Henle's Loop Research Articles

Tandem upregulation of ion transporters in thick ascending limb of Henle's loop of young Milan hypertensive strain of rats.

Milan hypertensive strain (MHS) of rat represents as one of the ideal rat models to study the genetic form of hypertension associated with aberrant renal salt reabsorption. In contrast to Milan normotensive strain (MNS), MHS rats possess missense mutations in three adducin genes and develop hypertension at 3 months old due to upregulation of sodium-chloride cotransporter (NCC). At pre-hypertensive stage (23-25 days old), MHS rats show enhanced protein abundance of Na+-K+-2Cl- cotransporter (NKCC2) but retain blood pressure comparable to MNS probably through enhanced GFR and reduced NCC and α-subunit of epithelial sodium channel (α-ENaC) expressed in distal convoluted tubule (DCT) and collecting duct (CD). In the present study, mRNA and protein expressions of ion transporters in thick ascending limb of Henle's loop (TAL) of young MHS rats were investigated. Protein abundance of core-glycosylated form of renal outer medullary potassium channel (ROMK) in inner stripe of outer medulla (ISOM) is remarkably increased in MHS rats at prehypertensive stage. Furthermore, basolaterally expressed Na+-K+-ATPase and Barttin were upregulated. These results may indicate that in TAL of MHS rats at this age, both total NKCC2 and core-glycosylated ROMK are upregulated in tandem potentially to balance the luminal potassium concentration. On the basolateral side, upregulation of Na+-K+-ATPase and CLC-Ka/b may energize the excretion of sodium and chloride out from the cells. These data may suggest the interplay of apical and basolateral ion transporters in TAL for the modulation of TAL function in favor of enhancing the transepithelial sodium reabsorption, although this seems compensated by NCC and ENaC expressed at the downstream nephron segments in young MHS rats.

Development Postnatal of the Kidney and Liver in Albino Rat Rattus Rattus, Histological and Biochemical Study

Objective: The primary goal of the current study was to look at histological, histochemical, and biochemical aspects of the development structure of rat's kidney and liver. Materials and Method: Seventy eight samples of kidney and liver were obtained from rats at days; 1, 5, 10, 15, 36 and 90 after birth and processed for histological, histochemical and biochemical techniques. Results: Up to the fifth day, new nephron anlages develop in the renal cortex. After ten days, the final anlages form into functional nephrons, and fifteen days after the corticis forms, the cortex seems mature. The collecting ducts and loops of Henle expand longitudinally to form the renal medulla. The immature medulla is structurally similar to the later inner stripe of the outer zone and cannot be split into distinct zones. Within ten days, the inner zone was developed, and within fifteen days, the outside zone's stripe. The kidney tubule's diameter increased with age, the renal medulla was mature at 36 days, the kidney was covered in a moderate amount of collagen fiber, and the henle loops were lengthy at certain ages, but short at adult age. Adults had larger corpuscle and nephron lumens. In contrast, the thick renal capsule at other ages contained less collagen fibers. A tiny renal gap encircling the glomeruli and a notably greater quantity of fibers were visible in adult Bowman's capsules. The liver was encased in a thin capsule on the first day of birth and a thick capsule between 36 and 90 days later. On day 15, the cells surrounding the central veins began to arrange themselves in a regular fashion. The hepatic parenchyma could be seen by symmetrical hepatic cords, which emerged from the central veins as radiating columns. After five days, Von Kuffer cells began to line the sinusoids next to the endothelial cells. On the tenth day, the hepatic sinusoids took on a more regular appearance, stretching between the hepatic cords and joining the central veins. The hepatic lobules with their central vein were visible at the age of 15 days. The capsule then thickened and elastic fibers started to show up. The reticular fibers also thickened and spread, and in 36 days, collagen fibers started to show up in the portal areas. Based on histochemical data, every PAS, AB, and PAS-AB stain was highly reacted with brush in renal and hepatic cells. In conclusion: The kidney and liver developed differently after birth and reached their mature, typical structures in 36 days.

Gold Nanoparticle Transport in the Injured Kidneys with Elevated Renal Function Biomarkers.

Renal function biomarkers such as serum blood urea nitrogen (BUN) and creatinine (Cr) serve as key indicators for guiding clinical decisions before administering kidney-excreted small-molecule agents. With engineered nanoparticles increasingly designed to be renally clearable to expedite their clinical translation, understanding the relationship between renal function biomarkers and nanoparticle transport in diseased kidneys becomes crucial to their biosafety in future clinical applications. In this study, renal-clearable gold nanoparticles (AuNPs) are used as X-ray contrast agents to noninvasively track their transport and retention in cisplatin-injured kidneys with varying BUN and Cr levels. The findings reveal that AuNP transport is significantly slowed in the medulla of severely injured kidneys, with BUN and Cr levels elevated to 10 times normal. In mildly injured kidneys, where BUN and Cr levels only four to five times higher than normal, AuNP transport and retention are not predictable by BUN and Cr levels but correlate strongly with the degree of tubular injury due to the formation of gold-protein casts in the Henle's loop of the medulla. These results underscore the need for caution when employing renal-clearable nanomedicines in compromised kidneys and highlight the potential of renal-clearable AuNPs as X-ray probes forassessing kidney injuries noninvasively.

The endocrine basis of the cardio-renal axis: New perspectives regarding corin.

The central role of natriuretic peptides (NPs) in the complex cardio-renal integrated physiology and organ failure has been revealed over the last four decades. Atrial natriuretic peptide (ANP), the oldest representative of the NPs family, is produced through conversion of proANP to the mature peptide by corin, a trans-membrane protease localized to the cardiac myocyte membrane. Similarly, brain natriuretic peptide (BNP) is generated by furin, which cleaves proBNP to BNP in myocytes. Though the components of NPs system, their synthesis and target organs are well established, understanding their role in the interplay between the heart and the kidney is steadily evolving. In this context, Feldman etal. (New England Journal of Medicine, 389, 1685) recently described patients with hypertension, cardiomyopathy, atrial arrhythmia and left atrial fibrosis, associated with a homozygous loss-of-function variant of the gene encoding corin (Cor-/-). Notably, reduced baseline urinary electrolyte and creatinine excretion have been observed in one of the studied patients. This renal excretory functional impairment could be attributed to the lack of cardiac-derived ANP in these patients, as implied by Feldman etal. Yet, in this mini-review we suggest that this aberrant renal manifestation may principally stem from lack of local ANP production at renal tissue, as corin is normally expressed in proximal tubules, Henle's loop and collecting ducts, with locally produced ANP provoking Na+ and water exertion. Collectively, it seems that beside the classic well-established cardio-renal axis, the renal NPs system functions as local endocrine machinery in the regulation of sodium excretion.

Novel in vivo optogenetic tools reveal autonomous pacemaker control of renal hemodynamics by macula densa cells

Macula densa (MD) cells, the tubular component of the juxtaglomerular apparatus (JGA) are generally considered as sensor cells detecting tubular salt (NaCl) concentration and based on this information provide feedback control of glomerular hemodynamics (tubuloglomerular feedback, TGF). However, the neuron-like differentiation and activity as non-traditional MD functions are emerging. This study addressed the paradigm-shifting hypothesis that rather than being simply the sensory arm of TGF, MD cells function as the nephron’s autonomous pacemaker and the primary driver and oscillator of glomerular hemodynamics. Single cell MD Ca2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of MD-GT mice that selectively express the ratiometric Ca2+ reporter GCaMP6f/tdTomato in MD cells. Optogenetic MD stimulation was induced in vivo by blue light (470nm) exposure of MD-Ai27 mouse kidneys that express the depolarizing channelrhodopsin ChR2 specifically in MD cells. Conversely, MD inhibition was induced by yellow light (560nm) exposure of MD-Ai39 mouse kidneys that selectively express the hyperpolarizing halorhodopsin NpHR in MD cells. Cell membrane expression of ChR2 (in MD-Ai27 mice) and NpHR (in MD-Ai39 mice) only in the MD among all renal cell types was confirmed using immunohistochemistry. Pacemaker-like regular Ca2+ oscillations (4-fold elevations in baseline Ca2+ and average frequency of 0.03 Hz) and their cell-to-cell propagation within the MD plaque via long cell processes were observed in MD-GT mice. These Ca2+ oscillations were MD cell autonomous, as they were preserved in freshly dissected single MD cells and in the in vitro dissected and microperfused JGA, and were exclusively confined to the MD area. Bulk and scRNA seq and MD transcriptome analysis identified the high MD expression of genes related to pacemaker function, cell membrane depolarization, voltage-dependent ion channels, Ca2+ signaling and synaptic transmission that was validated by immunohistochemistry. Blue light exposure of single glomeruli/JGAs using ROI scan in MD-Ai27 mouse kidneys that previously underwent superficial corticotomy (open loop nephrons) acutely and reversibly increased capillary and afferent (AA)/efferent arteriole (EA) blood flow by approx. 50% in the light-exposed glomerulus but not in adjacent glomeruli. In contrast, yellow light exposure of single glomeruli/JGAs in MD-Ai39 mouse kidneys acutely and reversibly decreased blood flow but increased glomerular capillary diameter (suggesting preferential EA vs AA vasoconstriction) in the exposed but not in adjacent single nephrons. In addition, yellow light stimulation of single glomeruli in MD-Ai39 mouse kidneys augmented the spontaneous glomerular blood flow oscillations in these open-loop nephrons (frequency of 0.03 Hz), but not in adjacent control nephrons. Altogether, these studies identified MD cells as the nephron’s central pacemaker and oscillator of single nephron blood flow, and emphasized the importance of MD cell membrane potential and Ca2+-dependent vasodilator release as a major driver of nephron function. DK064234 DK123564 DK135290. 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.

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Lipidomic Profiling of Kidney Cortical Tubule Segments Identifies Lipotypes with Physiological Implications.

A detailed knowledge of the lipid composition of components of nephrons is crucial for understanding physiological processes and the development of kidney diseases. However, the lipidomic composition of kidney tubular segments is unknown. We manually isolated the proximal convoluted tubule (PCT), the cortical thick ascending limb of Henle's loop, and the cortical collecting duct from 5 lean and obese mice and subjected the samples to shotgun lipidomics analysis by high-resolution mass spectrometry acquisition. Across all samples, more than 500 lipid species were identified, quantified, and compared. We observed significant compositional differences among the 3 tubular segments, which serve as true signatures. These intrinsic lipidomic features are associated with a distinct proteomic program that regulates highly specific physiological functions. The distinctive lipidomic features of each of the 3 segments are mostly based on the relative composition of neutral lipids, long-chain polyunsaturated fatty acids, sphingolipids, and ether phospholipids. These features support the hypothesis of a lipotype assigned to specific tubular segments. Obesity profoundly impacts the lipotype of PCT. In conclusion, we present a comprehensive lipidomic analysis of 3 cortical segments of mouse kidney tubules. This valuable resource provides unparalleled detail that enhances our understanding of tubular physiology and the potential impact of pathological conditions.

Macro and Micro Anatomical Comparative Study for Liver, Kidney and Testis Between the Albino Rat and Pigeon

The present study was designed to demonstrate the anatomical and histological variations between the certain organs in both the rat as a model of experimental mammals and pigeon as an avian model. Five albino rats and five pigeons were utilized, the liver, kidney and the testicles were taken from those animals after scarifying and specimens were taken from those organs, fixed in 10 % formalin for 24 hours before transferred for histological technique, the slices were stained with hematoxylin and eosin. The anatomical observation indicated that the liver of rat was formed by right, left lobes and intermediate, located in the anterior part of abdomen, in the pigeon, had two lobes without gall bladder. The kidney of the rat was bean shaped, brown reddish color with idented medial border present in the abdominal cavity near and around the lumbar vertebrae, the kidney of pigeon was lobulated into three and the kidney was lodged on the ventral aspect of the synsacrum.
 The testis of rat was oval in shape present in the scrotum, and in the pigeon located in the abdominal and pelvic cavity. The histological result indicated the liver of rat was with columns of polygonal liver cells surrounded by blood sinusoids draining in the central vein, while in the pigeon, the cells were present in groups also had blood sinusoids with Kupffer cells and central vein for draining the blood outside the liver, the kidney of both types had an cortex and medulla with presence of glomeruli in the cortex, both had proximal and distal convoluted tubules , in the pigeon there is no thin segment of Henle loops .
 The testis was occupied by seminiferous tubules in both types and each tubule was containing the different types of spermatic cells and spermatozoa, also Leydig cells were present in groups, spermatogenesis was intensive in pigeon in cases of seasonal phase in conclusion, variation for the visceral organs in both rats and pigeon were determined also histological differences were present, otherwise similarities were also observed.

The apical 70-pS potassium K channel in the thick ascending limb of Henle's loop is a large-conductance Na+-and Cl--activated, KNa1.1-like, channel.

Apical potassium channels are crucial for thick ascending limb (TAL) of Henle's loop transport function. The ROMK (KNCJ1) gene encodes a 30-pS K channel whose loss of function causes the reduced NaCl reabsorption in the TAL associated with Type 2 Bartter's syndrome. In contrast, the molecular basis of a functionally ROMK-related 70-pS K channel is still unclear. The aim of this study was to highlight new specific channel properties that may give insights on its molecular identity. Using the patch-clamp technique on the apical membrane of mouse split-open TAL tubules, we observed that 70-pS K channel activity, but not ROMK channel activity, increases with the internal Na+ and Cl- concentrations, with relative 50 % effective concentrations (EC50) and Hill coefficients (nH) of 40.6 mM (SD 1.65) and 2.4 (SD 0.28) for Na+, and of 29.3 mM (SD 2.35) and 2.2 (SD 0.39) for Cl-. Conversely, 70-pS K channel activity was inhibited by internal K+ with a relative EC50 of 64 mM (SD 13.5) and a nH of 3.5 (SD 2.3), and by internal NH4+ and Ca2+. The reevaluation of channel conductive properties revealed an actual inward conductance of ~ 170 pS, with multiple subconductance levels and an inward rectification, and a substantial permeability to NH4+ ( = 0.2). We conclude that the apical 70-pS K channel in TAL cells is a large-conductance Na+- and Cl--activated potassium channel functionally resembling a KNa1.1 channel and propose that ROMK determines its functional expression possibly at the level of channel protein synthesis or trafficking.

Disrupting Development: Unraveling the Interplay of Aryl Hydrocarbon Receptor (AHR) and Wnt/β-Catenin Pathways in Kidney Development Under the Influence of Environmental Pollutants.

Understanding the intricate molecular mechanisms governing aryl hydrocarbon receptor (AHR) and Wnt/β-Catenin pathways crosstalk is of paramount importance for elucidating normal development. We investigated the repercussions of aberrant activation of these signaling pathways on kidney development. HEK-293 cells were subjected to AHR and Wnt activators and inhibitors for 3 and 24h. Subsequently, pregnant adult female BALB/c mice were administered treatments at gestation day 9 (GD-9), and embryos were analyzed at GD-18 using a combination of cellular, molecular, stereological, and histopathological techniques. Our results demonstrated a noteworthy escalation in oxidative stress and gene expression endpoints associated with apoptosis. Moreover, stereological analyses exhibited alterations in cortex, proximal tubule, and kidney tissue vessels volumes. Remarkably, co-treatment with 6-formylindolo [3,2-b] carbazole (FICZ) and cadmium (Cd) resulted in a significant reduction in glomerulus volume, while elevating the volumes of distal tubule, Henle loop, and connective tissue, compared to the control group. Histopathological investigations further confirmed structural changes in the loop of Henle and proximal tubule, alongside a decline in glomerular volume. Additionally, the expression levels of AHR and Ctnnb1 genes significantly increased in the Cd-treated group compared to the control group. Enhanced expression of apoptosis-related genes, including Bcl-x, Bax, and Caspase3, along with alterations in mitochondrial membrane potential and cytochrome C release, was observed. In contrast, Gsk3 gene expression was significantly decreased. Our findings robustly establish that chemical pollutants, such as Cd, disrupt the AHR and Wnt/β-Catenin physiological roles during developmental stages by inhibiting the metabolic degradation of FICZ.

From Fish Physiology to Human Disease: The Discovery of the NCC, NKCC2, and the Cation-Coupled Chloride Cotransporters.

The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.

Tamm–Horsfall protein in humane urine: sex-dependent differences in the excretion and N-glycosylation pattern

Tamm–Horsfall protein (THP) is a highly N-glycosylated protein from epithelial cells of the ascending limb of Henle loop. It is secreted into the urine as part of the innate immune response against uropathogenic pathogens. As women are more likely to suffer from urinary tract infections, biomedical studies were conducted to investigate sex-differences in THP excretion, as well as differences in the THP N-glycosylation pattern. A total of 238 volunteers (92 men, 146 women, 69 with hormonal contraceptives) participated in this study, providing urine samples. Women showed a clear tendency to have higher THP concentration and excretion rates than men (p < 0.16). Regular intake of hormonal contraceptives had no significant influence on urinary THP concentration compared to no regular intake. The individual N-glycosylation pattern of THP in urine samples from randomly selected individuals (10 female, 10 male) was investigated after enzymatic release and MS analysis of the oligosaccharides. Female subjects tended to have an increased proportion of oligomannose type N-glycans and non-fucosylated glycans, whereas men had an increased proportion of fucosylated complex-type glycans. The higher level of oligomannose-type glycans in THP from women might be explained by a self-defence mechanism to overcome the higher infections pressure by the female anatomical properties.

Abstract B006: Dissecting metabolic alterations of clear cell renal cell carcinomas one cell at a time

Abstract Pseudohypoxia-related metabolic alterations are known to exist in clear-cell renal cell carcinoma (ccRCC). Here we characterized single-cell metabolic pathways with the 10Xmultiome assay comparing metabolic and compositional shifts in tumor vs. normal-adjacent samples. We analyzed 57 tumor and 6 normal-adjacent samples from the Dartmouth Renal Tumors Biobank collected between 1994-2009. Samples were enzymatically disassociated and stored at -80 C until 10X multiome processing. RNA counts were extracted using Seurat, cell types were deconvolved using Azimuth, and 85 KEGG metabolic pathways were interrogated using scMetabolism scores. Linear mixed-effects models compared the metabolic scores in 10 cell lineages (juxtaglomerular apparatus, glomeruli, proximal tubule system, nephron loop, distal tubules, collecting ducts, lymphoid, myeloid, endothelia, and other stroma cells) between tumor and normal adjacent adjusting for sex, patient age, stage and grade as fixed-effects and donor, and year of collection, as random-effects. P-values were calculated using Satterthwaite's method. The mean age at diagnosis was 61.7 yrs (SD: 12.5), and 67% were stages I/II. 79,804 tumor and 5,967 normal-adjacent cells were analyzed. 34,318 cells were nephron epithelia (juxtaglomerular, glomeruli, renal tubules), 5,947 were collecting ducts, 18,959 were immune, 13,445 were endothelial, and 13,112 were other stromal cells. Out of 85 KEGG metabolic pathways, nine were prioritized based on the kidney cancer literature; glycolysis, oxidative phosphorylation, TCA cycle, fatty acid biosynthesis, glutamine/glutamate, arachidonic acids, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and glutathione. When comparing vs. the corresponding normal adjacent cell type, in tumor cells: 1) glutamine/glutamate expression increased in lymphoid, endothelial, and stromal cells; 2) fatty acid biosynthesis increased in myeloid and reduced in lymphoid, collecting ducts-like, and nephron loop-like cells, 3) glutathione and glycolysis increased in glomeruli-like and nephron loops-like, 4) glycolysis increased in collecting ducts-like and endothelial, 5) oxidative phosphorylation and TCA cycle genes decreased in distal tubules-like, collecting ducts-like, and endothelia, 6) arachidonic acid metabolism increased in nephron loops-like and glomeruli-like, and 7) phenylalanine, tyrosine, and tryptophan biosynthesis decreased in nephron loops-like, distal tubules-like, and collecting ducts-like cells. Cell-specific metabolic shifts were observed between tumor and normal adjacent samples that cannot be explained only by sample heterogeneity or cell lineages. Oxidative phosphorylation and TCA cycle modifications were observed in tumor endothelia and cells resembling distal tubules and collecting ducts. Similarly, glutamate and fatty acid alterations were focused on tumor microenvironment immune cells. Further dissecting these findings will provide additional therapeutic avenues for newer targets, such as glutaminase inhibitors and combinations with current therapies. Citation Format: Lucas A. Salas, Ze Zhang, Laurent Perreard, Fred W. Kolling, Jason R. Pettus, Brock C. Christensen. Dissecting metabolic alterations of clear cell renal cell carcinomas one cell at a time [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B006.

Cyclosporin-induced hypertension is associated with the up-regulation of Na+-K+-2Cl- cotransporter (NKCC2).

The use of cyclosporin A (CsA) is hampered by the development of nephrotoxicity including hypertension, which is partially dependent on renal sodium retention. To address this issue, we have investigated in vivo sodium reabsorption in different nephron segments of CsA-treated rats through micropuncture study coupled to expression analyses of sodium transporters. To translate the findings in rats to human, kidney-transplanted patients having CsA treatment were enrolled in the study. Adult male Sprague-Dawley rats were treated with CsA (15 mg/kg/day) for 21days, followed by micropuncture study and expression analyses of sodium transporters. CsA-treated kidney-transplanted patients with resistant hypertension were challenged with 50mg furosemide. CsA-treated rats developed hypertension associated with reduced glomerular filtration rate. In vivo microperfusion study demonstrated a significant decrease in rate of absolute fluid reabsorption in the proximal tubule but enhanced sodium reabsorption in the thick ascending limb of Henle's loop (TAL). Expression analyses of sodium transporters at the same nephron segments further revealed a reduction in Na+-H+ exchanger isoform 3 (NHE3) in the renal cortex, while TAL-specific, furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and NHE3 were significantly upregulated in the inner stripe of outer medulla. CsA-treated patients had a larger excretion of urinary NKCC2 protein at basal condition, and higher diuretic response to furosemide, showing increased FeNa+, FeCl- and FeCa2+ compared with both healthy controls and FK506-treated transplanted patients. Altogether, these findings suggest that up-regulation of NKCC2 along the TAL facilitates sodium retention and contributes to the development of CsA-induced hypertension.

#4947 LMO7 IN URINE AS A SPECIFIC MARKER OF COMPROMISED RENAL EPITHELIAL INTEGRITY

Abstract Background and Aims Renal epithelial barrier (REB) are the physical isolator as well as physiological interface mediating body fluid and electrolytes homeostasis. Maintaining epithelial integrity is tightly related to REB biological function. LMO7 (LIM domain only 7) is a protein associated with the cell–cell junctional complex and is predominantly distributed in renal tubular epithelial cells. Previously, we reported that the LMO7 is associated with the maintenance of renal epithelial integrity, and that LMO7 releases to conditioned medium when the epithelial integrity was lost. In this study, we attempt to characterize roles of LMO7 and its functional interacting protein in maintaining epithelial integrity, and relevance of the LMO7 released from plasma membrane and the loss of tubular epithelial integrity. Method Renal ischemia-reperfusion injury (IRI) in mice as a model of acute kidney injury (AKI) were settled as a platform to investigate the the roles of LMO7 and its functional interacting protein in the pathological progression of compromised tubular epithelial integrity. Additionally, renal epithelial cells cultured in hypoxia as the pathogenic condition equivalent to AKI were also employed to investigate the role of LMO7. We also search the STRING database with LMO7 as query to find out its functional interacting proteins. Pathological pattern associated with LMO7 and its functional interacting protein in mice suffering from AKI as well as loss of tubular epithelial integrity were examined by immunofluorescent visualization. The LMO7 in urine as well as in conditioned medium derived from medium of hypoxia culture were quantified by Western blot analysis. Results Renal damage in IRI mice showed loss of epithelial integrity by histochemical staining, presence of LMO7 in urine, and high serum creatinine on the third day post IRI. Under the periodic acid-schiff (PAS) staining, loss of epithelial integrity is visible in proximal tubules, Henle's loops, and collecting ducts at the third day after reperfusion, and brush border impairment is observed in the outer stripe of the outer medulla. Most severe damages in renal tubules are present in Henle's loops where the epithelia express abundant LMO7. In the AKI mice, the LMO7 in renal tubules was apparently lower in Henle's loops. Additionally, the VANGL1, a LMO7 interacting protein participated in epithelial integrity and planar cell polarity in kidney, is also absent in Henle's loop of AKI mice. Hypoxia and glucose deprivation in cultured renal epithelial cells resulted in loss of epithelial integrity, and down-regulation of LMO7 and VANGL1. LMO7 was also identified in conditioned medium in which cells exposed to hypoxia but not normoxia. Additionally, the LMO7 was detected in urine of some patients with chronic kidney disease. Conclusion Presence of LMO7 in urine has been promising in experimental models holding compromised epithelial integrity. These approaches identified the role of LMO7 in maintaining epithelial integrity and that LMO7 being released into urine after renal epithelial damage as a novel marker for loss of REB.

#3508 FUROSEMIDE DECREASES KYNURENIC ACID SYNTHESIS IN RAT KIDNEY IN VITRO

Abstract Background and Aims Loop diuretics are highly potent agents, particularly beneficial in patients with impaired kidney function or heart failure exacerbation. Furosemide, due to fast onset and short time of action, remains the most commonly used drug from this class of diuretics. Inhibition of sodium-potassium-chloride cotransporter (NKCC2) in the thick ascending limb of Henle's loop is the main mechanism of furosemide's action. Other effects of furosemide, like modulation of potassium channels activity or activation of prostaglandin synthesis, were previously described. It was postulated that furosemide may exacerbate kidney injury in a dose dependent manner. Kynurenic acid (KYNA) is a tryptophan derivative synthesized from L-kynurenine (L-KYN) by kynurenine aminotransferases (KATs). KAT I and KAT II are the main KAT isoenzymes. Non-selective antagonism towards ionotropic glutamatergic receptors, as well as activation of aryl hydrocarbon receptors and the α7 nicotinic acetylcholine receptors are known mechanisms of KYNA’s action. KYNA was shown to regulate water and electrolyte balance through natriuretic effect. By some researchers KYNA, together with other tryptophan metabolites, is classified as uremic toxin. We aimed to analyze the influence of furosemide, widely used loop diuretic, on KYNA’s synthesis and the activity of both KATs in rat kidney in vitro. Method The influence of furosemide on KYNA’s production with KAT I and KAT II activity was tested in rat kidney homogenates in vitro after 2 hours incubation in the presence of KYNA precursor L-KYN and furosemide. Diuretic was examined at increasing concentrations: 1 μM, 10 μM, 50 μM, 100 μM, 500 μM and 1 mM. Enzymatic KYNA’s production was measured by high performance liquid chromatography (HPLC) with fluorometric detector. All applicable international, national and institutional guidelines for the care and use of animals were followed. Results Furosemide at 500 μM and 1 mM lowered KYNA formation in kidney homogenates in vitro to 80% (p &amp;lt; 0.05) and 70% (p &amp;lt; 0.05) of control value, respectively. At 1 mM concentration furosemide inhibited kidney KAT I activity in vitro to 38% (p &amp;lt; 0.01) of control value. Similarly, furosemide at 500 μM and 1 mM concentration decreased kidney KAT II activity in vitro to 53% (p &amp;lt; 0.01) and 18% (p &amp;lt; 0.001) of control value, respectively. Conclusion Our study presents a novel mechanism of action of furosemide. Inhibition of KYNA synthesis in the kidney by furosemide may have influence on kidney function.

#4708 RENAL ORGANOID FROM URINE-DERIVED HUMAN ADULT RENAL PROGENITOR CELLS CAN SPONTANEOUSLY GENERATE LONG RENAL TUBULES

Abstract Background and Aims Organoids are self-organizing 3D aggregations of cells that represent the structure and function of organs. Kidney organoids have the potential to advance the field of nephrology by providing a tool for the study of human kidney development and disease, by providing a tool for in vitro drug screening, and ultimately, for regenerative therapy. They can be derived from embryonic stem cells or induced pluripotent stem cells. We aimed to explore for the first time, the potentiality of human adult renal progenitor cells (ARPCs), isolated from urine of healthy subjects and patients, to generate spheroids and organoids for regenerative purpose. Method ARPCs were isolated from urine by immunolabeling. Optical microscopy, immunofluorescence experiments and cytofluorimetric analysis were used to characterize spheroids and organoids. CD133, NANOG, SSA4, OCT 3-4 and GATA-3, SOX2 stemness marker antibodies were used to study spheroids and CD13, Lotus tetragonolobus lectin, ZO-1, uromodulin, and aminopeptidase antibodies were used to investigate renal tubular markers. PKH26 was used to study cell migration from organoids to tubules. Real-time PCR was used to generate gene expression data of aquaporin profile in renal tubules. Chick embryo chorioallantoic membrane (CAM) assays were used as in vivo model for angiogenesis. Results We isolated ARPCs from urine of healthy subjects or CKD patients. Usually, organoids were generated using complicate induction protocols with several chemical factors. However, for the first time we generated renal spheroids and organoids under 3D culture conditions without any stimulation, such as chemokines or growth factors, starting from ARPCs isolated from subject urine. We showed that the spheroids express high levels of stem cell markers as CD133 and NANOG, functional and constitutional marker of ARPCs, and high levels of stem cell markers that normally are low or not at all expressed in ARPCs and are typical of embryonic stem cells: GATA-3, SSEA4, and Sox2. Moreover, the spheroids were able to induce angiogenesis when implanted in chick embryo chorioallantoic membrane. The urine-derived CD133+ organoids can spontaneously generate long renal tubules in 6–12 days, as showed by PKH26 tagging. The renal tubules were positive for the proximal tubule cell markers CD13 (aminopeptidase N), Lotus tetragonolobus lectin and ZO-1. Moreover, renal tubules expressed the aminopeptidase A (CD249) that is normally present in proximal tubules and glomeruli of the kidneys and that catalyzed the conversion of the Angiotensin II in Angiotensin III witch is important in the local regulation of blood pressure. Also uromodulin was expressed by tubules. It is normally present in epithelial cells of the thick ascending limb of Henle's loop, and after proteolytical cleavage, is secreted into urine. In addition, the renal tubules express several genes typical of renal tubules as AQP1 channel protein that facilitates the flow of water molecules into the cells of the proximal tubule and descending limb of the loop of Henle; γGlut enzyme used for the transport of amino acids across cell membranes; Na/H exchanger 1 (SLC9A1), transporter involved in the regulation of pH homeostasis, cell migration and cell volume; Na/Gluc-1 co-transporter SGLUT1, which promotes the passage of glucose across the membrane in renal tubular cells. In some cases, tubule structures were similar to that found in nephrons, including proximal convoluted tubule, Loop of Henle, Distal Convoluted tubule and collecting duct, as shown by optical microscopy. Conclusion For the first time we demonstrated that kidney organoids can be generated starting by urine-derived human adult renal progenitor cells from patients and for the first time we showed that these organoids can generate, spontaneously, without specific stimulation, long portions of renal tubules. These results open new perspectives in the field of the regenerative medicine as ARPCs can be isolated from urine of patients and used to generate spheroids for the study of human kidney development and disease, for in vitro drug screening, and ultimately, for regenerative therapy.

#5399 VONAFEXOR AS A NOVEL THERAPY FOR CHRONIC KIDNEY DISEASE

Abstract Background and Aims Chronic kidney diseases (CKD) are characterized by the progressive loss of renal function. The molecular mechanisms underlying this progression are still poorly characterized and probably multifactorial. Therefore, there is a real need to expand the very limited number of therapies able to slow down the rate of CKD progression. Recent findings indicate that NR1H4, a druggable nuclear receptor, might play an important role in CKD. The aim of this study was to investigate the benefit of Vonafexor, a novel small molecule and specific NR1H4 agonist in acquired and genetic models of CKD. Method Two complementary experimental models of CKD (the 75% excision of total renal mass (Subtotal Nephrectomy; Nx) and the Col4a3−/− mice, a model of Alport disease) were studied. NR1H4 and the expression of some of its downstream targets were evaluated by quantitative RT-PCR, western blot and immunohistochemistry. In the Nx model, mice were treated with Vonafexor by daily oral gavage from 5 to 8 weeks after the surgery. The possible beneficial effect was compared to that of Losartan and other NR1H4 agonists. In the Alport model, mice were treated by Vonafexor from 5 to 8 weeks after birth. In both models, group of mice were sacrificed at 5 weeks to evaluate the extent of renal lesions at baseline. Renal lesions were quantified using image J. Renal inflammation, myofibroblast activation and podocyte number were quantified after specific staining. Human biopsies were also studied. RNAseq was performed using Illumina NovaSeq6000. Results We observed that NR1H4 expression and OSTA and OSTB, two downstream targets of NR1H4, were significantly reduced in remnant kidneys of Nx FVB/N mice compared to sham-operated animals 8 weeks after surgery. Co-localization staining revealed that NR1H4 is mainly expressed in proximal tubules and, to a minor extent, in Henle loops. Renal lesions were already present at 5 weeks in both experimental models. Remarkably, Vonafexor stopped the progression of renal lesions, in terms of glomerulosclerosis and tubular dilatations and, more importantly, induced a regression of interstitial fibrosis 8 weeks after nephrectomy as compared to the 5-week baseline. Lymphocytes and macrophage infiltration, as well as activated myofibroblasts, were also significantly reduced. Similarly, podocyte loss was stopped by Vonafexor treatment. Of note, the beneficial effect of Vonafexor was unique compared to other classes of NR1H4 agonists and also higher than that of Losartan. The same beneficial curative effect was found in Col4a3−/− mice. RT-PCR confirmed the activation of several NR1H4 downstream targets in mice treated with Vonafexor, while RNAseq analysis revealed that Vonafexor impacted the pathological activation of pathways involved in cell cycle, cell signaling and metabolism. These results may be relevant to humans, since NR1H4 expression was significantly reduced in biopsies from patients with CKD of different etiologies as compared to control kidneys. Conclusion Altogether our results identified in Vonafexor a novel key drug to control CKD. This is consistent with recent results obtained in LIVIFY phase 2 clinical trial in which Vonafexor has already demonstrated beneficial effects on eGFR in NASH patients with mild to moderate CKD.

Acute kidney injury biomarker olfactomedin 4 predicts furosemide responsiveness.

Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. Olfactomedin 4 (OLFM4), a secreted glycoprotein expressed in neutrophils and stressed epithelial cells, is upregulated in loop of Henle (LOH) cells following AKI. We hypothesized that urine OLFM4 (uOLFM4) will increase in patients with AKI and may predict furosemide responsiveness. Urine from critically ill children was collected prospectively and tested for uOLFM4 concentrations with a Luminex immunoassay. Severe AKI was defined by KDIGO (stage 2/3) serum creatinine criteria. Furosemide responsiveness was defined as > 3mL/kg/h of urine output in the 4h after a 1mg/kg IV furosemide dose administered as part of standard of care. Fifty-seven patients contributed 178 urine samples. Irrespective of sepsis status or AKI cause, uOLFM4 concentrations were higher in patients with AKI (221ng/mL [IQR 93-425] vs. 36ng/mL [IQR 15-115], p = 0.007). uOLFM4 concentrations were higher in patients unresponsive to furosemide (230ng/mL [IQR 102-534] vs. 42ng/mL [IQR 21-161], p = 0.04). Area under the receiver operating curve for association with furosemide responsiveness was 0.75 (95% CI, 0.60-0.90). AKI is associated with increased uOLFM4. Higher uOLFM4 is associated with a lack of response to furosemide. Further testing is warranted to determine whether uOLFM4 could identify patients most likely to benefit from earlier escalation from diuretics to kidney replacement therapy to maintain fluid balance. A higher resolution version of the Graphical abstract is available as Supplementary information.

Study of Anatomical Description and Histological Structure of the kidney in Iraqi Birds.

Background:&#x0D; The current study aims to identify the morphological description and histological structure of kidney in bird. Morphologically the kidney consist of three lobes represented by the cranial, medial and caudal taking into account the difference in the shape and size of the lobes. &#x0D; According to histology, the kidney is surrounded in a delicate connective tissue capsule. The kidney's primary structural component is the lobule, which is made up of cortical and medullary tissue in each lobule. There are two parts to the kidney medulla and cortex. The cortex is larger than the medulla and has glomeruli, proximal convoluted tubules, and distal convoluted tubules, whereas the medulla has thin and thick segments of Henle's loop, collecting tubules, and collecting ducts.&#x0D; Conclusion:&#x0D; The kidneys in Iraqi birds are morphologically and histologically different.