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Proximal tubule (PT) transports most of the renal Ca2+, which was usually described as paracellular (passive). We found a regulated Ca2+ entry pathway in PT cells via the apical transient receptor potential canonical 3 (TRPC3) channel, which initiates transcellular Ca2+ transport. Although TRPC3 knockout (−/−) mice were mildly hypercalciuric and displayed luminal calcium phosphate (CaP) crystals at Loop of Henle (LOH), no CaP + calcium oxalate (CaOx) mixed urine crystals were spotted, which are mostly found in calcium nephrolithiasis (CaNL). Thus, we used oral calcium gluconate (CaG; 2%) to raise the PT luminal [Ca2+]o further in TRPC3−/− mice for developing such mixed stones to understand the mechanistic role of PT-Ca2+ signaling in CaNL. Expectedly, CaG-treated mice urine samples presented with numerous mixed crystals with remains of PT cells, which were pronounced in TRPC3−/− mice, indicating PT cell damage. Notably, PT cells from CaG-treated groups switched their mode of Ca2+ entry from receptor-operated to store-operated pathway with a sustained rise in intracellular [Ca2+] ([Ca2+]i), indicating the stagnation in PT Ca2+ transport. Moreover, those PT cells from CaG-treated groups demonstrated an upregulation of calcification, inflammation, fibrotic, oxidative stress, and apoptotic genes; effects of which were more robust in TRPC3 ablated condition. Furthermore, kidneys from CaG-treated groups exhibited fibrosis, tubular injury and calcifications with significant reactive oxygen species generation in the urine, thus, indicating in vivo CaNL. Taken together, excess PT luminal Ca2+ due to escalation of hypercalciuria in TRPC3 ablated mice induced surplus CaP crystal formation and caused stagnation of PT [Ca2+]i, invoking PT cell injury, hence mixed stone formation.

This study found that the thick ascending limb of Henle's loop is involved in the mechanisms of hypercalciuria and hypermagnesiuria in metabolic acidosis. Specifically, expression of claudin-16/19 and claudin-14 was altered via up-regulation of calcium-sensing receptor in NH4Cl-induced metabolic acidosis. Our novel findings contribute to understanding the regulatory role of paracellular tight junction proteins in the thick ascending limb.

Lithium, administered to patients of bipolar disorders, is mainly excreted into urine, and tubular reabsorption at the proximal tubule is involved in the renal handling of lithium. In this study, we examined the renal excretion of lithium in rats with Fanconi syndrome, characterized by defects of transports of various compounds at the proximal tubules, induced by maleic acid. After maleic acid was intravenously injected, mannitol and lithium chloride were infused in turn. Using samples of plasma and bladder urine during the mannitol infusion, renal parameters were determined. Pharmacokinetic parameters of lithium were obtained using samples during the lithium chloride infusion. Maleic acid decreased creatinine clearance and increased the fractional excretion of glucose and phosphate, suggesting the induction of Fanconi syndrome. In rats with Fanconi syndrome, plasma concentration of lithium was increased, and its renal clearance was decreased. No effect on the fractional excretion of lithium was exhibited. This study represents that the tubular reabsorption of lithium was impaired to the same degree with glomerular filtration in rats with experimental Fanconi syndrome and that the dysfunction of the tubular reabsorption of glucose and phosphate was more severe. It is possible that Fanconi syndrome inhibited the reabsorption of lithium at the proximal tubule and facilitated the reabsorption of lithium from the loop of Henle to the collecting duct.

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

Acidosis is associated with E. coli induced pyelonephritis but whether bacterial cell wall constituents inhibit HCO3 transport in the outer medullary collecting duct from the inner stripe (OMCDi) is not known. We examined the effect of lipopolysaccharide (LPS), on HCO3 absorption in isolated perfused rabbit OMCDi. LPS caused a ~ 40% decrease in HCO3 absorption, providing a mechanism for E. coli pyelonephritis-induced acidosis. Monophosphoryl lipid A (MPLA), a detoxified TLR4 agonist, and Wortmannin, a phosphoinositide 3-kinase inhibitor, prevented the LPS-mediated decrease, demonstrating the role of TLR4-PI3-kinase signaling and providing proof-of-concept for therapeutic interventions aimed at ameliorating OMCDi dysfunction and pyelonephritis-induced acidosis.

Na+–K+–Cl− cotransporter 2 (NKCC2; SLC12A1) is an integral membrane protein that comes as three splice variants and mediates the cotranslocation of Na+, K+, and Cl− ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, and through the involvement of other ion transport systems, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na+, Cl−, Ca2+, Mg2 +, and HCO3 − loads. The functional relevance of NKCC2 in human is illustrated by the many abnormalities that result from the inactivation of this transport system through the use of loop diuretics or in the setting of inherited disorders. The following presentation aims at discussing the physiological roles and molecular characteristics of Na+–K+–Cl− cotransport in the TALH and those of the individual NKCC2 splice variants more specifically. Many of the historical and recent data that have emerged from the experiments conducted will be outlined and their larger meaning will also be placed into perspective with the aid of various hypotheses., Na+–K+–Cl− cotransporter 2 (NKCC2) mediates the cotranslocation of Na+, K+, and Cl− ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na+, Cl−, Ca2+, Mg2 +, and HCO3 − load. The functional relevance of NKCC2 in humans is illustrated by the many abnormalities that result from the inactivation of this transport system.

Na+-coupled phosphate cotransporters from the SLC34 and SLC20 families of solute carriers mediate transepithelial transport of inorganic phosphate (Pi). NaPi-IIa/Slc34a1, NaPi-IIc/Slc34a3, and Pit-2/Slc20a2 are all expressed at the apical membrane of renal proximal tubules and therefore contribute to renal Pi reabsorption. Unlike NaPi-IIa and NaPi-IIc, which are rather kidney-specific, NaPi-IIb/Slc34a2 is expressed in several epithelial tissues, including the intestine, lung, testis, and mammary glands. Recently, the expression of NaPi-IIb was also reported in kidneys from rats fed on high Pi. Here, we systematically quantified the mRNA expression of SLC34 and SLC20 cotransporters in kidneys from mice, rats, and humans. In all three species, NaPi-IIa mRNA was by far the most abundant renal transcript. Low and comparable mRNA levels of the other four transporters, including NaPi-IIb, were detected in kidneys from rodents and humans. In mice, the renal expression of NaPi-IIa transcripts was restricted to the cortex, whereas NaPi-IIb mRNA was observed in medullary segments. Consistently, NaPi-IIb protein colocalized with uromodulin at the luminal membrane of thick ascending limbs of the loop of Henle segments. The abundance of NaPi-IIb transcripts in kidneys from mice was neither affected by dietary Pi, the absence of renal NaPi-IIc, nor the depletion of intestinal NaPi-IIb. In contrast, it was highly upregulated in a model of oxalate-induced kidney disease where all other SLC34 phosphate transporters were downregulated. Thus, NaPi-IIb may contribute to renal phosphate reabsorption, and its upregulation in kidney disease might promote hyperphosphatemia.

Uromodulin (UMO) is a multifunctional glycoprotein expressed in the epithelial cells of the thick ascending part of the loop of Henle. Currently a lot of data about mechanisms of biosynthesis, apical and basolateral transport of UMO, changes in urine and blood concentrations in different kidney compartments damage, roles of UMO in protecting kidneys from infections, maintaining mineral homeostasis, development of arterial hypertension and the participation of this glycoprotein in other physiological and pathological processes has been accumulated. The article discusses the clinical significance of UMO in the development and progression of chronic kidney disease, prognostic value of UMO urine and blood concentrations in terms of the risk of cardiovascular diseases and probability of acute kidney damage in patients with cardiovascular pathology. Briefly highlights issues of UMO gene mutation and development of autosomal dominant tubulointerstitial kidney disease.

Reptile kidneys maintain a constant extracellular environment within the body. They excrete waste products, maintain normal concentrations of salt and water, regulate acid-base balance, and produce hormones and vitamins. The kidneys contain nephrons consisting of glomeruli designed to filter the plasma, Bowman capsules that collect the filtrate, and tubules that resorb most of the filtered water and nutrients while excreting waste metabolites. A Loop of Henle is absent. Therefore, reptile kidneys cannot produce a hypertonic urine. The urinary bladder (if present) and cloaca excrete and absorb additional fluids and electrolytes. A renal portal system is present in all reptiles.

Gitelman and Bartter syndromes are rare inherited diseases that belong to the category of renal tubulopathies. The genes associated with these pathologies encode electrolyte transport proteins located in the nephron, particularly in the Distal Convoluted Tubule and Ascending Loop of Henle. Therefore, both syndromes are characterized by alterations in the secretion and reabsorption processes that occur in these regions. Patients suffer from deficiencies in the concentration of electrolytes in the blood and urine, which leads to different systemic consequences related to these salt-wasting processes. The main clinical features of both syndromes are hypokalemia, hypochloremia, metabolic alkalosis, hyperreninemia and hyperaldosteronism. Despite having a different molecular etiology, Gitelman and Bartter syndromes share a relevant number of clinical symptoms, and they have similar therapeutic approaches. The main basis of their treatment consists of electrolytes supplements accompanied by dietary changes. Specifically for Bartter syndrome, the use of non-steroidal anti-inflammatory drugs is also strongly supported. This review aims to address the latest diagnostic challenges and therapeutic approaches, as well as relevant recent research on the biology of the proteins involved in disease. Finally, we highlight several objectives to continue advancing in the characterization of both etiologies.

Dahl salt-sensitive (SS) rat kidneys produce less nitric oxide (NO) than those of salt-resistant (SR) rats. Thick ascending limb (TAL) NO synthase 3 (NOS3) is a major source of renal NO, and luminal flow enhances its activity. We hypothesized that flow-induced NO is reduced in TALs from SS rats primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal-salt (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused TALs and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35 ± 6 arbitrary units (AU)/min in TALs from SR rats but only 11 ± 2 AU/min in TALs from SS (P < 0.008). The superoxide scavenger tempol decreased the difference in flow-induced NO between strains by about 36% (P < 0.020). The NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) decreased flow-induced superoxide by 36 ± 8% in TALs from SS rats (P < 0.02) but had no effect in TALs from SR rats. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR rats: 44 ± 10 vs. SS: 9 ± 2 AU/min, P < 0.005). Tempol decreased the difference in flow-induced NO between strains by about 37% (P < 0.012). l-NAME did not significantly reduce flow-induced superoxide in either strain. HS increased NOS3 expression in TALs from SR rats but not in TALs from SS rats (P < 0.003). We conclude that 1) on NS, flow-induced NO is diminished in TALs from SS rats mainly due to NOS3 uncoupling such that it produces superoxide and 2) on HS, the difference is enhanced due to failure of TALs from SS rats to increase NOS3 expression. NEW & NOTEWORTHY The Dahl rat has been used extensively to study the causes and effects of salt-sensitive hypertension. Our study suggests that more complex processes other than simple scavenging of nitric oxide (NO) by superoxide lead to less NO production in thick ascending limbs of the Dahl salt-sensitive rat. The predominant mechanism involved depends on dietary salt. Impaired flow-induced NO production in thick ascending limbs most likely contributes to the Na(+) retention associated with salt-sensitive hypertension.

BACKGROUND Uromodulin modulates the sodium-potassium-two-chloride transporter in the thick ascending limb of the loop of Henle, and its overexpression in murine models leads to salt-induced hypertension. We hypothesized that individuals with higher baseline levels of urine uromodulin would have a greater increase in systolic blood pressure (SBP) for the same increase in sodium compared with those with lower uromodulin levels. METHODS We used data from 157 subjects randomized to the control diet of the Dietary Approaches to Stop Hypertension (DASH)-Sodium trial who were assigned to 30 days of low (1,500 mg/d), medium (2,400 mg/d), and high salt (3,300 mg/d) diets in random order. Blood pressure was measured prerandomization and then weekly during each feeding period. We evaluated the association of prerandomization urine uromodulin with change in SBP between diets, as measured at the end of each feeding period, using multivariable linear regression. RESULTS Baseline urine uromodulin stratified by tertiles was ≤17.64, 17.65–31.97, and ≥31.98 µg/ml. Across the tertiles, there were no significant differences in SBP at baseline, nor was there a differential effect of sodium diet on SBP across tertiles (low to high, P = 0.81). After adjusting for age, sex, body mass index, and race, uromodulin levels were not significantly associated with SBP change from low to high sodium diet (P = 0.42). CONCLUSIONS In a randomized trial of different levels of salt intake, higher urine uromodulin levels were not associated with a greater increase in blood pressure in response to high salt intake.

The process of kidney stone formation is complex and still not completely understood. Supersaturation and crystallization are the main drivers for the etiopathogenesis of uric acid, xanthine and cystine stones but this physicochemical concept fails to adequately explain the formation of calcium-based nephrolithiasis, which represents the majority of kidney stones. Contemporary concepts of the pathogenesis of calcium-based nephrolithiasis focus on a nidus-associated stone formation of calcium-based nephrolithiasis on Randall's plaques or on plugs of Bellini's duct. Randall's plaques originate from the interaction of interstitial calcium supersaturation in the renal papilla, vascular and interstitial inflammatory processes and mineral deposits of calcifying nanoparticles on the basal membrane of the thin ascending branch of the loop of Henle; however, plugs of Bellini's duct are assumed to be caused by mineral deposits on the wall of the collecting ducts. Aggregation and overgrowth are influenced by the interaction of matrix proteins with calcium supersaturated urine, by an imbalance between promoters and inhibitors of stone formation in the calyceal urine. Current research has elucidated many factors contributing to stone formation by revealing novel insights into the physiology of nephron and papilla, by analyzing vascular, inflammatory and calcifying processes in the renal medulla, by examining the proteome, the microbiome, promoters and inhibitors of stone formation in the urine and by conducting the first genome-wide association studies; however, more future research is mandatory to fill the gap of knowledge and hopefully, to obtain novel prophylactic, therapeutic and metaphylactic tools beyond the current state of knowledge.

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

In two rat models of acute paraoxon (POX) poisoning against the background of specific and nonspecific inhibition of blood plasma carboxyl esterase (CE) activity, the POX effect on morphology and functions of the rat kidneys was assessed within a period from 1 day to 6 weeks after the poisoning. During the first 3 days, rats developed transient glycosuria and increased urinary excretion of calbindin and sulfated glycosaminoglycans. One day after the poisoning, a positive reaction for protein deposits (AFOG+) was observed in the descending and ascending limbs of the loop of Henle and collecting ducts against the background of nonspecific CE inhibition. After 2 weeks, the AFOG+ reaction was detected in the proximal straight tubules along with desquamation of epithelial cells. Both 3 days and 2 weeks after the poisoning, regardless of the mode of CE inhibition, a morphometric analysis of individual components of the rat nephron revealed an increase in the size of the renal corpuscle and the glomerular capillary tuft within the Bowman’s capsule. Three days after the poisoning and at every subsequent observations, intoxicated rats exhibited an alteration in the luminal diameter of the proximal tubules and the luminal surface of the collecting ducts. Most pronounced alterations in the luminal diameter of the distal tubules were observed 3 days, 4 and 6 weeks after the poisoning.

The thick ascending limb of Henle’s loop (TAL) reabsorbs NaCl via the apical Na+-K+-2Cl−cotransporter (NKCC2). NKCC2 activity is regulated by surface NKCC2 levels. The second messenger cGMP decreases NKCC2 activity by decreasing surface NKCC2 levels. We found that surface NKCC2 undergoes constitutive degradation. Therefore, we hypothesized that cGMP decreases NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. We measured surface NKCC2 levels by biotinylation of surface proteins, immunoprecipitation of NKCC2, and ubiquitin in TALs. First, we found that inhibition of proteasomal degradation blunts the cGMP-dependent decrease in surface NKCC2 levels [vehicle: 100%, db-cGMP (500 µM): 70.3 ± 9.8%, MG132 (20 µM): 97.7 ± 5.0%, and db-cGMP + MG132: 103.3 ± 3.4%, n = 5, P < 0.05]. We then found that cGMP decreased the internalized NKCC2 pool and that this effect was prevented by inhibition of the proteasome but not the lysosome. Finally, we found that NKCC2 is constitutively ubiquitinated in TALs and that cGMP enhances the rate of NKCC2 ubiquitination [vehicle: 59 ± 14% and db-cGMP (500 µM): 111 ± 25%, n = 5, P < 0.05]. We conclude that NKCC2 is constitutively ubiquitinated and that cGMP stimulates NKCC2 ubiquitination and proteasomal degradation. Our data suggest that the cGMP-induced NKCC2 ubiquitination and degradation may contribute to the cGMP-induced decrease of the NKCC2-dependent NaCl reabsorption in TALs.

Basement membranes consist of various proteins, the major ones being laminin and collagen type IV. Primary defects in these two proteins have been extensively associated with kidney pathologies. This study aimed to establish baseline information on the immunohistochemical distribution of laminin and collagen type IV, and to corelate these with the ultrastructure of basal laminae in the uriniferous tubules of the dromedary camel. Tissue samples were taken from the kidneys of eight adult female camels, and processed for immunohistochemical and ultrastructural investigations. Strong intensity of collagen type IV was observed within the basement membranes of Bowman's capsule. The thickness of the basal lamina of the parietal layer of Bowman's capsule varied extensively depending on the region of the renal corpuscle; the thicker areas were always associated with cuboidal epithelial cells. The glomerular basement membrane revealed strong immunostaining of laminin, whereas the mesangial matrix was strongly immunoreactive to collagen type IV. Abundant amount of laminin was found in the basement membranes of proximal convoluted tubules, thin limbs of the loop of Henle, and collecting ducts. Dense immunostainings of laminin and collagen type IV were observed in the medullary regions of uriniferous tubule, in which numerous projections extended from the basal laminae into the subjacent connective tissue. Overall, the present study revealed marked variations in the distribution of the basement membrane markers laminin and collagen type IV in the uriniferous tubules of camel kidney. The results have also shown difference in the thickness of basal laminae. This variation in thickness, however, was unlikely to be influenced by the amount of laminin and collagen type IV.

Background: Inhibition of the Na+/glucose co-transporter 2 is a new therapeutic strategy for diabetes. It is unclear how proximal loss of Na+ (and glucose) affects the subsequent Na+ transporters in the proximal tubule (PT), thick ascending limb of loop of Henle (TAL), distal convoluted tubule (DCT) and collecting duct (CD). Methods: Mice on a high fat diet were administered 3 doses streptozotocin 6 days prior to oral dapagliflozin administration or vehicle for 18 days. A control group of lean mice were also included. Body weight and glucose were recorded at regular intervals during treatment. Renal Na+ transporters expression in nephron segments were analyzed by RT-qPCR and Western blot. Results: Dapagliflozin treatment resulted in a significant reduction in body weight and blood glucose compared to vehicle-treated controls. mRNA results showed that Na+-hydrogen antiporter 3 (NHE3), Na+/phosphate cotransporter (NaPi-2a) and epithelial Na+ channel expression was increased, Ncx1, ENaCβ and ENaCγ expression declined (p all < 0.05), respectively, in dapagliflozin-treated mice when compared with saline vehicle mice. Na-K-2Cl cotransporters and Na-Cl cotransporter mRNA expression was not affected by dapagliflozin treatment. Na+/K+-ATPase (Atp1b1) expression was also increased significantly by dapagliflozin treatment, but it did not affect Atp1a1 and glucose transporter 2 expression. Western blot analysis showed that NaPi-2a, NHE3 and ATP1b1 expression was upregulated in dapagliflozin-treated diabetic mice when compared with saline vehicle mice (p < 0.05). Conclusion: Our findings suggest that dapagliflozin treatment augments compensatory changes in the renal PT in diabetic mice.

Uromodulin is a key protein involved in the risk of CKD and hypertension, essentially produced by the cells lining the thick ascending limb of the loop of Henle ([1][1]). Uromodulin regulates sodium and potassium transport activity in the thick ascending limb, and its deletion in mice leads to salt

The glomerulus, the filtering part of the nephron, consists of a network of capillaries through which the blood flows under the influence of hydraulic forces generated by contractions of the heart. The nephron consists of a renal, or Malpighian, corpuscle, a proximal tubule, a loop of Henle, and a distal tubule. The interlobular arteries of the kidney branch extensively to form afferent arterioles which supply each glomerulus. The glomerulus nearly fills Bowman’s capsule. The specialized cells that constitute the visceral layer, called podocytes, are stellar in shape and possess cytoplasmic extensions that originate from the nucleated portion of the cell body. The Glomerular Basement Membrane is an extracellular matrix interposed between the epithelial foot processes and endothelium. The location and properties of the glomerular mesangial cells confer a central role for these cells in the regulation of renal function. Glomerular filtration is the initial step in urine formation.

Sepsis is the leading cause of acute kidney injury in critically ill patients. Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of septic kidney injury; however, the sites and mechanisms of renal TNF-α production during sepsis remain to be defined. In the present study, we showed that TNF-α expression is increased in medullary thick ascending limbs (MTALs) of mice with sepsis induced by cecal ligation and puncture. Treatment with lipopolysaccharide (LPS) for 3 h in vitro also increased MTAL TNF-α production. Sepsis and LPS increased MTAL TNF-α expression through activation of the myeloid differentiation factor 88 (MyD88)-IL-1 receptor-associated kinase 1-ERK signaling pathway. Pretreatment with monophosphoryl lipid A (MPLA), a nontoxic immunomodulator that protects against bacterial infection, eliminated the sepsis- and LPS-induced increases in MTAL TNF-α production. The suppressive effect of MPLA on TNF-α was mediated through activation of a phosphatidylinositol 3-kinase-dependent pathway that inhibits MyD88-dependent ERK activation. This likely involves MPLA-phosphatidylinositol 3-kinase-mediated induction of Tollip, which negatively regulates the MyD88-ERK pathway by inhibiting activation of IL-1 receptor-associated kinase 1. These regulatory mechanisms are similar to those previously shown to mediate the effect of MPLA to prevent sepsis-induced inhibition of MTAL [Formula: see text] absorption. These results identify the MTAL as a site of local TNF-α production in the kidney during sepsis and identify molecular mechanisms that can be targeted to attenuate renal TNF-α expression. The ability of MPLA pretreatment to suppress MyD88-dependent ERK signaling in the MTAL during sepsis has the dual beneficial effects of protecting tubule transport functions and attenuating harmful proinflammatory responses.

Empaglifolzin, canagliflozin, and dapagliflozin are SGLT2 (sodium-glucose linked transporter type 2) inhibitors for treatment of type 2 diabetes mellitus that also reduce blood pressure, mortality, and cardiovascular disease and slow the loss of glomerular filtration rate. SGLT2 inhibitors inhibit the coupled reabsorption of sodium and glucose from the proximal tubules, thereby increasing renal glucose and sodium excretion, but they have more widespread renal effects, including inhibition of the sodium:proton exchanger. They increase the delivery of sodium to the loop of Henle and can thereby activate the tubuloglomerular feedback response to correct glomerular hyperfiltration. There are multiple potential mechanisms whereby these drugs lower blood pressure and preserve kidney function that are the focus of this review.

The kidney controls body fluids, electrolyte and acid–base balance. Previously, we demonstrated that hyperpolarization-activated and cyclic nucleotide-gated (HCN) cation channels participate in ammonium excretion in the rat kidney. Since acid–base balance is closely linked to potassium metabolism, in the present work we aim to determine the effect of chronic metabolic acidosis (CMA) and hyperkalemia (HK) on protein abundance and localization of HCN3 in the rat kidney. CMA increased HCN3 protein level only in the outer medulla (2.74 ± 0.31) according to immunoblot analysis. However, immunofluorescence assays showed that HCN3 augmented in cortical proximal tubules (1.45 ± 0.11) and medullary thick ascending limb of Henle’s loop (4.48 ± 0.45) from the inner stripe of outer medulla. HCN3 was detected in brush border membranes (BBM) and mitochondria of the proximal tubule by immunogold electron and confocal microscopy in control conditions. Acidosis did not alter HCN3 levels in BBM and mitochondria but augmented them in lysosomes. HCN3 was also immuno-detected in mitoautophagosomes. In the distal nephron, HCN3 was expressed in principal and intercalated cells from cortical to medullary collecting ducts. CMA did not change HCN3 abundance in these nephron segments. In contrast, HK doubled HCN3 level in cortical collecting ducts and favored its basolateral localization in principal cells from the inner medullary collecting ducts. These findings further support HCN channels contribution to renal acid–base and potassium balance.

The thick ascending limb of the loop of Henle (TAL) is the first segment of the distal nephron, extending through the whole outer medulla and cortex, two regions with different composition of the peritubular environment. The TAL plays a critical role in the control of NaCl, water, acid, and divalent cation homeostasis, as illustrated by the consequences of the various monogenic diseases that affect the TAL. It delivers tubular fluid to the distal convoluted tubule and thereby affects the function of the downstream tubular segments. The TAL is commonly considered as a whole. However, many structural and functional differences exist between its medullary and cortical parts. The present review summarizes the available data regarding the similarities and differences between the medullary and cortical parts of the TAL. Both subsegments reabsorb NaCl and have high Na+-K+-ATPase activity and negligible water permeability; however, they express distinct isoforms of the Na+-K+-2Cl−cotransporter at the apical membrane. Ammonia and bicarbonate are mostly reabsorbed in the medullary TAL, whereas Ca2+and Mg2+are mostly reabsorbed in the cortical TAL. The peptidic hormone receptors controlling transport in the TAL are not homogeneously expressed along the cortical and medullary TAL. Besides this axial heterogeneity, structural and functional differences are also apparent between species, which underscores the link between properties and role of the TAL under various environments.

In mammals, three subtypes of V-receptors have been identified in the kidney. The effects of vasopressin, a hormone synthesized in the hypothalamus, are triggered by three distinct receptor isoforms: V2, V1a, and V1b. Stimulation of V2-receptors regulates urine osmotic concentration by increasing sodium reabsorption in the thick ascending limb of the loop of Henle and enhancing osmotic permeability of the epithelium cells in the collecting duct. Stimulation of V1a-receptors inhibits renal sodium reabsorption and induces natriuresis, comparable to the effect of the diuretic furosemide, in the thick ascending limb of the loop of Henle. Stimulation of V1b-receptors induces potassium secretion in the final parts of the distal segments and initial parts of the collecting ducts. In this review, we discuss the role of vasopressin and its interaction with V-receptor subtypes in natriuresis and for stabilizing the physicochemical parameters of the internal environment and water-salt homeostasis in humans. A better understanding of these systems and their regulation is necessary to facilitate identification of additional system components and mechanisms, clarify their contribution during various normal and pathological functional states, and suggest novel strategies for the development of therapeutic interventions.

The Reynolds number in the renal tubule is extremely low, consistent with laminar flow. Consequently, luminal flow can be described by the Hagen-Poiseuille laminar flow equation. This equation calculates the volumetric flow rate from the axial pressure gradient and flow resistance, which is dependent on the length and diameter of each renal tubule segment. Our goal was to calculate the pressure drop along each segment of the renal tubule and to determine the points of highest resistance. When the Hagen-Poiseuille equation was used for rat superficial nephrons based on known tubule flow rates, lengths, and diameters, it was found that the maximum pressure drop occurred in two segments: the thin descending limbs of Henle and the inner medullary collecting ducts. The high resistance in the thin descending limbs is due to their small diameters. The steep pressure drop observed in the inner medullary collecting ducts is due to the convergent structure of the tubules, which channels flow into fewer and fewer tubules toward the papillary tip. For short-looped nephrons, the calculated glomerular capsular pressure matched measured values, even with the high collecting duct flow rates seen in water diuresis, provided that tubule compliance was taken into account. In long-looped nephrons, the greater length of thin limb segments is likely compensated for by a larger luminal diameter. Simulation of the effect of proximal diuretics, namely acetazolamide or type 2 sodium-glucose transporter inhibitors, predicts a substantial back pressure in Bowman’s capsule, which may contribute to observed decreases in glomerular filtration rate.

Aminoglycosides are extensively used owing to advantages such as low cost and availability. Nephrotoxicity induced by antibiotics has been analyzed in different animal models. This study was aimed to investigate the effect of Vaccinium arctostaphylos L. fruit extract on gentamicin-induced nephrotoxicity in rats. In this experimental study, 25 rats were divided into five groups, including negative control (0.5 ml normal saline), positive control (80 mg/kg gentamicin and 0.5 ml normal saline), and three Vaccinium arctostaphylos L. fruit extract groups at 100, 200, and 400 mg/kg concentrations plus 80 mg/kg gentamicin. All administrations were done intraperitoneally for 7 days. Thirty days after the first injection, blood samples were taken from the heart. Serum creatinine (Cr), blood urea nitrogen (BUN), and sodium and potassium levels were measured. To prepare histological sections, the kidneys were kept in 10% formalin. Having prepared the histological sections, the volume of renal tubules (proximal and distal convoluted tubules and loop of Henle), renal vessels, interstitial tissue, and number and volume of renal glomeruli were measured. Vaccinium arctostaphylos L. fruit extract significantly increased serum urea (p = 0.023), creatinine (p = 0.009), volume of proximal convoluted tubules (p = 0.02), volume of distal convoluted tubules (p = 0.032), loop of Henle (p = 0.038), and glomeruli (p = 0.015). However, it significantly reduced the volume of vessels (p = 0.009) and interstitial tissue (p = 0.001). The 400 mg/kg concentration exerted the highest effect. Vaccinium arctostaphylos L. fruit extract has protective effects against gentamicin-induced renal impairments.

The kidneys are vital organs in the management of fluid balance, waste product removal, electrolyte homeostasis, acid–base balance and endocrine function. Waste products removed by the kidney include urea, uric acid, creatinine and other foreign products with similar physiochemical properties. Urea and uric acid are by products of protein metabolism and creatinine is generated by the metabolism of creatine compounds from muscle. The kidney regulates fluid and electrolyte balance through controlling the composition and volume of urine. In the proximal convoluted tubule and the loop of Henle, 90% of sodium, potassium, calcium and magnesium are reabsorbed. Acid–base balance is achieved by regulating the excretion of hydrogen ions and bicarbonate buffering. The kidney also has a number of endocrine functions including the production of renin and erythropoietin as well as hydroxylation of vitamin D. The kidneys receive 25% of cardiac output, generating 170–200 litres of ultrafiltrate per day. Urine output is approximately 1.5 litres per day, which is concentrated ultrafiltrate through selective reabsorption of solutes and water. In this article we will discuss tests frequently used to assess renal function.

Osteoblast activating peptide (OBAP) is a newly discovered peptide detected in the rat stomach, which has a major role in osteogenesis. Recently, we revealed its localization in the parietal cells of the rat stomach. There have been no data regarding OBAP expression in the kidney, despite its role in calcium reabsorption in renal tubules. The current study aimed to inspect the expression of OBAP in the kidney of twelve 10-week-old male C3H/HeNJc1 mice using immunohistochemistry, and immunoelectron microscopic localization. The immunohistochemical investigation revealed an OBAP positive reaction mainly in the medulla, which was stronger than the cortex of the kidney and was concentrated in the distal convoluted tubules (DCT), connecting tubules (CT), and the thick limbs of the loop of Henle (HL). Moreover, we clarified that the OBAP was co-distributed with ghrelin and calbindin (markers of the DCT). Interestingly, immunoelectron microscopy demonstrated that OBAP was concentrated in the mitochondrial inner membrane of the DCT and CT. Based on these results, it was concluded that the mitochondria of the DCT, CT, and HL of the mice kidney generate OBAP. Furthermore, our results suggest that OBAP might have a role in the regulation of calcium reabsorption by the renal tubule; however, further investigations are required to clarify this potential role.

In general, the mammalian whole body mass-specific metabolic rate correlates positively with maximal urine concentration (Umax) irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass-specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na+-K+-ATPase activity and Na+-K+-ATPase α1-gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na+-K+-ATPase activity (~70%) compared with two rat strains (Sprague-Dawley and Munich-Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na+-K+-ATPase α1-protein (~4- to 6-fold) and mRNA (~13-fold) and higher TAL mitochondrial volume density (~20%) occur in the kangaroo rat compared with both rat strains. Rat TAL Na+-K+-ATPase α1-protein expression is relatively unaffected by body hydration status or, shown previously, by dietary Na+, arguing against confounding effects from two unavoidably dissimilar diets: grain-based diet without water (kangaroo rat) or grain-based diet with water (rat). We conclude that higher TAL Na+-K+-ATPase activity contributes to relationships between whole body mass-specific metabolic rate and high Umax. More vigorous TAL Na+-K+-ATPase activity in kangaroo rat than rat may contribute to its steeper Na+and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin.

The nephron segments in the inner medulla are part of the urine concentrating mechanism. Depending on the diuretic state, they are facing a large range of extracellular osmolality. We investigated whether water homeostasis affects tubular transport and permeability properties in inner medullary descending thin limb (IMdTL) and ascending thin limb (IMaTL). Three experimental groups of rats under different diuretic states were investigated on metabolic cages: waterload, furosemide-induced diuresis, and control (antidiuresis). Urine production and osmolalities reflected the 3-day treatment. To functionally investigate tubular epithelial properties, we performed experiments in freshly isolated inner medullary thin limbs from these animals. Tubular segments were acutely dissected and investigated for trans- and paracellular properties by in vitro perfusion and electrophysiological analysis. IMdTL and IMaTL were distinguished by morphological criteria. We confirmed absence of transepithelial electrogenic transport in thin limbs. Although diffusion potential measurements showed no differences between treatments in IMdTLs, we observed increased paracellular cation selectivity under waterload in IMaTLs. NaCl diffusion potential was −5.64 ± 1.93 mV under waterload, −1.99 ± 1.72 mV under furosemide-induced diuresis, and 0.27 ± 0.40 mV under control. The corresponding permeability ratio PNa/Clwas 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.

Tamm–Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP(−/−) mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP(−/−) mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP(−/−) mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP(−/−) mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.

Previous in vitro studies demonstrated that aldosterone rapidly activates sodium-hydrogen exchangers 1 and 3 (NHE 1 and 3). In vitro investigations revealed that protein kinase C (PKC) regulates NHE properties. We previously demonstrated that aldosterone rapidly enhances PKCα protein abundance in the rat kidney. There are no reports of renal PKCβ (I and II) protein levels related to the regulation by aldosterone. There are also no in vivo data regarding the rapid effects of aldosterone on renal protein levels of NHE (1 and 3) and PKCβ (I and II), simultaneously. In the current study, rats received normal saline solution or aldosterone (150 μg/kg BW, i.p.). After 30 minutes, abundance and immunoreactivity of these proteins were determined by Western blot analysis and immunohistochemistry, respectively. Aldosterone increased NHE1 and NHE3 protein abundance to 152% and 134%, respectively (P<0.05). PKCβI protein level was enhanced by 30%, whereas PKCβII declined slightly. Aldosterone increased NHE protein expression mostly in the medulla. PKCβI immunostaining intensity was increased in the glomeruli, renal vasculature, and thin limb of the loop of Henle, while PKCβII was reduced. This is the first in vivo study to simultaneously demonstrate that aldosterone rapidly elevates PKCβI and NHE (1 and 3) protein abundance in the rat kidney. Aldosterone-induced NHE (1 and 3) protein levels may be related to PKCβI activation.

Purpose of review The kidney Na-K-2Cl cotransporter (NKCC2) is essential for urinary concentration and renal electrolyte handling. Loss of function mutations in the NKCC2 gene cause urinary salt and potassium wasting, whereas excessive NKCC2 function has been linked to high blood pressure. Loop diuretics, targeting the transporter, are instrumental for relieving edema or hypertension. This review focuses on intrinsic mechanisms regulating NKCC2 activity at the posttranslational level, namely its trafficking and phosphorylation. Recent findings Protein networks mediating cellular turnover of NKCC2 have recently received major attention. Several key components of its apical trafficking were identified, including respective chaperones, SNARE protein family members and raft-associated proteins. NKCC2 internalization has been characterized qualitatively and quantitatively. Kinase and phosphatase pathways regulating NKCC2 activity have been clarified and links between NKCC2 phosphorylation and trafficking proposed. Constitutive and inducible NKCC2 trafficking and phosphorylation mechanisms have been specified with focus on endocrine control of thick ascending limb (TAL) function by vasopressin. Summary Proper NKCC2 trafficking and phosphorylation are critical to the TAL function in the physiological context of urinary concentration and extracellular volume regulation. Clarification of the underlying mechanisms and respective protein networks may open new therapeutic perspectives for better management of renal electrolyte disorders and blood pressure control.

About 50% of the Na+ reabsorbed in thick ascending limbs traverses the paracellular pathway. Nitric oxide (NO) reduces the permselectivity of this pathway via cGMP, but its effects on absolute Na+ ([Formula: see text]) and Cl- ([Formula: see text]) permeabilities are unknown. To address this, we measured the effect of l-arginine (0.5 mmol/l; NO synthase substrate) and cGMP (0.5 mmol/l) on [Formula: see text] and [Formula: see text] calculated from the transepithelial resistance (Rt) and [Formula: see text]/[Formula: see text] in medullary thick ascending limbs. Rt was 7,722 ± 1,554 ohm·cm in the control period and 6,318 ± 1,757 ohm·cm after l-arginine treatment (P < 0.05). [Formula: see text]/[Formula: see text] was 2.0 ± 0.2 in the control period and 1.7 ± 0.1 after l-arginine (P < 0.04). Calculated [Formula: see text] and [Formula: see text] were 3.52 ± 0.2 and 1.81 ± 0.10 × 10-5 cm/s, respectively, in the control period. After l-arginine they were 6.65 ± 0.69 (P < 0.0001 vs. control) and 3.97 ± 0.44 (P < 0.0001) × 10-5 cm/s, respectively. NOS inhibition with Nω-nitro-l-arginine methyl ester (5 mmol/l) prevented l-arginine´s effect on Rt Next we tested the effect of cGMP. Rt in the control period was 7,592 ± 1,470 and 4,796 ± 847 ohm·cm after dibutyryl-cGMP (0.5 mmol/l; db-cGMP) treatment (P < 0.04). [Formula: see text]/[Formula: see text] was 1.8 ± 0.1 in the control period and 1.6 ± 0.1 after db-cGMP (P < 0.03). [Formula: see text] and [Formula: see text] were 4.58 ± 0.80 and 2.66 ± 0.57 × 10-5 cm/s, respectively, for the control period and 9.48 ± 1.63 (P < 0.007) and 6.01 ± 1.05 (P < 0.005) × 10-5 cm/s, respectively, after db-cGMP. We modeled NO´s effect on luminal Na+ concentration along the thick ascending limb. We found that NO´s effect on the paracellular pathway reduces net Na+ reabsorption and that the magnitude of this effect is similar to that due to NO´s inhibition of transcellular transport. Fil: Monzón, Casandra Margarita. Case Western Reserve University; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Occhipinti, Rossana. Case Western Reserve University; Estados Unidos Fil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Garvin, Jeffrey L.. Case Western Reserve University; Estados Unidos

Calcineurin dephosphorylates nuclear factor of activated T cells transcription factors, thereby facilitating T cell-mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation but may cause side effects, including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar rats, AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured thick ascending limb cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial action of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.

We have previously shown that TNF-α produced by renal epithelial cells inhibits Na+-K+-2Cl− cotransporter (NKCC2) activity as part of a mechanism that attenuates increases in blood pressure in response to high NaCl intake. As the role of TNF-α in the kidney is still being defined, the effects of low salt intake on TNF-α and NKCC2B expression were determined. Mice given a low-salt (0.02% NaCl) diet (LSD) for 7 days exhibited a 62 ± 7.4% decrease in TNF-α mRNA accumulation in the renal cortex. Mice that ingested the LSD also exhibited an ~63% increase in phosphorylated NKCC2 expression in the cortical thick ascending limb of Henle’s loop and a concomitant threefold increase in NKCC2B mRNA abundance without a concurrent change in NKCC2A mRNA accumulation. NKCC2B mRNA levels increased fivefold in mice that ingested the LSD and also received an intrarenal injection of a lentivirus construct that specifically silenced TNF-α in the kidney (U6-TNF-ex4) compared with mice injected with control lentivirus. Administration of a single intrarenal injection of murine recombinant TNF-α (5 ng/g body wt) attenuated the increases of NKCC2B mRNA by ~50% and inhibited the increase in phosphorylated NKCC2 by ~54% in the renal cortex of mice given the LSD for 7 days. Renal silencing of TNF-α decreased urine volume and NaCl excretion in mice given the LSD, effects that were reversed when NKCC2B was silenced in the kidney. Collectively, these findings demonstrate that downregulation of renal TNF-α production in response to low-salt conditions contributes to the regulation of NaCl reabsorption via an NKCC2B-dependent mechanism.

Stable and reproducible kidney cellular models could accelerate our understanding of diseases, help therapeutics development, and improve nephrotoxicity screenings. Generation of a reproducible in vitro kidney models has been challenging owing to the cellular heterogeneity and structural complexity of the kidney. We generated mixed immortalized cell lines that stably maintained their characteristic expression of renal epithelial progenitor markers for the different lineages of kidney cellular compartments via the BMP7 signaling pathway from a mouse and a human whole kidney. These cells were used to generate functional and matured kidney spheroids containing multiple renal lineages, such as the proximal tubule, loop of Henle, distal tubules, and podocytes, using extracellular matrix and physiological force, named spheroid-forming unit (SFU). They expressed all apical and basolateral transporters that are important for drug metabolism and displayed key functional aspects of the proximal tubule, including protein endocytosis and increased gamma-glutamyltransferase activity, and cyclic AMP responded to external cues, such as parathyroid hormone. Following exposure, cells fluxed and took up drugs via proximal tubule-specific apical or basolateral transporters, and displayed increased cell death and expression of renal injury marker. Here, we developed a new differentiation method to generate kidney spheroids that structurally recapitulate important features of the kidney effectively and reproducibly using mixed immortalized renal cells, and showed their application for renal toxicity studies.

The structure of the kidneys of sparrows living in wet and arid zones was compared to each other. A stereological study was conducted to quantify the components of the bird’s kidneys. A total of 10 female sparrows living in wet and arid zones were collected. The length of the kidneys was measured before blocking. The transverse serial sections were taken with a thickness of 5 I¼m. The sections were stained with haematoxylin and eosin (H & E) as well as periodic acid Schiff- alcian blue (PAS- AL). The kidneys of the sparrows consisted mostly of a cortex with only a small portion as medulla. The medulla was arranged in the form of cones with different lengths, which were randomly distributed within the kidney. The medullary nephron tubules were arranged sequentially. Thick and thin limbs of loop of Henle were separated by the collecting ducts. The histological structure and absolute volumes of the kidneys and components of the nephron were quantified. The absolute volumes of the structures within the right and left kidneys were not significantly different. The volume of the whole kidney (p

LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-extracellular signal-regulated kinase (ERK) pathway that is upregulated by sepsis. Pretreatment with the nontoxic immunomodulator monophosphoryl lipid A (MPLA) prevents inhibition by LPS through activation of a TLR4-TIR-domain-containing adaptor-inducing interferon-β (TRIF)-phosphatidylinositol 3-kinase (PI3K) pathway that prevents LPS-induced ERK activation. Here, we identified the molecular mechanisms that underlie the protective inhibitory interaction between the MPLA-PI3K and LPS-ERK pathways. Treatment of mouse MTALs with LPS in vitro increased phosphorylation of IL-1 receptor-associated kinase (IRAK)-1, a critical mediator of LPS signaling downstream of TLR4-MyD88. Activation of ERK by LPS was eliminated by a selective IRAK-1 inhibitor, establishing IRAK-1 as the upstream mediator of ERK activation. Pretreatment of MTALs with MPLA in vitro prevented LPS-induced IRAK-1 activation; this effect was dependent on PI3K. Treatment of MTALs with MPLA increased expression of Toll-interacting protein (Tollip), an inducible protein that negatively regulates LPS signaling by inhibiting IRAK-1. The MPLA-induced increase in Tollip protein level was prevented by PI3K inhibitors. In coimmunoprecipitation experiments, MPLA increased the amount of Tollip stably bound to IRAK-1, an interaction that inhibits IRAK-1 activation. These results support a mechanism whereby MPLA increases Tollip expression in the MTAL through a PI3K-dependent pathway. Tollip, in turn, inhibits LPS-induced TLR4 signaling by suppressing activation of IRAK-1, thereby preventing activation of ERK that inhibits [Formula: see text] absorption. These studies show that MPLA induces reprogramming of MTAL cells that protects against LPS stimulation and identify IRAK-1 and Tollip as new therapeutic targets to prevent renal tubule dysfunction in response to infectious and inflammatory stimuli.

In rats, intramuscular injection of oxytocin (0.25 nmol/100 g body weight) increased sodium excretion from 19±5 to 120±11 μmol/min. A significant correlation (p

The apical Na+-K+-2Cl− cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ([Formula: see text]) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges [Formula: see text] and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that [Formula: see text] stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with [Formula: see text] produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% ( P < 0.05). [Formula: see text]-stimulated surface NKCC2 expression was blocked by the [Formula: see text] scavenger tempol (50 µM). Scavenging H2O2 with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, P < 0.05). Inhibition of endogenous NO production with Nω-nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% ( P < 0.05). Scavenging [Formula: see text] with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, P < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked [Formula: see text]-stimulated surface NKCC2 expression ( P < 0.05). [Formula: see text] did not affect NKCC2 phosphorylation at Thr96/101 or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that [Formula: see text] increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. [Formula: see text]-stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.

The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir, Kir1.1) channels. It is primarily expressed in two regions of the kidney, the cortical collecting duct (CCD) and the thick ascending loop of Henle (TALH). At the CCD it tightly regulates potassium secretion while controlling potassium recycling in TALH. As loss-of-function mutations lead to salt wasting and low blood pressure, it has been surmised that inhibitors of ROMK would represent a target for new and improved diuretics for the treatment of hypertension and heart failure. In this review, we discuss and provide an overview of the medicinal chemistry approaches toward the development of small molecule ROMK inhibitors over the past decade.

Hv1 is a voltage-gated proton channel highly expressed in immune cells where, it acts to maintain NAD(P)H oxidase activity during the respiratory burst. We have recently reported that Hv1 is expressed in cells of the medullary thick ascending limb (mTAL) of the kidney and is critical to augment reactive oxygen species (ROS) production by this segment. While Hv1 is associated with NOX2 mediated ROS production in immune cells, the source of the Hv1 dependent ROS in mTAL remains unknown. In the current study, the rate of ROS formation was quantified in freshly isolated mTAL using dihydroethidium and ethidium fluorescence. Hv1 dependent ROS production was stimulated by increasing bath osmolality and ammonium chloride (NH4Cl) loading. Loss of either p67phox or NOX4 did not abolish the formation of ROS in mTAL. Hv1 was localized to mitochondria within mTAL, and the mitochondrial superoxide scavenger mitoTEMPOL reduced ROS formation. Rotenone significantly increased ROS formation and decreased mitochondrial membrane potential in mTAL from wild-type rats, while treatment with this inhibitor decreased ROS formation and increased mitochondrial membrane potential in mTAL from Hv1−/− mutant rats. These data indicate that NADPH oxidase is not the primary source of Hv1 dependent ROS production in mTAL. Rather Hv1 localizes to the mitochondria in mTAL and modulates the formation of ROS by complex I. These data provide a potential explanation for the effects of Hv1 on ROS production in cells independent of its contribution to maintenance of cell membrane potential and intracellular pH., Graphical abstract Image 1

Sodium-glucose cotransporter type-2 inhibitors (SGLT2is) reduce glomerular hyperfiltration in diabetic people with early diabetic nephropathy. The objective of this report was to assess changes in glomerular filtration rate in healthy cats after treatment with a SGLT2i. Eight healthy research adult castrated male cats were used in a randomized, controlled, cross-over study design. We induced isolated renal tubular glucosuria by dosing cats with the SGLT2i dapagliflozin. The cats received by mouth 10 mg dapagliflozin or control every 24 h in each of the 4, 5-d trial periods that were separated by a 7-d washout period. We assessed glomerular filtration rate (iohexol clearance method), serum urea, creatinine, symmetric dimethylarginine, and 24-h sodium and chloride urinary excretion on the fifth day of each trial period. We analyzed the data with a mixed linear model that included the fixed effects of treatment (treated and control) and trial period, and the random effect of the cat. Compared with controls, cats treated with dapagliflozin had a significant increase in mean (±SE) glomerular filtration rate (3.1 ± 0.2 vs 2.5 ± 0.2 mL/kg/min; P = 0.01), whereas there were no significant differences in serum urea, creatinine and symmetric dimethylarginine, and 24-h urine sodium and chloride excretion. We propose that dapagliflozin-mediated delivery of sodium and glucose distal from the proximal convoluted tubule induced compensatory increased sodium absorption at the thick ascending loop of Henle that resulted in decreased sodium delivery to the distal tubule leading to tubuloglomerular feedback–mediated glomerular hyperfiltration. Future studies should determine if SGLT2is' renoprotective effect in people can be enhanced with the addition of a Na+-K+-Cl− diuretic and whether dapagliflozin will be useful in mitigating proteinuria and hypertension that follow glomerular hyperfiltration in diabetic companion animals in a similar mechanism as in people.

Fil: Ibeh, Cliff-Lawrence. Veterans Affairs Medical Center. Research Service Center. Calcium Signaling Laboratory; Estados Unidos Fil: Yiu, Allen J. Veterans Affairs Medical Center. Research Service Center. Calcium Signaling Laboratory; Estados Unidos Fil: Yiu, Allen J. The George Washington University. Division of Renal Diseases & Hypertension. Department of Medicine; Estados Unidos Fil: Kanaras, Yianni L. Veterans Affairs Medical Center. Research Service Center. Calcium Signaling Laboratory; Estados Unidos Fil: Paal, Edina. Veterans Affairs Medical Center. Pathology and Laboratory Service; Estados Unidos Fil: Birnbaumer, Lutz. Research Triangle Park. National Institute of Environmental Health Sciences. Division of Intramural Research; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jose, Pedro A. The George Washington University. Division of Renal Diseases & Hypertension. Department of Medicine; Estados Unidos Fil: Jose, Pedro A. The George Washington University. Department of Pharmacology and Physiology; Estados Unidos Fil: Bandyopadhyay, Bidhan C. Veterans Affairs Medical Center. Research Service Center. Calcium Signaling Laboratory; Estados Unidos Fil: Bandyopadhyay, Bidhan C. The George Washington University. Division of Renal Diseases & Hypertension. Department of Medicine; Estados Unidos Fil: Bandyopadhyay, Bidhan C. The George Washington University. Department of Pharmacology and Physiology; Estados Unidos Abstract: Calcium phosphate (CaP) crystals, which begin to form in the early segments of the loop of Henle (LOH), are known to act as precursors for calcium stone formation. The proximal tubule (PT), which is just upstream of the LOH and is a major site for Ca2+ reabsorption, could be a regulator of such CaP crystal formation. However, PT Ca2+ reabsorption is mostly described as being paracellular. Here, we show the existence of a regulated transcellular Ca2+ entry pathway in luminal membrane PT cells induced by Ca2+-sensing receptor (CSR, also known as CASR)-mediated activation of transient receptor potential canonical 3 (TRPC3) channels. In support of this idea, we found that both CSR and TRPC3 are physically and functionally coupled at the luminal membrane of PT cells. More importantly, TRPC3-deficient mice presented with a deficiency in PT Ca2+ entry/transport, elevated urinary [Ca2+], microcalcifications in LOH and urine microcrystals formations. Taken together, these data suggest that a signaling complex comprising CSR and TRPC3 exists in the PT and can mediate transcellular Ca2+ transport, which could be critical in maintaining the PT luminal [Ca2+] to mitigate formation of the CaP crystals in LOH and subsequent formation of calcium stones.

Mitogen-activated protein kinases (MAPKs) are intracellular molecules regulating a wide range of cellular functions, including proliferation, differentiation, apoptosis, cytoskeleton remodeling and cytokine production. MAPK activity has been shown in normal kidney, and its over-activation has been demonstrated in several renal diseases. The extracellular signal-regulated protein kinases (ERK 1,2) signalling pathway is the first described MAPK signaling. Intensive investigations have demonstrated that it participates in the regulation of ureteric bud branching, a fundamental process in establishing final nephron number; in addition, it is also involved in the differentiation of the nephrogenic mesenchyme, indicating a key role in mammalian kidney embryonic development. In the present manuscript, we show that ERK1,2 signalling mediates several cellular functions also in mature kidney, describing its role along the nephron and demonstrating whether it contributes to the regulation of ion channels and transporters implicated in acid-base and electrolytes homeostasis.