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fusion and formation of a slit diaphragm-like structure in insect nephrocytes. Development 136: 2335–2344, 2009 Kestilä M, Lenkkeri U, Männikkö M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K: Positionally cloned gene for a novel glomerular protein–nephrin–is mutated in congenital nephrotic syndrome. Mol Cell 1: 575–582, 1998 D’Agati VD, Kaskel FJ, Falk RJ: Focal segmental glomerulosclerosis. N Engl J Med 365: 2398–2411, 2011 Nielsen R, Christensen EI: Proteinuria and events beyond the slit. Pediatr Nephrol 25: 813–822, 2010 Christensen E, Verroust PJ, Nielsen R: Receptor-mediated endocytosis in renal proximal tubule. Pflugers Arch 458: 1039–1048, 2009 Zhang F, Zhao Y, Chao Y, Muir K, Han Z: Cubilin and Amnionless mediate protein reabsorption in Drosophila nephrocytes. J Am Soc Nephrol 24: 209–216, 2013 Zhang F, Zhao Y, Han Z: An in vivo functional analysis system for renal gene discovery in Drosophila pericardial nephrocytes. J Am Soc Nephrol 24: 191–197, 2013 Ferrandon D, Jung AC, Criqui MC, Lemaitre B, Uttenweiler-Joseph S, Michaut L, Reichhart JM, Hoffmann JA: A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway. EMBO J 17: 1217–1227, 1998 Murphy S, Xu J, Kochanek K: Deaths: Preliminary data for 2010. Natl Vital Stat Rep 60: 1–51, 2012 Pavenstädt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 83: 253–307, 2003 Yaoita E, Kurihara H, Sakai T, Ohshiro K, Yamamoto T: Phenotypic modulation of parietal epithelial cells of Bowman’s capsule in culture. Cell Tissue Res 304: 339–349, 2001
See related articles, “Cubilin and Amnionless Mediate Protein Reabsorption in Drosophila Nephrocytes,” and “An In Vivo Functional Analysis System for Renal Gene Discovery in Drosophila Pericardial Nephrocytes,” on pages 209–216 and 191–197, respectively.
Salt and Pepper Distribution of Cell Types in the Collecting Duct Rosemary V. Sampogna and Qais Al-Awqati Department of Medicine, College of Physicians & Surgeons of Columbia University, New York, New York J Am Soc Nephrol 24: 163–165, 2013. doi: 10.1681/ASN.2012121183
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 Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Qais Al-Awqati, Department of Medicine, College of Physicians & Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032. Email:
[email protected] Copyright © 2013 by the American Society of Nephrology
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cell type. With a bit of training, it is easy to distinguish colon from ileum or proximal tubule from thick ascending limb. Rare epithelia, however, exhibit what has come to be known as a salt and pepper type. These are composed of one major cell type, with others sprinkled throughout that clearly differ in structure and function. The classic example of this epithelium is the skin of fish, amphibians, and reptiles, in which so-called chloride cells are present in a distinctive pattern. In mammals, the collecting tubule of the kidney is such an epithelium; remarkably, the sprinkled cells (the intercalated cells) are similar to the chloride cells in structure and in function. Development of organs can be considered a straightforward march starting with a multipotent progenitor that can produce all types of the cells of the organ, followed by stepwise differentiation with restricted potencies to produce only one or another segment. Within this context, the presence of a mosaic pattern in a single segment raises an interesting question: How do the minority cells develop? Do they invade from the interstitium, or does the progenitor cell type give rise to both? Because the distribution often seems random, how would the progenitor cell know where and when to stop making the majority cell and instead specify the minority cell? When both cell types arise from the same progenitor, the key mechanism to cause the mosaic pattern is lateral inhibition. In this process one cell with a given developmental fate sends a direct signal to its neighbor, causing it to assume a different fate. The notch signaling pathway mediates the molecular mechanism whereby the sending cell expresses a notch ligand (Deltalike or Jagged in mammals) and the receiving cell expresses the notch receptor. Upon ligand binding, the extracellular domain of the notch receptor undergoes endocytosis within the signalsending cell. In the signal-receiving cell, the notch intracellular domain (NICD) is generated by a series of proteolytic steps through g-secretase. The NICD is then translocated to the nucleus of this cell and ultimately induces expression of several transcription factors of the HES family (hairy and enhancer of split-1) that are usually repressors. Recently, Jeong et al.1 deleted one component of the notch signaling pathway in the collecting tubule and found an increase in percentage of intercalated cells and a concomitant decrease in that of principal cells. When NICD was expressed in these mutant mice in the collecting duct, all of the cells were found to be principal cells with no intercalated cells. Similar results were found in the Xenopus skin.2 These studies suggest that active Notch signaling allows the intercalated cells to appear. Not clear, however, were the identities of the sending and receiving cells because the inductions of the mutation in these two studies were performed without the use of cell-type– specific agents. Earlier studies showed that although the ureteric bud expresses three of the notch receptors, it does not express any of the ligands at embryonic day 15.5 (i.e., before the intercalated cell–specific proteins are expressed).3 This observation suggests that expression of the notch ligand in the adjacent principal cell results in the suppression of the principal cell fate and the appearance of the intercalated cell Editorial
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fate and is the target of the signaling pathway that produces the intercalated cells. However, more direct studies will have to be done to verify this notion. In this issue of JASN, Wu et al.4 used an aquaporin 2-Cre to delete the histone methyltransferase, Dot1l, to probe the role of epigenetic mechanisms in the lineage specification of the collecting tubule. This enzyme adds a methyl group to lysine 79 of H3 histone, imparting many functions during the cell cycle, cardiac development, and angiogenesis. In aquaporin 2–expressing cells, Cre expression deletes Dot1l, thereby blocking histone methylation. The authors used antibodies for these specific methylation events and showed that all aquaporin 2–expressing cells (principal cells in the adult mice) express the Cre recombinase and lack the histone methyl group. Remarkably, the mice present a phenotype identical to the notch inhibition mentioned above: that is, a higher proportion of intercalated cells are present in the collecting tubule. But what interests the authors is the finding that in the mutant mice, the methylation events are absent not only in the principal cells but also in almost 75% of the intercalated cells. They conclude that because this subset of intercalated cells does not express Cre, the lack of methylation indicates they must have been derived from principal cells. This is certainly a legitimate conclusion and overturns an older study showing that isolated b intercalated cells in culture can generate both principal and a intercalated cells.5 Could Dot1l function through notch signaling? At present no evidence suggests this, but the similarity of the findings is striking. There are probably many potential direct targets given the severe phenotypes of Dot1l null mice, including growth impairment, cardiac malformations, angiogenesis defects, and early embryonic lethality.6,7 That Dot1l may signal through common developmental pathways is also supported by evidence of its participation in Wnt-dependent mouse intestinal development and homeostasis.8 The collecting tubule is derived from the embryonic ureteric bud, which grows out of the Wolffian duct during embryonic day 11 (E11) in mice. At about E15, aquaporin 2 is expressed;9 approximately 2 days later, expression of the intercalated cell– specific subunits of the H1ATPase begins in the developing collecting tubule,10 as does Foxi1, the transcription factor necessary for intercalated cell specification.11 Hence, it appears that aquaporin 2 is present well before the intercalated cell appears supporting the conclusion that intercalated cells derive from aquaporin-2 expressing cells. However, there is an issue perhaps of definition. Strictly speaking, the present results show that intercalated cells are derived from cells that express aquaporin 2. Although aquaporin 2 is specifically expressed in the mature principal cell, one has to ask whether that is all there is to being a principal cell. What about other channels, such as epithelial Na1 channel, renal outer medullary potassium, or maxi-potassium, not to mention V2 receptors or mineralocorticoid receptors? But when do such cells become canonical principal cells? Finally, the authors (as well as Jeong et al.1) found that the mice had polyuria, ascribed by both groups to the reduced 164
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percentage of principal cells. But neither group ascertained whether there were actually fewer principal cells or just a reduced percentage. A reduction of only 20% in the number of principal cells seems too small to cause such a large functional defect resulting in polyuria. It would have been necessary to measure the length of the collecting tubule, as was done by Duong Van Huyen et al., who found that an acid diet in adult mice caused proliferation of the a intercalated cell in the outer medullary collecting duct with a concomitant decrease in the percentage of principal cells; however, they reported no polyuria.12 Moreover, microdissected outer medullary collecting duct segments were lengthened and noted to be sinuous in acidotic mice. Because Dot1l conditional deletions must have occurred at the time of induction of aquaporin 2 (E15) (i.e., timed at the middle of nephrogenesis), one possibility is that the mutation might also produce a subtle dysplasia of the principal cells that might cause the polyuria and urine-concentrating defects observed in these animals. DISCLOSURES None.
REFERENCES 1. Jeong HW, Jeon US, Koo BK, Kim WY, Im SK, Shin J, Cho Y, Kim J, Kong YY: Inactivation of Notch signaling in the renal collecting duct causes nephrogenic diabetes insipidus in mice. J Clin Invest 119: 3290–3300, 2009 2. Quigley IK, Stubbs JL, Kintner C: Specification of ion transport cells in the Xenopus larval skin. Development 138: 705–714, 2011 3. Chen L, Al-Awqati Q: Segmental expression of Notch and Hairy genes in nephrogenesis. Am J Physiol Renal Physiol 288: F939–F952, 2005 4. Wu H, Chen L, Zhou Q, Zhang X, Berger S, Bi J, Lewis DE, Xia Y, Zhang W: Aqp2-expressing cells give rise to renal intercalated cells. J Am Soc Nephrol. 24: 243–252, 2013 5. Fejes-Tóth G, Náray-Fejes-Tóth A: Differentiation of renal beta-intercalated cells to alpha-intercalated and principal cells in culture. Proc Natl Acad Sci U S A 89: 5487–5491, 1992 6. Nguyen AT, Zhang Y: The diverse functions of Dot1 and H3K79 methylation. Genes Dev 25: 1345–1358, 2011 7. Jones B, Su H, Bhat A, Lei H, Bajko J, Hevi S, Baltus GA, Kadam S, Zhai H, Valdez R, Gonzalo S, Zhang Y, Li E, Chen T: The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet 4: e1000190, 2008 10.1371/ journal.pgen.1000190 8. Mahmoudi T, Boj SF, Hatzis P, Li VSW, Taouatas N, Vries RG, Teunissen H, Begthel H, Korving J, Mohammed S, Heck AJ, Clevers H: The leukemiaassociated Mllt10/Af10-Dot1l are Tcf4/b-catenin coactivators essential for intestinal homeostasis. PLoS Biol 8: e1000539, 2010 10.1371/journal. pbio.1000539 9. Parreira KS, Debaix H, Cnops Y, Geffers L, Devuyst O: Expression patterns of the aquaporin gene family during renal development: Influence of genetic variability. Pflugers Arch 458: 745–759, 2009 10. Jouret F, Auzanneau C, Debaix H, Wada GH, Pretto C, Marbaix E, Karet FE, Courtoy PJ, Devuyst O: Ubiquitous and kidney-specific subunits of vacuolar H1-ATPase are differentially expressed during nephrogenesis. J Am Soc Nephrol 16: 3235–3246, 2005 11. Blomqvist SR, Vidarsson H, Fitzgerald S, Johansson BR, Ollerstam A, Brown R, Persson AE, Bergström G: Gö, Enerbäck S. Distal renal tubular acidosis in mice that lack the forkhead transcription factor Foxi1. J Clin Invest 113: 1560–1570, 2004
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12. Duong Van Huyen JP, Cheval L, Bloch-Faure M, Belair MF, Heudes D, Bruneval P, Doucet A: GDF15 triggers homeostatic proliferation of acidsecreting collecting duct cells. J Am Soc Nephrol 19: 1965–1974, 2008
See related article, “Aqp2-Expressing Cells Give Rise to Renal Intercalated Cells,” on pages 243–252.
Poverty and Mortality in Hemodialysis Patients William M. McClellan* and Chandler B. McClellan† *Departments of Epidemiology and Medicine, Rollins School of Public Health, Emory School of Medicine, Atlanta, Georgia; and †Andrew Young School of Public Policy, Georgia State University, Atlanta, Georgia J Am Soc Nephrol 24: 165–167, 2013. doi: 10.1681/ASN.2012121176
A vexing observation among individuals with ESRD is the decreased mortality associated with attributes that, in healthy populations, confer increased risk of death. Examples include the apparent protective benefit of obesity, higher levels of LDL cholesterol, elevated systolic and diastolic BP, and increased levels of parathyroid hormone.1,2 Potential explanations for this survival paradox include unmeasured confounders, misspecification of exposures, selective survival with informative censoring, selection bias due to conditioning on a diseased population,3 and a true, unbiased protective benefit of the otherwise harmful attribute.4,5 Self-reported black race is an interesting example of this conundrum. As summarized by Kimmel and colleagues in this issue of JASN, increased risk of mortality is consistently reported for black populations in the United States, a disadvantage that stands in stark contrast to their longer survival after the onset of hemodialysis.6 The authors sought to clarify this unexpected survival advantage by exploring the degree to which spatial measures of material disadvantage and racial segregation might mediate these survival differences. Material disadvantage was estimated using the Gini coefficient for a personal income in a county, which measures of the degree of inequality of the income distribution in the population. It varies from 0, which represents perfect equality of income distribution, to 1, which represents maximal income inequality. The race-specific median income of an individual patient’s zip code of residence was used as a surrogate for individual income. A third measure, the dissimilarity index, was used to measure racial segregation in the county of residence. The index varies from 0, an equal distribution of Published online ahead of print. Publication date available at www.cjasn.org. Correspondence: Dr. William M. McClellan, Department of Epidemiology, Rollins School of Public Health, Emory University School of Medicine, 1518 Clifton Road, Atlanta, GA 30322. Email:
[email protected] Copyright © 2013 by the American Society of Nephrology
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blacks and whites in a county, to 1, indicative of a racially homogeneous population. The degree of racial segregation was used to assess perceived discrimination and related stress that an individual might experience in a community. It was hypothesized that these spatial measures would explain part of the observed excess mortality observed in white, compared with black, ESRD patients. The results of this interesting article are provocative and instructive. First, neither measures of income inequality nor racial segregation attenuated the reduction in mortality among blacks compared with whites. After controlling for individual risk factors at the start of ESRD, including two surrogates for individual socioeconomic status (health insurance status and employment status), blacks were 73% less likely to die during follow-up (hazard ratio [HR], 0.73; 95% confidence interval [95% CI], 0.72, 0.75). Serial addition of income inequality and segregation measures actually accentuated the protective benefit of race, and after accounting for individual-level attributes and spatial characteristics, blacks were 30% less likely to die (HR, 0.70; 95% CI, 0.69, 0.71) during follow-up. Race-specific median income, income inequality, and segregation were independently associated with mortality after controlling for race and other covariates. Finally, in multivariable models stratified by race, there was a graded, inverse association between median zip code income and mortality such that HRs were lower as income level increased in both races. Taken together, these results lead to the conclusion that community measures of income, income inequality, and racial segregation as measured in this study cannot explain racial differences in survival among ESRD patients. This interesting article raised important additional issues. Mortality in blacks and whites appears to be influenced differently by income inequality and racial segregation, because statistically significant interactions between race and both county income inequality and residential segregation were found. Kimmel et al.6 found that higher levels of income inequality were associated with higher mortality among whites but not blacks, whereas higher levels of racial segregation were associated with substantially higher mortality in blacks and not whites. Although such interaction may be artifactual, it may also signal modification of mortality risk among blacks and whites by unappreciated mechanisms that should be further studied. The effect of income inequality on mortality in blacks is of similar magnitude as that reported by a recent meta-analysis.7 These meta-analytics support a posited threshold effect for income inequality, with Gini values ,0.3 less likely to confer increased risk of adverse outcomes. It is interesting to speculate this may be one mechanism through which race-specific income inequality may have increased mortality risk in whites, but not blacks, in this study. Because interactions may exist between individual and county measures of income inequality that could not be examined, it is possible that black/white mortality differentials may be modified by individual incomes and these effects might differ by county income inequality. Another consideration is the complexity inherent in measures of income inequality like the Gini index. The influence of income inequality on mortality may be mediated by either direct effects Editorial
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