 vasa recta. In the mouse, SLC4A11-positive and AQP1-positive DTLs tend to be clustered adjacent to the bundle region, but also lie more centrally within the interbundle region (Fig. 2). SLC4A11 expression is more predominant in the DTL apical membrane compared to the basolateral membrane ( Fig. 3). SLC4A11 is also expressed in cells of the OMCD in the ISOM, including principal and intercalated cells; the latter are identified by their lack of AQP2 expression (Fig. 3). In these cells, SLC4A11 is predominantly apical.

Expression of SLC4A11 in mouse outer medullary descending vasa recta (DVR)
The urea transporter UT-B is selectively expressed in DVR, and AQP1 is expressed in some of these vascular segments. As shown in Figure 4, SLC4A11 is co-expressed with UT-B and AQP1 in the mouse DVR. The endothelium of DVR is thinner than the epithelium of DTLs; therefore, SLC4A11 and AQP1 expression appear weaker in DVR compared to DTLs (Fig. 4).
The endothelial plasmalemma vesicle-associated protein (PV1, PLVAP) is a marker of fenestrated capillaries in the medulla. In the kidney, all medullary ascending vasa recta, including the branching ascending vasa recta are fenestrated and express PV1; however, these vessels do not express detectable AQP1 or SLC4A11 (Fig. 5).
Expression of SLC4A11 in the cortex and outer stripe of the mouse outer medulla In the mouse, whereas SLC4A11 is abundantly expressed in the ISOM, relatively lower or no expression was observed in the nephron segments of the cortex and OSOM (Fig. 6).

Expression of SLC4A11 in mouse inner medullary long-looped DTLs
The inner medullary SLC4A11 expression for the mouse is shown in Figure 7. SLC4A11 colocalizes with AQP1 in all AQP1-positive DTL segments. The AQP1-null segments, DVR (UTB-positive segments), and IMCDs do not express detectable SLC4A11.
Expression of SLC4A11 in rat outer medullary long-looped DTLs AQP1 is expressed in the upper portions of the descending segments of rat long-looped nephrons ("upper DTLs") (Fig. 8). The "lower DTL" segments do not express detectable AQP1. As shown in Figure 8, all of the SLC4A11-positive segments in the rat OM colocalize with AQP1. Therefore, in the rat, SLC4A11 is expressed primarily along the AQP1-positive segment of the outer medullary DTL and is not expressed in the AQP1-null segments of DTLs nor in the DVR or OMCD.

Expression of SLC4A11 in rat inner medullary long-looped DTLs
The SLC4A11 expression in a tissue section from the upper 30% of the rat IM is shown in Figure 9. SLC4A11 colocalizes with AQP1 in all segments. The AQP1-null segments, DVR (UTB-positive segments) and IMCDs do not express detectable SLC4A11. The absence of SLC4A11 expression in the AQP1-null segment is further illustrated for the rat IM, in which all ATLs were selectively labeled using an antibody against the Cl channel ClCK. 2-5% of the ClCK-positive segments are known as prebend descending segments, which extend along the terminal~200 lm of the DTL (Pannabecker 2008). Assuming equivalence of descending and ascending segments in any random tissue section, the ratio of AQP1-positive-to-AQP1-null DTLs in this section is approximately 4:3. We are unaware of an antibody that selectively labels mouse ATLs. The expression of SLC4A11 protein along the tubular and vascular segments of the mouse kidney is summarized schematically in Figure 1. SLC4A11 is expressed predominantly in the DTL upper and outer medullary DVR. A very weak expression was observed in cells of some, but not all, OMCD segments in the mouse. Little or no SLC4A11 expression was detected in the rat OMCD or DVR.

Discussion
Our new data on SLC4A11 expression in the kidney complement previous studies of DTL Na +, urea and water permeability indicating that the upper and lower DTLs of long-looped nephrons are functionally distinct (Nawata et al. 2014). In previous studies, we have documented the complex heterogeneity of AQP1 and UT-B expression in the rodent OM. In most rodent and human short-looped DTLs, little or no AQP1 is expressed along the entire length (Zhai et al. 2007). In the DVR, AQP1 and the urea transporter UT-B are expressed along the entire length (Nawata and Pannabecker 2018). SLC4A11 is expressed in AQP1-positive DTLs in the mouse and rat OM and IM. The absolute colocalization of SLC4A11 with AQP1 confirms the absence of SLC4A11 in the AQP1-null DTL. This absence is clearly illustrated for the rat IM, in which the relative abundance of AQP1-positive and AQP1-null DTLs can be determined by comparison to the abundance of ascending thin limbs. SLC4A11 colocalized with AQP1 and UT-B in the outer medullary DVR in the mouse but not rat, whereas no SLC4A11 was detected in the inner medullary DVR nor in the AVR in either species. SLC4A11 staining was also detected in the OMCD principal and intercalated cells of the mouse but not rat, and no SLC4A11 was detected in the upper 30% of the rat or mouse inner medulla. Relatively few intercalated cells occur in this region of the collecting duct. Two previous studies using immunohistochemistry have reported expression of SLC4A11 in additional nephron segments of the mouse, rat, and human kidney compared to those that we have identified (Damkier et al. 2007;Gr€ oger et al. 2010). These include the glomerular  podocytes and cortical proximal tubules in the human and the glomeruli, proximal tubules of the outer stripe of the OM and IMCD of the mouse or rat kidneys. The differences between those two studies and ours may reflect, at least in part, both qualitative and quantitative differences that arise from variable detectability levels. In the previous rat study, SLC4A11 tissue gene expression increased gradually from cortex to the outer medulla to the inner medulla; however, for the rat and human, protein expression was only shown in the principal cells of the IMCD from the deep papilla (Damkier et al. 2007). In the latter study, the papillary IMCD can be identified by their large diameter and by the sparse double-layered epithelium. We speculate that, for the mouse, rat and human, while SLC4A11 protein expression occurs in the cortical collecting ducts and OMCD, it is expressed relatively more abundantly in collecting ducts of the lower inner medulla (and only in principal cells). Our study did not investigate this region of the medulla. SLC4A11 protein expression differences potentially play a role in variable solute transepithelial transport and countercurrent mechanisms between the three species.
Recent studies have provided strong evidence that SLC4A11 functions as an ammonia and alkaline pH stimulated H + transporter and that Na +, Cl -, and K + are not transported ions. This finding coupled with our new expression data are compatible with the hypothesis that in the upper DTL and initial DVR, SLC4A11 plays a potential role in the countercurrent transport of ammonia absorbed from the outer medullary thick ascending limb. Furthermore, in the OMCD (inner stripe), apical SLC4A11 may modulate the rate of luminal ammonia secretion.  In the mammalian kidney, the ability to produce a concentrated urine is associated with the generation of a corticomedullary osmolality gradient (increasing from cortex to medullary tip). The underlying processes that generate this gradient differ in the outer versus the inner medulla. In the outer medulla, it is generally believed that there is a countercurrent-generating process resulting from active transport of NaCl by the mTAL. This segment has a very low water permeability (Rocha and Kokko 1973) and its active transport processes increase the peritubular osmolality of the anatomically adjacent long-looped DTLs in the OM (Kriz et al. 1972). These tubules express AQP1 and have a high water permeability unlike short-loop descending thin limbs (Kim et al. 2016). Previous studies have also provided support for a countercurrent ammonia recycling system in the OM. In this scheme, a portion of the ammonia absorbed by the mTAL is coupled to ammonia secretion by the DTL of long-looped nephrons in the OM (Fig. 10). Of the tubule components involved in outer medullary ammonia recycling, the transport processes mediating mTAL ammonia absorption (and their regulation) are well understood (By et al. 1994;Mount 2014). The luminal absorption of ammonia via NKCC2 (apical) and basolateral secretion via NHE4 ensures that the