Urine-concentrating Mechanism Research Articles

The SLC6A18 Transporter Is Most Likely a Na-Dependent Glycine/Urea Antiporter Responsible for Urea Secretion in the Proximal Straight Tubule: Influence of This Urea Secretion on Glomerular Filtration Rate.

Urea is the major end-product of protein metabolism in mammals. In carnivores and omnivores, a large load of urea is excreted daily in urine, with a concentration that is 30-100 times above that in plasma. This is important for the sake of water economy. Too little attention has been given to the existence of energy-dependent urea transport that plays an important role in this concentrating activity. This review first presents functional evidence for an energy-dependent urea secretion that occurs exclusively in the straight part of the proximal tubule (PST). Second, it proposes a candidate transmembrane transporter responsible for this urea secretion in the PST. SLC6A18 is expressed exclusively in the PST and has been identified as a glycine transporter, based on findings in SLC6A18 knockout mice. We propose that it is actually a glycine/urea antiport, secreting urea into the lumen in exchange for glycine and Na. Glycine is most likely recycled back into the cell via a transporter located in the brush border. Urea secretion in the PST modifies the composition of the tubular fluid in the thick ascending limb and, thus, contributes, indirectly, to influence the "signal" at the macula densa that plays a crucial role in the regulation of the glomerular filtration rate (GFR) by the tubulo-glomerular feedback. Taking into account this secondary active secretion of urea in the mammalian kidney provides a new understanding of the influence of protein intake on GFR, of the regulation of urea excretion, and of the urine-concentrating mechanism.

Physiological Functions of Aquaporin-3 in Mediating Water and Solutes

Aquaporins (AQP), are a family of membrane channel proteins that facilitate the transmembrane diffusion of water and some small non-charged solutes such as glycerol and urea by osmosis or solute gradient. AQP3, an aquaglyceroporin, is expressed in the kidney, skin, and testes, etc. Mice lacking AQP3 have symptoms of diabetes insipidus. In addition, the absence of AQP3 leads to dry skin in mice. AQP4 is permeable to water only. To understand the specific function of the water permeability and small solute transport mediated by AQP3, we generated AQP3 knock-out (AQP3 KO) mice and AQP4 knock-in mice (AQP4 KI) with AQP4 replacing AQP3 in situ. In the basal condition, knock-out of AQP3 in mice led to increased urine volume, decreased urine osmolality and tubule dilation. Interestingly, in AQP4 KI mice, these phenomena were reversed, showing improved urine concentration and tubular morphology. However, there was no significant difference in non-urea solute concentrations between both genotypes. Subsequently, we conducted acute urea loading tests and high-protein diet experiments to further increase the production and excretion of urea in mice. The experimental results showed that both urea loading and high-protein diet had little impact on urine osmolality and urinary urea concentration in AQP3 KO mice. But the urine osmolality and urinary urea concentration in AQP4 KI mice were altered by exogenous administration of urea. Our data indicates that the absence of AQP3 prevents urea accumulation in the kidney, disrupting urine concentration mechanism. The knock-in of AQP4 can restore AQP3 mediated water channel function and help to regulate the urine concentration ability. The water permeability, other than solute transport, of AQP3 plays important role in urine concentrating mechanism. AQP3 KO mice exhibited significant epidermal thickening, excessive keratinization, decreased skin hydration and elasticity. The replacement of AQP4 in situ did not improve these phenomena, indicating that solute transport mediated by AQP3 may play a role in maintaining normal skin function. Therefore, we constructed a mouse model of psoriasis induced by imiquimod (IMQ). It was found that AQP3 expression was reduced in the lesional skin of wildtype (WT) mice, while AQP3 KO mice and AQP4 KI mice exhibited dry skin, excessive epidermal proliferation, and increased inflammatory response. Local treatment with urea (10%) could alleviate these symptoms. These experimental results confirmed the important role of AQP3 in the pathogenesis of psoriasis and the potential role of urea in the treatment of psoriasis. Our study suggests the diverse physiological functions of AQP3 in different tissues. This research was supported by Beijing Natural Science Foundation (7232249). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterization of gene expression in the kidney of renal tubular cell-specific NFAT5 knockout mice.

Nuclear factor of activated T cells 5 (NFAT5; also called TonEBP/OREBP) is a transcription factor that is activated by hypertonicity and induces osmoprotective genes to protect cells against hypertonic conditions. In the kidney, renal tubular NFAT5 is known to be involved in the urine concentration mechanism. Previous studies have suggested that NFAT5 modulates the immune system and exerts various effects on organ damage, depending on organ and disease states. Pathophysiological roles of NFAT5 in renal tubular cells, however, still remain obscure. We conducted comprehensive analysis by performing transcription start site (TSS) sequencing on the kidney of inducible and renal tubular cell-specific NFAT5 knockout (KO) mice. Mice were subjected to unilateral ureteral obstruction to examine the relevance of renal tubular NFAT5 in renal fibrosis. TSS sequencing analysis identified 722 downregulated TSSs and 1,360 upregulated TSSs, which were differentially regulated ≤-1.0 and ≥1.0 in log2 fold, respectively. Those TSSs were annotated to 532 downregulated genes and 944 upregulated genes, respectively. Motif analysis showed that sequences that possibly bind to NFAT5 were enriched in TSSs of downregulated genes. Gene Ontology analysis with the upregulated genes suggested disorder of innate and adaptive immune systems in the kidney. Unilateral ureteral obstruction significantly exacerbated renal fibrosis in the renal medulla in KO mice compared with wild-type mice, accompanied by enhanced activation of immune responses. In conclusion, NFAT5 in renal tubules could have pathophysiological roles in renal fibrosis through modulating innate and adaptive immune systems in the kidney.NEW & NOTEWORTHY TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.

Biomolecular condensates in kidney physiology and disease.

The regulation and preservation of distinct intracellular and extracellular solute microenvironments is crucial for the maintenance of cellular homeostasis. In mammals, the kidneys control bodily salt and water homeostasis. Specifically, the urine-concentrating mechanism within the renal medulla causes fluctuations in extracellular osmolarity, which enables cells of the kidney to either conserve or eliminate water and electrolytes, depending on the balance between intake and loss. However, relatively little is known about the subcellular and molecular changes caused by such osmotic stresses. Advances have shown that many cells, including those of the kidney, rapidly (within seconds) and reversibly (within minutes) assemble membraneless, nano-to-microscale subcellular assemblies termed biomolecular condensates via the biophysical process of hyperosmotic phase separation (HOPS). Mechanistically, osmotic cell compression mediates changes in intracellular hydration, concentration and molecular crowding, rendering HOPS one of many related phase-separation phenomena. Osmotic stress causes numerous homo-multimeric proteins to condense, thereby affecting gene expression and cell survival. HOPS rapidly regulates specific cellular biochemical processes before appropriate protective or corrective action by broader stress response mechanisms can be initiated. Here, we broadly survey emerging evidence for, and the impact of, biomolecular condensates in nephrology, where initial concentration buffering by HOPS and its subsequent cellular escalation mechanisms are expected to have important implications for kidney physiology and disease.

Urine osmolality predicts worsening renal function and poor prognosis in acute decompensated heart failure

Abstract Background/Purpose Worsening renal function (WRF) can sometimes occur in the patients with acute decompensated heart failure (ADHF) and increase the risk of morbidity and mortality (1). In a previous study, it was reported that fractional excretion of sodium (FENa) reflects net sodium reabsorption from nephron segments and predicts WRF during treating ADHF (2). On the other hand, recently the new drugs which approach urine concentration mechanism and affect urine osmolality (U-OSM), such as tolvaptan and sodium-glucose cotransporter-2 inhibitor, have begun to be widely used as treatment of heart failure. Thus, we focused on U-OSM, which reflects not only sodium handling but also water excretion controlled by the collecting duct, and evaluated the association between WRF and U-OSM. Moreover, previous studies have demonstrated that FENa, fractional excretion of urea nitrogen and transtubular potassium concentration gradient are markers for long-term prognosis in patients with ADHF (3–5). Therefore, we also studied whether U-OSM can predict prognosis in ADHF. Methods A total of 157 patients admitted to our hospital because of a primary diagnosis of ADHF from February 2020 through July 2021 were retrospectively reviewed. U-OSM in the spot urinary samples were examined within 72 hours after admission. U-OSM was calculated based on the following validated formula (6): U-OSM = 1.07 × {2 × [urine sodium (mEq/L)] + [urine urea nitrogen (mg/dL)]/2.8 + [urine creatinine (mg/dl)] × 2/3} + 16.2. The primary outcome was the occurrence of WRF during hospitalization. WRF was defined as increased serum creatinine ≥0.3 mg/dL from baseline (7). The secondary outcome was the occurrence of ADHF readmission and all-cause death within 180 days after discharge. Results Primary Outcome. WRF developed in 46% of all patients. In the patients that developed WRF during hospitalization, U-OSM was significantly lower than in the patients without WRF (366±106 mOsm/L versus 430±128 mOsm/L; P<0.001). Receiver operating characteristic curve analysis revealed the optimal cutoff values of U-OSM was 403 mOsm/L (AUC 0.64; 95% CI: 0.56–0.72; P<0.001) to predict the WRF (Figure 1). On multivariable logistic regression analysis, U-OSM (OR, 1.99, 95% CI: 1.27–3.12; p=0.003) and serum creatinine (OR, 1.00, 95% CI: 0.99–1.00; P=0.009) were independent predictors of WRF. Secondary Outcome. There were 34 patients (22%) readmitted and 9 patients (6%) died within 180 days after discharge. ROC curve analysis revealed the optimal cutoff values of U-OSM as 349 mOsm/L (C-statistic 0.74; 95% CI: 0.65–0.83; P<0.001) to predict ADHF readmission and all-cause death within 180 days (Figure 2A). On Kaplan-Meier analysis, the secondary outcome was significantly higher in patients with U-OSM<349 mOsm/L (u-OSM≥349, 57%, U-OSM<349, 43%; HR, 0.99; 95% CI: 0.99–1.00, P<0.001) (Figure 2B). Conclusion U-OSM on admission may be a predictor of WRF and a prognostic marker in ADHF patients. Funding Acknowledgement Type of funding sources: None.

Analysis of the Hypoxic Response in a Mouse Cortical Collecting Duct-Derived Cell Line Suggests That Esrra Is Partially Involved in Hif1α-Mediated Hypoxia-Inducible Gene Expression in mCCDcl1 Cells.

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.

Resemblance in Physiological Systems during Phylogeny and Ontogeny

Although it is not certain whether some characteristics of the mammalian fetus/embryo may repeat the adult stage of ancestral vertebrates, comparison of these two time‐dependent processes should provide new insight into the molecular and functional evolution and its significance. Here we discuss two important biological models: 1) Renin‐angiotensin system (RAS) and 2) Urine concentration mechanism.Renin is a hormone and substrate‐dependent enzyme secreted mostly by the juxtaglomerular (JG) cells of the afferent arteriole located at the vascular pole of the glomerulus. Renin acts on renin substrates and forms angiotensin (Ang) I which is subsequently converted to Ang II by Ang converting enzyme (ACE). Renin containing cells evolved early both in phylogeny and ontogeny, whereas, the macula densa (tubule component), and extracellular mesangium appeared at later stages.Renin containing cells are distributed more widely intrarenally and/or extrarenally in early stages during phylogeny and ontogeny. Granulated cells and renin expression become progressively restricted to JG areas with phylogenic and ontogenic advancement. Kidney renin activities tend to be higher in primitive fishes than in more advanced, while the relative level of extrarenal renin expression to that of the kidney is higher in embryos than in more mature chicken and mice. Extra renal renin is noted in primitive vertebrates. Also, in both the mammalian fetus and nonmammalian vertebrates, the vasopressor and vascular action of Ang II evolved early. A single application of ACE inhibitors decreases the basal level of blood pressure and increase plasma renin activity in conscious adult teleost fish. Ang II stimulates, whereas ACE inhibitors inhibit, arteriolar branching in zebrafish. Likewise, the RAS is important during early ontogeny of mammals in vascular growth and development.Distal tubules of freshwater trout show secondary active NaCl cotransport mechanism without accompaniment of water and act as the so‐called “diluting segment.” Teleost fish cannot concentrate urine because they lack structural and functional countercurrent mechanisms. Only birds and mammals can form urine clearly hyperosmotic to plasma and conserve body water. The structure and function of adult quail kidneys have similarities to those of developing rodents. Both have a loop of Henle, consisting of descending limb and thick ascending limb, but they lack the thin ascending limb. Countercurrent urine concentration of adult quail and developing rodents heavily depends on NaCl recycling without participation of urea transport. Moreover, in both adult quail and developing rodent kidneys, aquaporin 2 gene expression and protein are present in the apical membrane of the collecting tubules, but the water conservation effect of neurohypophysial hormones is smaller than that of adult rodent kidneys. Hence, comparing these two time‐dependent processes may provide new insights into the interpretation of existing findings and provide perspective for future investigations.

Comprehensive analysis of NFAT5 associated gene expression in the renal collecting duct

BackgroundThe renal cortico‐medullary osmotic gradient is a crucial factor for the urine concentration mechanism and is mainly generated by sodium chloride and urea. Due to this, the cells in the renal inner medulla are challenged with a hypertonic environment. The adaptation of these cells is mediated by the action of the tonicity‐responsive enhancer‐binding factor (TonEBP, also known as nuclear factor of activated T cells 5, NFAT5). NFAT5 induces the expression of genes that lead to accumulation of organic osmolytes within the cells. The cortico‐medullary osmotic gradient also induces a specific spatial gene expression pattern within the kidneys. So far, a systematic analysis of the contribution of NFAT5 on gene expression in the kidneys was missing, which was accomplished here.MethodsWe performed next generation RNA sequencing (NGS) using NFAT5 deficient and wild type control primary cultivated inner medullary collecting duct (IMCD) cells. As a second approach, we used renal cortex and renal inner medulla from principal cell specific NFAT5 deficient mice and control mice for NGS analysis. Differentially expressed genes were analyzed by pathway, functional and gene enrichment analysis.ResultsIn primary IMCD cells hyperosmolality induced massive changes in gene expression. Aquaproin‐2 (Aqp2) was one of the transcripts with the highest level of induction. Loss of NFAT5 abolished hyperosmolality‐induced expression of Aqp2. Most of the hyperosmolality induced transcripts had the highest expression in the inner medulla. Loss of NFAT5 affected the expression of more than 3,000 genes in renal cortex and more than 5,000 genes in the inner medulla, compared to control kidneys. Again, loss of NFAT5 was associated with decreased expression of Aqp2. We observed increased expression of kidney injury marker genes, like lipocalin‐2 or kidney injury molecule 1. Gene enrichment analysis also indicated that loss of NFAT5 is associated with a renal inflammation‐like phenotype.ConclusionThis is the first study addressing the role of NFAT5 on hypertonicty‐induced changes in gene expression in the kidney. NFAT5 is the master regulator of osmolality induced Aqp2 gene expression and in vivoloss of function induces a kidney injury‐like phenotype through currently unknown mechanisms.

A 3-Dimentional Framework for Studying the Effects of Structural and Physical Properties of the Renal Outer Medulla on Urine Concentrating Mechanism

Background: Many theories and mathematical simulations have been proposed concerning urine concentrating mechanism (UCM). Due to significant effect of the tubule and vessel architecture in concentrating mechanism, the numerical analysis of UCM through a 3-Dimensional structure might be the answer to find a better consistency between the experimental and theoretical results. Methods: In this paper we have investigated the effects of structural characteristics of the tubules and vessels on the urine concentrating mechanism in the outer medulla (OM) by developing a simple three-dimensional mathematical model. This model is a framework to attain a converged numerical solution for the momentum and species transport equations along with their stiff and coupled boundary conditions based on standard expressions for trans-tubular solutes and water transports on tubule’s membrane. Results: The model structure and the number of the involved tubules have been assumed to be as simple as possible. The effects of slip boundary condition on membrane, Darcy permeability and solute’s diffusivity of the intermediate media on UCM have been studied. It has been shown that this approach can simply simulate preferential interactions and tubule’s confinement by radial diffusion coefficients. Conclusions: All in all, the feasibility of the idea of completely 3-D modeling by employing the concept of diffusion coefficient and Darcy permeability has been explored and validated.

First person – Jessica Kabutomori

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Jessica Kabutomori is first author on ‘Water transport mediated by murine urea transporters: implications for urine concentration mechanisms’, published in BIO. Jessica is a masters student in the lab of Raif Musa Aziz at the Department of Physiology and Biophysics, University of São Paulo, investigating water transport across murine urea transporters UT-A2, UT-A3 and UT-B, towards the goal of better understanding the role(s) of these proteins in the kidney.

The urea transporter UT-A1 plays a predominant role in a urea-dependent urine-concentrating mechanism

Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues that play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter (UT)-B, UT-A1/A3, or all UTs leads to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UT-A1-knockout mouse model. Phenotypically, daily urine output in UT-A1-knockout mice was nearly 3-fold that of WT mice and 82% of all-UT-knockout mice, and the UT-A1-knockout mice had significantly lower urine osmolality than WT mice. After 24-h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1-knockout mice were unable to increase urine-concentrating ability. Compared with all-UT-knockout mice, the UT-A1-knockout mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UT-A1 represents a promising diuretic target.

Discovery of novel diarylamides as orally active diuretics targeting urea transporters

Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.

Water transport mediated by murine urea transporters: implications for urine concentration mechanisms.

ABSTRACTUrea transporters (UTs) facilitate urea diffusion across cell membranes and play an important role in the urinary concentration mechanisms in the kidney. Herein, we injected cRNAs encoding for c-Myc-tagged murine UT-B, UT-A2 or UT-A3 (versus water-injected control) in Lithobates oocytes and evaluated oocyte surface protein expression with biotinylation and immunoblotting, urea uptake using [14C] counts and water permeability (Pf) by video microscopy. Immunoblots of UT-injected oocyte membranes revealed bands with a molecular weight consistent with that of a UT monomer (34 kDa), and UT-injected oocytes displayed significantly increased and phloretin-sensitive urea uptake and Pf when compared to day-matched control oocytes. Subtracting the water-injected urea uptake or Pf values from those of UT-injected oocytes yielded UT-dependent values*. We demonstrate for the first time that UT-A2 and UT-A3 can transport water, and we confirm that UT-B is permeable to water. Moreover, the [14C] urea*/Pf* ratios fell in the sequence mUT-B>mUT-A2>mUT-A3, indicating that UTs can exhibit selectivity to urea and/or water. It is likely that specific kidney regions with high levels of UTs will exhibit increased urea and/or water permeabilities, directly influencing urine concentration. Furthermore, UT-mediated water transport activity must be considered when developing UT-inhibitors as novel diuretics.This article has an associated First Person interview with the first author of the paper.

Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2.

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

Solute and water transport along an inner medullary collecting duct undergoing peristaltic contractions.

The mechanism by which solutes accumulate in the inner medulla of the mammalian kidney has remained incompletely understood. That persistent mystery has led to hypotheses based on the peristaltic contractions of the pelvic wall smooth muscles. It has been demonstrated the peristaltic contractions propel fluid down the collecting duct in boluses. In antidiuresis, boluses are sufficiently short that collecting ducts may be collapsed most of the time. In this study, we investigated the mechanism by which about half of the bolus volume is reabsorbed into the collecting duct cells despite the short contact time. To accomplish this, we developed a dynamic mathematical model of solute and water transport along a collecting duct of a rat papilla undergoing peristaltic contractions. The model predicts that, given preexisting axial concentration gradients along the loops of Henle, ∼40% of the bolus volume is reabsorbed as the bolus flows down the inner medullary collecting duct. Additionally, simulation results suggest that while the contraction-induced luminal hydrostatic pressure facilitates water extraction from the bolus, that pressure is not necessary to concentrate the bolus. Also, neither the negative interstitial pressure generated during the relaxation phase nor the concentrating effect of hyaluronic acid has a significant effect on bolus concentration. Taken together, these findings indicate that the high collecting duct apical water permeability allows a substantial amount of water to be extracted from the bolus, despite its short transit time. However, the potential role of the peristaltic waves in the urine-concentrating mechanism remains to be revealed.

3D simulation of solutes concentration in urinary concentration mechanism in rat renal medulla

In this work, a mathematical model was developed to simulate the urinary concentration mechanism. A 3-D geometry was derived based on the detail physiological pictures of rat kidney. The approximate region of each tubule was obtained from the volume distribution of structures based on Walter Pfaller's monograph and Layton's region-based model. Mass and momentum balances were applied to solve for the change in solutes concentration and osmolality. The osmolality of short and long descending nephrons at the end of the outer medulla was obtained to be 530 mOsmol/kgH2O and 802 mOsmol/kgH2O, respectively, which were in acceptable agreement with experimental data. The fluid osmolality of the short and long ascending nephrons was also compatible with experimental data. The osmolality of CD fluid at the end of the inner medulla was determined to be 1198 mOsmol/kgH2O which was close the experimental data (1216 ± 118). Finally, the impact of the position of each tubule on the fluid osmolality and solutes concentration were obvious in the results; for example, short descending limb a1, which is the closest tubule to the collecting duct, had the highest urea concentration in all tubules. This reflects the important effect of the 3D modeling on the precise analysis of urinary concentration mechanism.

Mammalian urine concentration: a review of renal medullary architecture and membrane transporters.

Mammalian kidneys play an essential role in balancing internal water and salt concentrations. When water needs to be conserved, the renal medulla produces concentrated urine. Central to this process of urine concentration is an osmotic gradient that increases from the corticomedullary boundary to the inner medullary tip. How this gradient is generated and maintained has been the subject of study since the 1940s. While it is generally accepted that the outer medulla contributes to the gradient by means of an active process involving countercurrent multiplication, the source of the gradient in the inner medulla is unclear. The last two decades have witnessed advances in our understanding of the urine-concentrating mechanism. Details of medullary architecture and permeability properties of the tubules and vessels suggest that the functional and anatomic relationships of these structures may contribute to the osmotic gradient necessary to concentrate urine. Additionally, we are learning more about the membrane transporters involved and their regulatory mechanisms. The role of medullary architecture and membrane transporters in the mammalian urine-concentrating mechanism are the focus of this review.

ILDR1 is important for paracellular water transport and urine concentration mechanism

Whether the tight junction is permeable to water remains highly controversial. Here, we provide evidence that the tricellular tight junction is important for paracellular water permeation and that Ig-like domain containing receptor 1 (ILDR1) regulates its permeability. In the mouse kidney, ILDR1 is localized to tricellular tight junctions of the distal tubules. Genetic knockout of Ildr1 in the mouse kidney causes polyuria and polydipsia due to renal concentrating defects. Microperfusion of live renal distal tubules reveals that they are impermeable to water in normal animals but become highly permeable to water in Ildr1 knockout animals whereas paracellular ionic permeabilities in the Ildr1 knockout mouse renal tubules are not affected. Vasopressin cannot correct paracellular water loss in Ildr1 knockout animals despite normal effects on the transcellular aquaporin-2-dependent pathway. In cultured renal epithelial cells normally lacking the expression of Ildr1, overexpression of Ildr1 significantly reduces the paracellular water permeability. Together, our study provides a mechanism of how cells transport water and shows how such a mechanism may be exploited as a therapeutic approach to maintain water homeostasis.

A mathematical model of the rat kidney: K+-induced natriuresis.

A model of the rat nephron (Weinstein. Am J Physiol Renal Physiol 308: F1098-F1118, 2015) has been extended with addition of medullary vasculature. Blood vessels contain solutes from the nephron model, plus additional species from the model of Atherton et al. (Am J Physiol Renal Fluid Electrolyte Physiol 247: F61-F72, 1984), representing hemoglobin buffering. In contrast to prior models of the urine-concentrating mechanism, reflection coefficients for DVR are near zero. Model unknowns are initial proximal tubule pressures and flows, connecting tubule pressure, and medullary interstitial pressures and concentrations. The model predicts outer medullary (OM) interstitial gradients for Na+, K+, CO2, and [Formula: see text], such that at OM-IM junction, the respective concentrations relative to plasma are 1.2, 3.0, 2.7, and 8.0; within IM, there is high urea and low [Formula: see text], with concentration ratios of 11 and 0.5 near the papillary tip. Quantitative similarities are noted between K+ and urea handling (medullary delivery and permeabilities). The model K+ gradient is physiologic, and the urea gradient is steeper due to restriction of urea permeability to distal collecting duct. Nevertheless, the predicted urea gradient is less than expected, suggesting reconsideration of proposals of an unrecognized reabsorptive urea flux. When plasma K+ is increased from 5.0 to 5.5 mM, Na+ and K+ excretion increase 2.3- and 1.3-fold, respectively. The natriuresis derives from a 3.3% decrease in proximal Na+ reabsorption and occurs despite delivery-driven increases in Na+ reabsorption in distal segments; kaliuresis derives from a 30% increase in connecting tubule Na+ delivery. Thus this model favors the importance of proximal over distal events in K+-induced diuresis.

Investigating the role of the renal sodium gradient in defence against urinary tract infection

BackgroundTissue-resident macrophages and dendritic cells respond to immunological challenges within an organ. The kidney is a unique environment for these cells, with extreme but variable hypersalinity in the medulla, generated by urine concentration mechanisms. Lower urinary tract infections are one of the commonest human bacterial infections, yet renal involvement is rare. Mechanical forces, such as anterograde urine flow can be protective. We aimed to determine whether additional tissue-specific mechanisms protect the kidney. MethodsWe characterised macrophages and dendritic cells (mononuclear phagocytes [MNP]) in human kidneys, and evaluated their role in defence against uropathogenic Escherichia coli (UPEC). We used murine models and mouse and human cells in culture to investigate the factors controlling MNP positioning in the medulla and, together with human epidemiological datasets, to determine the importance of the renal sodium gradient in defence against urinary tract infection. FindingsWe found that physiological hypersalinity provided a cue to renal tubular epithelial cells, stimulating TonEBP-dependent production of chemokines (CCL2 and CX3CL1) that orchestrate the recruitment of CD14+ macrophages to the medulla. These CD14+ macrophages were adept at phagocytosis of UPEC, and their bactericidal and neutrophil chemotactic function was further enhanced by hypersalinity. In murine models, we confirmed that these macrophages were derived from monocytes and that their medullary localisation was CCR2-dependent. Finally, we demonstrated the relevance of these observations in vivo, showing that disruption of the renal sodium gradient in patients and mice led to aberrant chemokine expression and macrophage localisation, and susceptibility to pyelonephritis. InterpretationWe have shown a novel mechanism whereby changes in the tissue environment generated by the homoeostatic function of an organ stimulate epithelial–macrophage crosstalk to optimise defence against infection. FundingMedical Research Council, Kidney Research UK.