin or laminin, but not fibronectin or collagen type IV, formed hemispheric epithelial structures whose outermost layer terminally differentiated to an epithelium that resembled the visceral endoderm. Hensin induced columnar epithelia, whereas laminin formed squamous epithelia. At the egg cylinder stage, the distal visceral endoderm is columnar, and these cells begin to migrate anteriorly to create the anterior visceral endoderm, which assumes a squamous shape. Hensin expression coincided with the dynamic appearance and disappearance of columnar cells at the egg cylinder stage of the embryo. These expression patterns, and the fact that hensin null embryos (and those already reported for laminin) die at the onset of egg cylinder formation, support the view that hensin and laminin are required for terminal differentiation of columnar and squamous epithelial phenotypes during early embryogenesis.

Stem cells in the kidney. The kidney is derived from the ureteric bud and the metanephrogenic mesenchyme, and these two progenitor cells differentiate into more than 26 different cell types in the adult kidney. The ureteric bud contains the precursor of the epithelial cells of the collecting duct and the renal mesenchyme contains precursors of all the epithelia of the rest of the nephron, endothelial cell precursors and stroma cells, but the relatedness among these cells is unclear. A single metanephric mesenchymal cell can generate all the epithelial cells of the nephron (except the collecting duct), indicating that the kidney contains epithelial stem cells. It is currently unknown whether these stem cells also are present in the adult kidney but experience in other organs makes this likely. It also is unclear whether embryonic renal epithelial stem cells can generate other cell types, but preliminary studies in our laboratory suggest that they can differentiate into myofibroblasts, smooth muscle, and perhaps endothelial cells, indicating that they are pluripotent renal stem cells. The important problem to be solved now is the identification and location of adult renal stem cells. This article discusses work done in other organs and in renal development that we believe may be useful for the resolution of this problem.

Branching morphogenesis is a central event during the development of kidneys, lungs, and other organs. We previously generated a monoclonal antibody, 3D2-E9, that inhibited branching morphogenesis and caused widespread apoptosis. We now report the purification of its antigen and cloning of its full-length cDNA. Its cDNA encodes an integral membrane protein that contains four cadherin-like ectodomains and a thrice tandemly repeated region enriched in threonine, serine, and proline, similar to those of mucins. We thus term this protein mu-protocadherin, reflecting the hybrid nature of its extracellular region. mu-Protocadherin is expressed in two forms that are developmentally regulated, with the shorter isoform lacking the mucin-like repeats. Expression of the long isoform in heterologous cells results in adhesion of the expressing cells, suggesting that it is a new cell adhesion molecule. mu-Protocadherin contains both N and O glycosylations. It is expressed at lateral and basal surfaces of epithelia during kidney and lung development and is located in coated pits. Colocalization of mu-protocadherin with beta-catenin was noted primarily at the junction of the lateral and basal membrane. The cytoplasmic domain contains four proline-rich regions, similar to SH3 binding regions. Thus, it is likely that adhesive interactions mediated by mu-protocadherin induce signaling events that regulate branching morphogenesis.

The intercalated cell of the collecting tubule exists in a spectrum of types. The alpha form secretes acid by an apical H+ ATPase and a basolateral Cl:HCO3 exchanger which is an alternatively spliced form of the red cell band 3 (kAE1), while the beta form secretes HCO3 by having these transporters on the reverse membranes. In a clonal cell line of the beta form we found that seeding density causes this conversion. A new protein, termed hensin, was deposited in the extracellular matrix of high-density cells which on purification reversed the polarity of the transporters. Hensin also induced the expression of the microvillar protein, villin, and caused the appearance of the apical terminal web proteins, cytokeratin 19 and actin, all of which led to the development of an exuberant microvillar structure. In addition, hensin caused the beta cells to assume a columnar shape. All of these studies demonstrate that the conversion of polarity in the intercalated cell, at least in vitro, represents terminal differentiation and that hensin is the first protein in a new pathway that mediates this process. Hensin, DMBT1, CRP-ductin, and ebnerin are alternately spliced products from a single gene located on human chromosome 10q25–26, a region often deleted in several cancers, especially malignant gliomas. Hensin is expressed in many epithelial cell types, and it is possible that it plays a similarly important role in the differentiation of these epithelia as well.

When an intercalated epithelial cell line was seeded at low density and allowed to reach confluence, it located the anion exchanger band 3 in the apical membrane and an H+-ATPase in the basolateral membrane. The same clonal cells seeded at high density targeted these proteins to the reverse location. Furthermore, high density cells had vigorous apical endocytosis, and low density cells had none. The extracellular matrix of high density cells was capable of inducing apical endocytosis and relocation of band 3 to the basolateral membrane in low density cells. A 230-kDa extracellular matrix (ECM) protein termed hensin, when purified to near-homogeneity, was able to reverse the phenotype of the low density cells. Antibodies to hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. We show here that hensin was synthesized by both low density and high density cells. Whereas both phenotypes secreted soluble hensin into their media, only high density cells localized it in their ECM. Analysis of soluble hensin by sucrose density gradients showed that low density cells secreted monomeric hensin, and high density cells secreted higher order multimers. When 35S-labeled monomeric hensin was added to high density cells, they induced its aggregation suggesting that the multimerization was catalyzed by surface events in the high density cells. Soluble monomeric or multimeric hensin did not induce apical endocytosis in low density cells, whereas the more polymerized hensin isolated from insoluble ECM readily induced it. These multimers could be disaggregated by sulfhydryl reagents and by dimethylmaleic anhydride, and treatment of high density ECM by these reagents prevented the induction of endocytosis. These results demonstrate that hensin, like several ECM proteins, needs to be precipitated in the ECM to be functional.

In the kidney, there is a close and intricate association between epithelial and endothelial cells, suggesting that a complex reciprocal interaction may exist between these two cell types during renal ontogeny. Thus, we examined whether metanephrogenic mesenchymal cells secrete endothelial mitogens. With an endothelial mitogenic assay and sequential chromatography of the proteins in the media conditioned by a cell line of rat metanephrogenic mesenchymal cells (7.1.1 cells), we isolated a protein whose amino acid analysis identified it as hepatoma-derived growth factor (HDGF). Media conditioned with Cos-7 cell transfected with HDGF cDNA stimulated endothelial DNA synthesis. With immunoaffinity purified antipeptide antibodies, we found that HDGF was widely distributed in the renal anlage at early stages of development but soon concentrated at sites of active morphogenesis and, except for some renal tubules, disappeared from the adult kidney. From a 7.1.1 cells cDNA library, a clone of most of the translatable region of HDGF was obtained and used to synthesize digoxigenin-labeled riboprobes. In situ hybridization showed that during kidney development mRNA for HDGF was most abundant at sites of nephron morphogenesis and in ureteric bud cells while in the adult kidney transcripts disappeared except for a small population of distal tubules. Thus, HDGF is an endothelial mitogen that is present in embryonic kidney, and its expression is synchronous with nephrogenesis.

Although many polarized proteins are sorted to the same membrane domain in all epithelial tissues, there are some that exhibit a cell type–specific polarity. We recently found that band 3 (the anion exchanger AE1) was present in the apical membrane of a renal intercalated cell line when these cells were seeded at low density, but its targeting was reversed to the basolateral membrane under the influence of an extracellular matrix protein secreted when the cells were seeded at high density. Because apical and basolateral lipids differ in epithelia, we asked what effect might these lipids have on band 3 function. This question is especially interesting since apical anion exchange in these cells is resistant to disulfonic stilbene inhibitors while basolateral anion exchange is quite sensitive. Furthermore, the apical anion exchanger cannot be stained by antibodies that readily identify the basolateral protein.We used short chain sphingolipid analogues and found that sphingomyelin was preferentially targeted to the basolateral domain in the intercalated cell line. The ganglioside GM1 (Gal 1β1, 3GalNAcβ1, 4Gal-NeuAcα2, 3Galβ1, 4Glc ceramide) was confined to the apical membrane as visualized by confocal microscopy after addition of fluorescent cholera toxin to filter grown cells. We reconstituted erythrocyte band 3 into liposomes using apical and basolateral types of lipids and examined the inhibitory potency of 4,4′-dinitorsostilbene-2,2′-disulfonic acid (DNDS; a reversible stilbene) on 35SO4/SO4 exchange. Although anion exchange in sphingomyelin liposomes was sensitive to inhibition, the addition of increasing amounts of the ganglioside GM1 reduced the potency of the inhibitor drastically. Because these polarized lipids are present in the exofacial surface of the bilayer, we propose that the lipid structure might influence the packing of the transmembrane domains of band 3 in that region, altering the binding of the stilbenes to these chains. These results highlight the role of polarized lipids in changing the function of unpolarized proteins or of proteins whose locations differ in different epithelia.

The intercalated cell of the collecting tubule of the mammalian kidney is specialized for the transport of H+ and HCO3. They exist in two forms; one specialized for acid secretion and the other secretes HCO3 into the urine. We discovered many years ago that feeding animals an acid diet converts the HCO3 secreting form to an acid secreting type. Here I discuss the molecular basis of this transformation. The conversion of the cell types is mediated by an extracellular matrix protein hensin (also known as DMBT1). However much remains to be identified in the differentiation of these cells.

The idea that the kidney is an organ needs to be tempered by the realization that it is the nephron that is the organ. Each nephron, as far as we know, is independent from other nephrons, and the kidney is to a first approximation a collection of these mini-organs put together in a complex three-dimensional assembly. The nephrons of all mammals are remarkably similar in size, function, and origin (as far as we know), and the differences among species are largely if not entirely due to the number of these units in the assembly, with mice having 10,000 and whales having 250 million in each kidney. But while the epithelial character of the nephron garners most of the attention, one needs to be reminded that the kidney has a very extensive vascular network with multiple distinct morphological and functional domains, as well as an abundant interstitial cell population that unfortunately is still poorly understood (see Kaissiling and Le Hir 1 for a review). While the taxonomy of the renal epithelial cells is nearly complete, that of the cells of the vascular and interstitial compartments awaits detailed characterization. Hence, analysis of renal regeneration after injury, as well as search for putative renal stem cells in the adult kidney, has essentially been restricted to the epithelial compartment. However, as epithelial cells are likely instructed by mesenchymal signals and epithelia-mesenchymal cross-talk is critical for renal epithelial differentiation and function, 2 there is a need for deeper understanding of the cell types that comprise the renal vascular and interstitial compartments, and their roles in kidney homeostasis and repair from injury. For example, in many organs including the kidney, mesenchymal cells with characteristics of precursor/stem cells have been found to reside near or in the vascular wall, [3] ; [4] ; [5] but the exact origin and normal function of these cells is unknown.

The intercalated cell is an epithelial cell of the renal collecting tubule that is specialized for H+ and HCO3- transport. These cells exist as two types, alpha and beta. The alpha-cell secretes H+ into the lumen by an apical H(+)-ATPase and a basolateral Cl-/HCO3- exchanger that is a form of band 3 protein (AE1). The beta-cell secretes HCO3- into the lumen by an apical Cl-/HCO3- exchanger and a basolateral H(+)-ATPase. In a previous study, it was suggested that a reversal in epithelial polarity of these cells occurs during the response of the kidney to an acid load (G.J. Schwartz, J. Barasch, and Q. Al-Awqati. Nature Lond. 318: 368-371,1985). Recent studies, however have shown that there are many other subtypes where the distribution of these two proteins does not fit into this neat bipolar classification. This group of investigators recently generated an immortalized cell line of the beta-intercalated cell and found that the apical Cl-/HCO3- exchanger is also AE1. Furthermore, when these cells were seeded at high densities, the polarized targeting of the apical band 3 was reversed to the basolateral membrane. This was produced by the secretion of extracellular matrix protein that by themselves were capable of reversing the polarity of band 3 (J. S. van Adelsberg, J. C. Edwards, J. Takito, B. Kiss, and Q. Al-Awqati. Cell 76: 1053-1061, 1995). A large new extracellular matrix protein, hensin, was identified and found to be present exclusively in the collecting tubule. The extensive recent literature on the biology of alpha- and beta-intercalated cells is reviewed here and found to be compatible with the idea of the reversal of polarity as a mechanism for the regulation of H+ secretion by the tubule

Chronic colonization and infection of the lung with Pseudomonas aeruginosa is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. We found that polarized CF bronchial and pancreatic epithelia bound P. aeruginosa in a reversible and dose-dependent manner. There was significantly greater binding to CF bronchial and pancreatic cells than to their matched pairs rescued with the wild-type CF transmembrane conductance regulator. Bound P. aeruginosa were easily displaced by unlabeled P. aeruginosa but not by Escherichia coli, an organism that does not cause significant pulmonary disease in CF. In contrast, Staphylococcus aureus, a frequent pathogen in CF, could effectively displace bound P. aeruginosa from its receptor. We found undersialylation of apical proteins and a higher concentration of asialoganglioside 1 (aGM1) in apical membranes of CF compared with rescued epithelia. Incubation of P. aeruginosa with aGM1 reduced its binding, as did treatment of the epithelia with the tetrasaccharide moiety of this ganglioside (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc). Finally, an antibody to aGM1 effectively displaced P. aeruginosa from its binding site and blocked binding of S. aureus to CF cells but not to rescued cells. These results show that the tetrasaccharide of aGM1 is a receptor for P. aeruginosa and S. aureus and that its increased abundance in the apical membrane of CF epithelia makes it a likely contributor to the pathogenesis of bacterial infections in the CF lung.

Podocytes are specialized cells that play an integral role in the renal glomerular filtration barrier via their foot processes. The foot processes form a highly organized structure, the disruption of which causes nephrotic syndrome. Interestingly, several similarities have been observed between mechanisms that govern podocyte organization and mechanisms that mediate neuronal synapse development. Dynamin, synaptojanin, and endophilin are functional partners in synaptic vesicle recycling via interconnected actions in clathrin-mediated endocytosis and actin dynamics in neurons. A role of dynamin in the maintenance of the kidney filtration barrier via an action on the actin cytoskeleton of podocytes was suggested. Here we used a conditional double-KO of dynamin 1 (Dnm1) and Dnm2 in mouse podocytes to confirm dynamin’s role in podocyte foot process maintenance. In addition, we demonstrated that while synaptojanin 1 (Synj1) KO mice and endophilin 1 (Sh3gl2), endophilin 2 (Sh3gl1), and endophilin 3 (Sh3gl3) triple-KO mice had grossly normal embryonic development, these mutants failed to establish a normal filtration barrier and exhibited severe proteinuria due to abnormal podocyte foot process formation. These results strongly implicate a protein network that functions at the interface between endocytosis and actin at neuronal synapses in the formation and maintenance of the kidney glomerular filtration barrier.

There is a specter haunting nephrologic research today, the specter of decline. Perhaps I am simply parroting a recent spate of books arguing that everything is in a state of decline: countries, the United States in particular, Western civilization, quality of life, and manners. Declinism, made

Thyroid parafollicular (PF) cells are neural crest-derived endocrine cells that secrete serotonin and calcitonin. The secretory vesicles of PF cells acidify when secretion is induced by increased extracellular Ca2+ or TSH. We tested the hypothesis that acidification is regulated by secretogogue-gated Cl- channels in vesicular membranes. Cl- channel (p64) immunoreactivity was enriched in purified PF vesicles. X-Ray microanalysis showed a change in chlorine level in PF vesicles in response to secretogogue-stimulation of isolated cells. Secretogogue stimulation also altered the degree of p64 channel phosphorylation. Protein kinase and phosphatase inhibitors antagonized secretogogue-induced vesicle acidification and secretion; however, secretion could occur even when acidification was blocked. We conclude that acidification of PF vesicles is regulated by a gatable Cl- channel in vesicle membranes and that protein phosphorylation and dephosphorylation are involved in channel activation. Acidification of vesicles is not required for exocytosis.

The intercalated epithelial cell exists in two interconvertible forms in vivo, one where band 3 protein is apical and the other where it is basolateral. We seeded an immortalized clone of these cells at low density and found that band 3 was apical at confluence. There was little or no apical endocytosis. But when the cells were plated at high density, band 3 was basolateral, and there was vigorous apical endocytosis. Extracellular matrix produced by high density cells was able to retarget band 3 in low density cells and to induce apical endocytosis, as did a 230 kd protein partially purified from this matrix. Therefore, polarized targeting of some proteins is determined by external cues that might determine their polarity by reorganizing the cytoplasm.

The intercalated cell of collecting ducts of the kidney is of two forms, the α form secretes acid, whereas the β form secretes HCO3. Here, we review recent work that shows that the α form is derived from the β form and that the pathway is mediated by an extracellular matrix protein called hensin/DMBT1.

There are two types of intercalated cells of the renal collecting duct; one secretes H+ and the other secretes HCO3-. The H(+)-secreting form has an apical vacuolar H(+)-ATPase and a basolateral Cl/HCO3 exchanger that cross-reacts with antibodies to band 3, the product of the AE1 gene. The HCO3(-)-secreting form has a basolateral vacuolar H(+)-ATPase and an apical Cl/HCO3 exchanger, whose identity has not been established previously. Apical membrane vesicles of beta intercalated cells purified from rabbit kidney cortex contain both an electroneutral Cl/HCO3 exchange activity and polypeptides that react with antibodies to band 3 on Western blots. Furthermore, both primary cultures of HCO3(-)-secreting intercalated cells and an immortalized cell line derived from these cells express AE1 and have an apical Cl/HCO3 exchanger. Apical membranes purified from these cells contain a 100-kDa polypeptide that cross-reacts with antibody to the cytoplasmic domain of band 3. These data suggest that the apical Cl/HCO3 exchanger of HCO3(-)-secreting intercalated cells is band 3.

Acid–base transport in the renal collecting tubule is mediated by two canonical cell types: the β-intercalated cell secretes HCO 3 by an apical Cl:HCO 3 named pendrin and a basolateral vacuolar (V)-ATPase. Acid secretion is mediated by the α-intercalated cell, which has an apical V-ATPase and a basolateral Cl:HCO 3 exchanger (kAE1). We previously suggested that the β-cell converts to the α-cell in response to acid feeding, a process that depended on the secretion and deposition of an extracellular matrix protein termed hensin (DMBT1). Here, we show that deletion of hensin from intercalated cells results in the absence of typical α-intercalated cells and the consequent development of complete distal renal tubular acidosis (dRTA). Essentially all of the intercalated cells in the cortex of the mutant mice are canonical β-type cells, with apical pendrin and basolateral or diffuse/bipolar V-ATPase. In the medulla, however, a previously undescribed cell type has been uncovered, which resembles the cortical β-intercalated cell in ultrastructure, but does not express pendrin. Polymerization and deposition of hensin (in response to acidosis) requires the activation of β1 integrin, and deletion of this gene from the intercalated cell caused a phenotype that was identical to the deletion of hensin itself, supporting its critical role in hensin function. Because previous studies suggested that the conversion of β- to α-intercalated cells is a manifestation of terminal differentiation, the present results demonstrate that this differentiation proceeds from HCO 3 secreting to acid secreting phenotypes, a process that requires deposition of hensin in the ECM.

During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.

Chloride channels are present in the plasma and intracellular membranes of most cells. Previously, using the ligand indanyloxyacetic acid (IAA), we purified four major proteins from bovine kidney cortex membrane vesicles. These proteins gave rise to chloride channel activity when reconstituted into phospholipid vesicles. Two of these proteins (97 and 27 kDa) were found to be drug-binding proteins by N-terminal sequence analysis. Antibodies raised to the 64-kDa protein stained only this protein on immunoblots, and only this protein was present after purification on an immunoaffinity column. In addition, these same antibodies were able to deplete IAA-94 inhibitable chloride channel activity from solubilized kidney membranes. Of fractions obtained from the gel filtration of solubilized kidney membranes, only those containing this 64-kDa protein exhibited measurable chloride channel activity. Immunoblots of a variety of species and cell types, both epithelial and nonepithelial, revealed that this protein is ubiquitous and highly conserved. Immunocytochemistry in CFPAC-1 cells revealed staining for this protein on the apical plasma membrane and in the membranes of intracellular organelles. These results demonstrate that the integral membrane protein p64 is a component of chloride channels present in both epithelial plasma membrane and the membranes of intracellular organelles.

At least fourteen epithelial cell types of the mammalian nephron develop from the metanephric mesenchyme. To distinguish whether this single embryological primor-dium contains a heterogenous population of committed renal cell lines or a multipotent stem cell, the lac-Z gene was introduced into individual renal progenitors by retroviral mediated gene transfer. The differentiated fate of Zac-Z-tagged daughters derived from single metanephric mesenchymal cells was characterized after cytodifferentiation. We found that the metanephric mesenchyme contains multipotent stem cells that can generate at least three distinct cell types; glomerular, proximal and distal epithelia. After induction the fate of this multipotent cell becomes restricted to populate a single nephron segment.