Architecture of kangaroo rat inner medulla: segmentation of descending thin limb of Henle's loop

Abstract

We hypothesize that the inner medulla of the kangaroo rat Dipodomys merriami, a desert rodent that concentrates its urine to more than 6,000 mosmol/kgH(2)O water, provides unique examples of architectural features necessary for production of highly concentrated urine. To investigate this architecture, inner medullary nephron segments in the initial 3,000 μm below the outer medulla were assessed with digital reconstructions from physical tissue sections. Descending thin limbs of Henle (DTLs), ascending thin limbs of Henle (ATLs), and collecting ducts (CDs) were identified by immunofluorescence using antibodies that label segment-specific proteins associated with transepithelial water flux (aquaporin 1 and 2, AQP1 and AQP2) and chloride flux (the chloride channel ClC-K1); all tubules and vessels were labeled with wheat germ agglutinin. In the outer 3,000 μm of the inner medulla, AQP1-positive DTLs lie at the periphery of groups of CDs. ATLs lie inside and outside the groups of CDs. Immunohistochemistry and reconstructions of loops that form their bends in the outer 3,000 μm of the inner medulla show that, relative to loop length, the AQP1-positive segment of the kangaroo rat is significantly longer than that of the Munich-Wistar rat. The length of ClC-K1 expression in the prebend region at the terminal end of the descending side of the loop in kangaroo rat is about 50% shorter than that of the Munich-Wistar rat. Tubular fluid of the kangaroo rat DTL may approach osmotic equilibrium with interstitial fluid by water reabsorption along a relatively longer tubule length, compared with Munich-Wistar rat. A relatively shorter-length prebend segment may promote a steeper reabsorptive driving force at the loop bend. These structural features predict functionality that is potentially significant in the production of a high urine osmolality in the kangaroo rat.

Fig. 1.

Immunolocalization of loops of Henle and collecting ducts (CDs) in inner medullary transverse sections. A and B: kangaroo rat, 500 and 2,000 μm below the outer medulla (K rat 2 in Fig. 4). C and D: Munich-Wistar rat, 500 and 2,000 μm below the outer medulla (MW rat 5 in Fig. 4). Antibodies were applied at equal concentrations, and images were acquired at equivalent intensity scaling. Structures not labeled with aquaporin 1 (AQP1), aquaporin 2 (AQP2), or the chloride channel ClC-K antibodies are shown in off-white, labeled with wheat germ agglutinin. Scale bars: 100 μm and 20 μm (inset). Boxed areas are enlarged in right corner insets. Arrows identify AQP1-positive descending vasa recta (DVR).

Fig. 2.

Three-dimensional reconstruction of kangaroo rat inner medullary loops of Henle and CDs. Descending thin limbs of Henle (DTLs) lie primarily at the periphery of groups of CDs, whereas ascending thin limbs of Henle (ATLs) are intermixed among the CDs. Tubules are oriented in a corticopapillary direction, with the upper edge of the image near the outer medullary-inner medullary boundary. Animal sex was female. Comparable architecture has been reported for the male Munich-Wistar rat (32). WGA, wheat germ agglutinin. Scale bar: 125 μm.

Fig. 3.

Ratios of AQP1-positive DTL and ATL profiles in transverse sections at 500 and 2,000 μm below the outer medullary-inner medullary boundary for kangaroo rat (n = 3 males, 3 females) and Munich-Wistar rat (n = 4 males). Kangaroo rat values are not significantly different from each other. Munich-Wistar rat values are significantly different from kangaroo rat values at each level and from each other at the two levels (two-way ANOVA/Scheffé tests, P < 0.05). These data are included in Fig. 4.

Fig. 4.

Ratios of AQP1-positive DTL and ATL profiles in transverse sections at various depths below the outer medullary-inner medullary boundary for kangaroo rat and Munich-Wistar rat. Kangaroo rat males, 1–3; females, 4–6. Lines fit by least squares method; y = −1E-04x + 0.69 (kangaroo rat, upper line); y = −1E-04x + 0.42 (Munich-Wistar rat, lower line).

Fig. 5.

AQP1-positive fractional length of inner medullary DTLs of kangaroo rat and Munich-Wistar rat. The AQP1-positive length is variably proportional to the length of the inner medullary thin limb segment, measured from the outer medullary-inner medullary boundary to the bend of the loop. Lines are fit to exponentials; y = −9E-06x² + 0.058x − 23.73 (kangaroo rat), y = −2E-06x² + 0.023x − 10.52 (Munich-Wistar rat).

Fig. 6.

Lengths of prebend segments in kangaroo rat and Munich-Wistar rat inner medullas. Prebend segments were reconstructed from loops with inner medullary lengths between about 400 and 2,800 μm. The prebend length is relatively constant regardless of loop length. Munich-Wistar rat, filled triangles; Kangaroo rat, open triangles. Kangaroo rat males, 1–3; females, 4 and 5. Trend line equations for these five medullas are y = −0.026x + 193.99 (MW rat 1); y = −0.014x + 220.38 (MW rat 2); y = −0.004x + 207.47 (MW rat 3); y = 0.019x + 76.95 (K rat 1); y = 0.002x + 113.05 (K rat 4). See text for additional details.

Fig. 7.

Segmentation of inner medullary loops of Henle in kangaroo rat and Munich-Wistar rat. AQP1 is expressed throughout a longer proportion of the inner medullary DTL in the kangaroo rat, compared with the Munich-Wistar rat. ClC-K1 expression begins at a lower level above the bend in the kangaroo rat, compared with the Munich-Wistar rat (arrows).