The thick ascending limb (TAL) of Henle's loop drives paracellular Na + , Ca 2+ , and Mg 2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Förster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na + over Mg 2+ , whereas TJs dominated by cldn16 favor Mg 2+ over Na + ; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na + and a spatially distinct site for reabsorption of divalent cations such as Ca 2+ and Mg 2+ .T he kidney regulates the salt and water balance of the body by filtration and subsequent reabsorption or secretion of ions and water. Thereby it controls blood pressure and maintains acid-base homeostasis. The thick ascending limb (TAL) of Henle's loop drives reabsorption of Na + , Cl − , Ca 2+ , and Mg 2+ from the tubular fluid into the blood. Na + and Cl − are reabsorbed via the transcellular pathway, involving the renal-specific isoform of the Na + /K + /2Cl − cotransporter (NKCC2) in the apical epithelial cell membrane and Na + /K + -ATPase and chloride channel ClC-Kb in the basolateral membrane. K + is circulated via NKCC2 and the renal outer medullary K + channel ROMK1, across the apical cell membrane. These transport processes generate a lumen-positive transepithelial potential that drives additional paracellular reabsorption of Na + as well as the reabsorption of divalent cations, mainly Ca 2+ and Mg 2+ . Paracellular transport is regulated by the tight junction (TJ) in a size-, charge-, and water-selective manner. The main functional constituent of the TJ is the family of claudins with 27 members in mammals. Claudins consist of a four-transmembrane helix bundle, two extracellular segments that expand into the paracellular cleft, and intracellular N and C termini. Claudins interact in cis (within the same plasma membrane) and in trans (with claudins in the plasma membrane of neighbori...

The thick ascending limb of Henle's loop (TAL) drives an important part of the reabsorption of divalent cations. This reabsorption occurs via the paracellular pathway formed by the tight junction (TJ), which in the TAL shows cation selectivity. Claudins, a family of TJ proteins, determine the permeability and selectivity of this pathway. Mice were fed with normal or high-Ca(2+) diet, and effects on the reabsorptive properties of cortical and medullary TAL segments were analysed by tubule microdissection and microperfusion. Claudin expression was investigated by immunostaining and quantitative PCR. We show that the TAL adapted to high Ca(2+) load in a sub-segment-specific manner. In medullary TAL, transcellular NaCl transport was attenuated. The transepithelial voltage decreased from 10.9 ± 0.6 mV at control diet to 8.3 ± 0.5 mV at high Ca(2+) load, thereby reducing the driving force for Ca(2+) and Mg(2+) uptake. Cortical TAL showed a reduction in paracellular Ca(2+) and Mg(2+) permeabilities from 8.2 ± 0.7 to 6.2 ± 0.5 ∙ 10(-4) cm/s and from 4.8 ± 0.5 to 3.0 ± 0.2 · 10(-4) cm/s at control and high-Ca(2+) diet, respectively. Expression, localisation and regulation of claudins 10, 14, 16 and 19 differed along the corticomedullary axis: Towards the cortex, the main site of divalent cation reabsorption in TAL, high-Ca(2+) intake led to a strong upregulation of claudin-14 within TAL TJs while claudin-16 and -19 were unaltered. Towards the inner medulla, only claudin-10 was present in TAL TJ strands. In summary, high-Ca(2+) diet induced a reduction of divalent cation reabsorption via a diminution of NaCl transport and driving force in mTAL and via decreased paracellular permeabilities in cTAL. We reveal an important regulatory pattern along the corticomedullary axis and improve the understanding how the kidney disposes of detrimental excess Ca(2+).

Renal tubular transport mechanisms are optimized to be energy efficient and tailored to local gradients and transport rates. The combined transcellular action of ion channels, transporters, and pumps, together with the paracellular pathway, enables kidney function. Monogenetic diseases and mouse models indicate that both trans- and paracellular proteins can become disease-causing candidates and may be targets for future therapeutic approaches. Recent advances in tight junction research have provided new insights into their structure, function, and regulation. The thick ascending limb (TAL) is a nephron segment with specific requirements for the paracellular pathway. It has to fuel the generation of the corticomedullary concentration gradient, to be watertight, and to provide a highly selective permeability for Na and divalent cations. Tight junction composition and function in the TAL is organized along the corticomedullary axis. Even on the level of a seemingly homogeneous tubular epithelium like the TAL, there is a separation of tight junction protein expression in the strands between the respective tricellular nexus of the junctional network. Here, we highlight some new insights from our recent work and that of others in this context. In addition, we provide some perspectives for the further study of paracellular transport mechanisms.

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a coreceptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF 165 -induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF 165 -induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF 121 , an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.

The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.

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 P 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.

The accumulation of senescent cells is an important contributor to kidney aging, chronic renal disease, and poor outcome after kidney transplantation. Approaches to eliminate senescent cells with senolytic compounds have been proposed as novel strategies to improve marginal organs. While most existing senolytics induce senescent cell clearance by apoptosis, we observed that ferroptosis, an iron‐catalyzed subtype of regulated necrosis, might serve as an alternative way to ablate senescent cells. We found that murine kidney tubular epithelial cells became sensitized to ferroptosis when turning senescent. This was linked to increased expression of pro‐ferroptotic lipoxygenase‐5 and reduced expression of anti‐ferroptotic glutathione peroxidase 4 (GPX4). In tissue slice cultures from aged kidneys low dose application of the ferroptosis‐inducer RSL3 selectively eliminated senescent cells while leaving healthy tubular cells unaffected. Similar results were seen in a transplantation model, in which RSL3 reduced the senescent cell burden of aged donor kidneys and caused a reduction of damage and inflammatory cell infiltration during the early post‐transplantation period. In summary, these data reveal an increased susceptibility of senescent tubular cells to ferroptosis with the potential to be exploited for selective reduction of renal senescence in aged kidney transplants.

Background: The risk of cardiovascular events rises after acute kidney injury. Leukocytes promote atherosclerotic plaque growth and instability. We have established a model of enhanced remote atherosclerosis after renal ischemia reperfusion (IR) injury and investigate the underlying inflammatory mechanisms. Methods: Atherosclerotic lesions and inflammation were investigated in native and bone marrow-transplanted LDL receptor-deficient (LDLr-/-) mice after unilateral renal IR injury using histology, flow cytometry, and gene expression analysis. Results: Aortic root atherosclerotic lesions were significantly larger after renal IR injury than in controls. A gene expression screen revealed enrichment for chemokines and their cognate receptors in aortas of IR-injured mice in early atherosclerosis, and of T cell-associated genes in advanced disease . Confocal microscopy revealed increased aortic macrophage proximity to T cells. Differential aortic inflammatory gene regulation in IR-injured mice largely paralleled the pattern in the injured kidney. Single-cell analysis identified renal cell types that produced soluble mediators upregulated in the atherosclerotic aorta. The analysis revealed a marked early increase in Ccl2, which CCR2+ myeloid cells mainly expressed. CCR2 mediated myeloid cell homing to the post-ischemic kidney in a cell-individual manner. Reconstitution with Ccr2-/- bone marrow dampened renal post-ischemic inflammation, reduced aortic Ccl2 and inflammatory macrophage marker CD11c, and abrogated excess aortic atherosclerotic plaque formation after renal IR. Conclusions: Our data introduce an experimental model of remote proatherogenic effects of renal IR and delineate myeloid CCR2 signaling as a mechanistic requirement. Monocytes should be considered as mobile mediators when addressing systemic vascular sequelae of kidney injury.