Randall’s plaque in stone formers originates in ascending thin limbs

Randall’s plaque, an attachment site over which calcium oxalate stones form, begins in the basement membranes of thin limbs of the loop of Henle. The mechanism of its formation is unknown. Possibly, enhanced delivery of calcium out of the proximal tubule, found in many stone formers, increases reabsorption of calcium from the thick ascending limb into the interstitium around descending vasa recta, which convey that calcium into the deep medulla, and raises supersaturations near thin limbs (“vas washdown”). According to this hypothesis, plaque should form preferentially on ascending thin limbs, which do not reabsorb water. We stained serial sections of papillary biopsies from stone-forming patients for aquaporin 1 (which is found in the descending thin limb) and the kidney-specific chloride channel ClC-Ka (which is found in the ascending thin limb). Plaque (which is detected using Yasue stain) colocalized with ClC-Ka, but not with aquaporin 1 (χ2 = 464, P < 0.001). We conclude that plaque forms preferentially in the basement membranes of ascending thin limbs, fulfilling a critical prediction of the vas washdown theory of plaque pathogenesis. The clinical implication is that treatments such as a low-sodium diet or thiazide diuretics that raise proximal tubule calcium reabsorption may reduce formation of plaque as well as calcium kidney stones.

Diuretic state affects ascending thin limb tight junctions

The nephron segments in the inner medulla are part of the urine concentrating mechanism. Depending on the diuretic state, they are facing a large range of extracellular osmolality. We investigated whether water homeostasis affects tubular transport and permeability properties in inner medullary descending thin limb (IMdTL) and ascending thin limb (IMaTL). Three experimental groups of rats under different diuretic states were investigated on metabolic cages: waterload, furosemide-induced diuresis, and control (antidiuresis). Urine production and osmolalities reflected the 3-day treatment. To functionally investigate tubular epithelial properties, we performed experiments in freshly isolated inner medullary thin limbs from these animals. Tubular segments were acutely dissected and investigated for trans- and paracellular properties by in vitro perfusion and electrophysiological analysis. IMdTL and IMaTL were distinguished by morphological criteria. We confirmed absence of transepithelial electrogenic transport in thin limbs. Although diffusion potential measurements showed no differences between treatments in IMdTLs, we observed increased paracellular cation selectivity under waterload in IMaTLs. NaCl diffusion potential was −5.64 ± 1.93 mV under waterload, −1.99 ± 1.72 mV under furosemide-induced diuresis, and 0.27 ± 0.40 mV under control. The corresponding permeability ratio PNa/Cl was 1.53 ± 0.21 (waterload), 1.22 ± 0.18 (furosemide-induced diuresis), and 0.99 ± 0.02 (control), respectively. Claudins are main constituents of the tight junction responsible for paracellular selectivity; however, immunofluorescence did not show qualitative differences in claudin 4, 10, and 16 localization. Our results show that IMaTLs change tight junction properties in response to diuretic state to allow adaptation of NaCl reabsorption.

Urine concentrating mechanism: impact of vascular and tubular architecture and a proposed descending limb urea-Na+ cotransporter

We extended a region-based mathematical model of the renal medulla of the rat kidney, previously developed by us, to represent new anatomic findings on the vascular architecture in the rat inner medulla (IM). In the outer medulla (OM), tubules and vessels are organized around tightly packed vascular bundles; in the IM, the organization is centered around collecting duct clusters. In particular, the model represents the separation of descending vasa recta from the descending limbs of loops of Henle, and the model represents a papillary segment of the descending thin limb that is water impermeable and highly urea permeable. Model results suggest that, despite the compartmentalization of IM blood flow, IM interstitial fluid composition is substantially more homogeneous compared with OM. We used the model to study medullary blood flow in antidiuresis and the effects of vascular countercurrent exchange. We also hypothesize that the terminal aquaporin-1 null segment of the long descending thin limbs may express a urea-Na+ or urea-Cl− cotransporter. As urea diffuses from the urea-rich papillary interstitium into the descending thin limb luminal fluid, NaCl is secreted via the cotransporter against its concentration gradient. That NaCl is then reabsorbed near the loop bend, raising the interstitial fluid osmolality and promoting water reabsorption from the IM collecting ducts. Indeed, the model predicts that the presence of the urea-Na+ or urea- Cl− cotransporter facilitates the cycling of NaCl within the IM and yields a loop-bend fluid composition consistent with experimental data.

Three-dimensional architecture of inner medullary vasa recta

The manner in which vasa recta function and contribute to the concentrating mechanism depends on their three-dimensional relationships to each other and to tubular elements. We have examined the three-dimensional architecture of vasculature relative to tubular structures in the central region of rat kidney inner medulla from the base through the first 3 mm by combining immunohistochemistry and semiautomated image acquisition techniques with graphical modeling software. Segments of descending vasa recta (DVR), ascending vasa recta (AVR), descending thin limb (DTL), ascending thin limb (ATL), and collecting duct (CD) were identified with antibodies against segment-specific proteins associated with solute and water transport (urea channel B, PV-1, aquaporin-1, ClC-K1, aquaporin-2, respectively) by immunofluorescence. Results indicate: 1) DVR, like DTLs, are excluded from CD clusters that we have previously shown to be the organizing element for the inner medulla; 2) AVR, like ATLs, are nearly uniformly distributed transversely across the entire inner medulla outside of and within CD clusters; 3) DVR and AVR outside CD clusters appear to be sufficiently juxtaposed to permit good countercurrent exchange; 4) within CD clusters, about four AVR closely abut each CD, surrounding it in a highly symmetrical fashion; and 5) AVR abutting each CD and ATLs within CD clusters form repeating nodal interstitial spaces bordered by a CD on one side, one or more ATLs on the opposite side, and one AVR on each of the other two sides. These relationships may be highly significant for both establishing and maintaining the inner medullary osmotic gradient.

Altered expression of selected genes in kidney of rats with lithium-induced NDI

Lithium treatment is associated with development of nephrogenic diabetes insipidus, caused in part by downregulation of collecting duct aquaporin-2 (AQP2) and AQP3 expression. In the present study, we carried out cDNA microarray screening of gene expression in the inner medulla (IM) of lithium-treated and control rats, and selected genes were then investigated at the protein level by immunoblotting and/or immunohistochemistry. The following genes exhibited significantly altered transcription and mRNA expression levels, and these were compatible with the changes in protein expression. 11β-Hydroxysteroid dehydrogenase type 2 protein expression in the IM was markedly increased (198 ± 25% of controls, n = 6), and immunocytochemistry demonstrated an increased labeling of IM collecting duct (IMCD) principal cells. This indicated altered renal mineralocorticoid/glucocorticoid responses in lithium-treated rats. The inhibitor of cyclin-dependent kinases p27 (KIP) protein expression was significantly decreased or undetectable in the IMCD cells, pointing to increased cellular proliferation and remodeling. Heat shock protein 27 protein expression was decreased in the IM (64 ± 6% of controls, n = 6), likely to be associated with the decreased medullary osmolality in lithium-treated rats. Consistent with this, lens aldose reductase protein expression was markedly decreased in the IM (16 ± 2% of controls, n = 6), and immunocytochemistry revealed decreased expression in the thin limb cells in the middle and terminal parts of the IM. Ezrin protein expression was upregulated in the IM (158 ± 16% of controls, n = 6), where it was predominantly expressed in the apical and cytoplasmic domain of the IMCD cells. Increased ezrin expression indicated remodeling of the actin cytoskeleton and/or altered regulation of IMCD transporters. In conclusion, the present study demonstrates changes in gene expression not only in the collecting duct but also in the thin limb of the loop of Henle in the IM, and several of these genes are linked to altered sodium and water reabsorption, cell cycling, and changes in interstitial osmolality.