Salt and Pepper Distribution of Cell Types in the Collecting Duct
Authors: Rosemary V. Sampogna, Qais Al-Awqati
Journal: Journal of the American Society of Nephrology (2013)

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 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 (Delta-like 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 signal-sending cell. In the signal-receiving cell, the notch intracellular domain (NICD) is generated by a series of proteolytic steps through γ-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 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 β intercalated cells in culture can generate both principal and α 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 H+ATPase 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 Na+ 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 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 α 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. Disclosure None.