The calcium-sensing receptor has only a parathyroid hormone-dependent role in the acute response of renal phosphate transporters to phosphate intake.

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

Expression of claudin-8 is induced by aldosterone in renal collecting duct principal cells.

Fine tuning of Na+ reabsorption takes place along the aldosterone-sensitive distal nephron, which includes the collecting duct (CD), where it is mainly regulated by aldosterone. In the CD, Na+ reabsorption is mediated by the epithelial Na+ channel and Na+ pump (Na+-K+-ATPase). Paracellular ion permeability is mainly dependent on tight junction permeability. Claudin-8 is one of the main tight junction proteins expressed along the aldosterone-sensitive distal nephron. We have previously shown a coupling between transcellular Na+ reabsorption and paracellular Na+ barrier. We hypothesized that aldosterone controls the expression levels of both transcellular Na+ transporters and paracellular claudin-8 in a coordinated manner. Here, we show that aldosterone increased mRNA and protein levels as well as lateral membrane localization of claudin-8 in cultured CD principal cells. The increase in claudin-8 mRNA levels in response to aldosterone was prevented by preincubation with 17-hydroxyprogesterone, a mineralocorticoid receptor antagonist, and by inhibition of transcription with actinomycin D. We also showed that a low-salt diet, which stimulated aldosterone secretion, was associated with increased claudin-8 abundance in the mouse kidney. Reciprocally, mice subjected to a high-salt diet, which inhibits aldosterone secretion, or treated with spironolactone, a mineralocorticoid receptor antagonist, displayed decreased claudin-8 expression. Inhibition of glycogen synthase kinase-3, Lyn, and Abl signaling pathways prevented the effect of aldosterone on claudin-8 mRNA and protein abundance, suggesting that signaling of protein kinases plays a permissive role on the transcriptional activity of the mineralocorticoid receptor. This study shows that signaling via multiple protein kinases working in concert mediates aldosterone-induced claudin-8 expression in the CD.NEW & NOTEWORTHY In this study, we showed that aldosterone modulates claudin-8 expression in cultured collecting duct principal cells and in the mouse kidney. The upregulation of claudin-8 expression in response to aldosterone is dependent on at least glycogen synthase kinase-3, Lyn, and Abl signaling pathways, indicating the participation of multiple protein kinases to the effect of aldosterone.

Effects of SGLT2 inhibitor and dietary NaCl on glomerular hemodynamics assessed by micropuncture in diabetic rats.

Inhibitors of the main proximal tubular Na-glucose cotransporter (SGLT2) mitigate diabetic glomerular hyperfiltration and have been approved by the United States Food and Drug Administration for slowing the progression of diabetic kidney disease. It has been proposed that SGLT2 inhibitors improve hard renal outcomes by reducing glomerular capillary pressure (PGC) via a tubuloglomerular feedback (TGF) response to a decrease in proximal reabsorption (Jprox). However, the effect of SGLT2 inhibition on PGC has not been measured. Here, we studied the effects of acute SGLT2 blockade (ertugliflozin) on Jprox and glomerular hemodynamics in two-period micropuncture experiments using streptozotocin-induced diabetic rats fed high- or low-NaCl diets. PGC was measured by direct capillary puncture or computed from tubular stop-flow pressure (PSF). TGF is intact while measuring PGC directly but rendered inoperative when measuring PSF. Acute SGLT2 inhibitor reduced Jprox by ∼30%, reduced PGC by 5-8 mmHg, and reduced glomerular filtration rate (GFR) by ∼25% (all P < 0.0001) but had no effect on PSF. The decrease in PGC was larger with the low-NaCl diet (8 vs. 5 mmHg, P = 0.04) where PGC was higher to begin with (54 vs. 50 mmHg, P = 0.003). Greater decreases in PGC corresponded, unexpectedly, to lesser decreases in GFR (P = 0.04). In conclusion, these results confirm expectations that PGC would decline in response to acute SGLT2 inhibition and that a functioning TGF system is required for this. We infer a contribution of postglomerular vasorelaxation to the TGF responses where decreases in PGC were large and decreases in GFR were small.NEW & NOTEWORTHY It has been theorized that Na-glucose cotransporter (SGLT2) blockade slows progression of diabetic kidney disease by reducing physical strain on the glomerulus. This is the first direct measurement of intraglomerular pressure during SGLT2 blockade. Findings confirmed that SGLT2 blockade does reduce glomerular capillary pressure, that this is mediated through tubuloglomerular feedback, and that the tubuloglomerular feedback response to SGLT2 blockade involves preglomerular vasoconstriction and postglomerular vasorelaxation.

Comparative study of a novel selective urate reabsorption inhibitor "dotinurad" among patient groups with different stages of renal dysfunction.

BACKGROUND: Dotinurad is a selective urate reabsorption inhibitor (SURI), which selectively inhibits URAT1 to lower serum uric acid levels in patients with hyperuricemia. Herein, the effects of dotinurad were compared among patient groups with different stages of renal dysfunction. METHODS: Patient data from four clinical trials were pooled and divided into four groups according to the stage of renal dysfunction to compare the effects of dotinurad at different stages. The grouping (stages G1-G3b) was based on the estimated glomerular filtration rate (eGFR) of the patients. In addition, patient data from a long-term study (34 or 58 weeks) were evaluated in the same manner. RESULTS: In the pooled analysis, the percentage of patients achieving a serum uric acid level of ≤ 6.0 mg/dL was 64.7-100.0% at a dose of 2 or 4 mg. In the long-term analysis, the percentage of patients achieving a serum uric acid level of ≤ 6.0 mg/dL was 60.0-100.0% at a dose of 2 or 4 mg. Although the outcomes in stage G3b were worse due to higher baseline serum uric acid levels, satisfactory outcomes were observed in all stages. Even in stages G3a and G3b, when renal function declined, the eGFR remained constant throughout the dose period. CONCLUSION: The efficacy of dotinurad was confirmed in hyperuricemic patients with normal renal function (stage G1) and mild to moderate renal dysfunction (stage G2-G3b). Dotinurad was found to be effective in the treatment of hyperuricemia in patients with mild to moderate renal dysfunction.

Effect of sodium-glucose cotransporter-2 inhibitors on renal handling of electrolytes.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are the latest introduction into the armamentarium of diabetes care in the present decade. By virtue of their beneficial effects, such as blood pressure-lowering, bodyweight reduction and significant renal and cardioprotective effects which extends beyond their glycaemic control effects, SGLT2i have become one of the most preferred oral antihyperglycaemic agents of recent times. However, they can influence tubular handling of electrolytes that can result in some electrolyte disturbances such as alteration in the serum levels of magnesium, potassium and phosphate levels. Some of these changes are mild or transient and may not have significant clinical implications. The underlying putative mechanism(s) responsible for disturbances of electrolytes are yet to be deciphered. In this review, we aim to describe electrolytes and acid-base abnormalities due to SGLT2i as well as to elucidate the underlying mechanism.

Dietary fructose enhances angiotensin II-stimulated Na+ transport via activation of PKC-α in renal proximal tubules.

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II's ability to stimulate Na+ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation and increases in intracellular Ca2+. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na+ reabsorption, and intracellular Ca2+ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-ß, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca2+ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (-15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (-0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group (P < 0.046). ANG II did not stimulate PKC-ß in either group. ANG II increased Na+ transport by 454 ± 87 nmol·min-1·mg protein-1 in fructose group, and the PKC-α/ß inhibitor Gö6976 blocked this increase (-96 ± 205 nmol·min-1·mg protein-1, P < 0.045). ANG II increased intracellular Ca2+ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group (P < 0.035). The intracellular Ca2+ chelator BAPTA blocked the ANG II-induced increase in Na+ transport in the fructose group. We concluded that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca2+.

Adenosine inhibits the basolateral Cl- ClC-K2/b channel in collecting duct intercalated cells.

Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A1 receptors (A1Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K+ conductance mediated by Kir4.1/5.1 channels to mediate K+ recycling and to set up a favorable driving force for Na+/K+ exchange (47). Intercalated cells express the Cl- ClC-K2/b channel mediating transcellular Cl- reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on Kir4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A1R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A1R agonist N6-cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and Kir4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A1Rs. We propose that inhibition of transcellular Cl- reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.

Competing Effects of Renin Angiotensin System Blockade and Sodium-Glucose Cotransporter-2 Inhibitors on Erythropoietin Secretion in Diabetes.

BACKGROUND: Anaemia is a common finding in diabetes, particularly in those patients with albuminuria or renal dysfunction and is associated with impaired erythropoietin (EPO) secretion. This review focuses on mechanisms involved in the regulation of erythropoiesis in diabetic patients in an effort to elucidate the competing effects of the renin angiotensin system (RAS) blockade and sodium-glucose cotransporter-2 (SGLT2) inhibitors on haemoglobin concentration and hematocrit values. SUMMARY: The RAS shows significant activation in diabetic subjects. Angiotensin II, its active octapeptide, causes renal tubulointerstitial hypoxia, which stimulates hypoxia-inducible factors (HIF) and increases EPO secretion and erythropoiesis. As expected, drugs that inactivate RAS, such as angiotensin converting enzyme inhibitors or angiotensin receptor blockers (ACEi/ARB) are associated with a significant hematocrit-lowering effect and/or anaemia in various clinical conditions, including diabetes. Dual blockade by a combination of ACEi and ARB in diabetic patients achieves a better RAS inhibition, but at the same time a worse drop of haemoglobin concentration. Increased glucose reabsorption by SGLTs in diabetic subjects generates a high-glucose environment in renal tubulointerstitium, which may impair HIF-1, damage renal erythropoietin-producing cells (REPs) and decrease EPO secretion and erythropoiesis. SGLT2 inhibitors, which inhibit glucose reabsorption, may attenuate glucotoxicity in renal tubulointerstitium, allowing REPs to resume their function and increase EPO secretion. Indeed, EPO levels increase within a few weeks after initiation of therapy with all known SGLT2 inhibitors, followed by increased reticulocyte count and a gradual elevation of haemoglobin concentration and hematocrit level, which reach zenith values after 2-3 months. Key Messages: The competing effects of RAS blockade and SGLT2 inhibitors on erythropoiesis may have important clinical implications. The rise of hematocrit values by SGLT2 inhibitors given on top of RAS blockade in recent outcome trials may significantly contribute to the cardiorenal protection attained. The relative contribution of each system to erythropoiesis and outcome remains to be revealed in future studies.

Mineralized tissues in hypophosphatemic rickets.

Hypophosphatemic rickets is caused by renal phosphate wasting that is most commonly due to X-linked dominant mutations in PHEX. PHEX mutations cause hypophosphatemia indirectly, through the increased expression of fibroblast growth factor 23 (FGF23) by osteocytes. FGF23 decreases renal phosphate reabsorption and thereby increases phosphate excretion. The lack of phosphate leads to a mineralization defect at the level of growth plates (rickets), bone tissue (osteomalacia), and teeth, where the defect facilitates the formation of abscesses. The bone tissue immediately adjacent to osteocytes often remains unmineralized ("periosteocytic lesions"), highlighting the osteocyte defect in this disorder. Common clinical features of XLH include deformities of the lower extremities, short stature, enthesopathies, dental abscesses, as well as skull abnormalities such as craniosynostosis and Chiari I malformation. For the past four decades, XLH has been treated by oral phosphate supplementation and calcitriol, which improves rickets and osteomalacia and the dental manifestations, but often does not resolve all aspects of the mineralization defects. A newer treatment approach using inactivating FGF23 antibodies leads to more stable control of serum inorganic phosphorus levels and seems to heal rickets more reliably. However, the long-term benefits of FGF23 antibody treatment remain to be elucidated.

Olsalazine Sodium Increases Renal Urate Excretion by Modulating Urate Transporters in Hyperuricemic Animals.

Hyperuricemia is mainly the result of relative underexcretion of urate. Urate is mainly eliminated by kidney and several important transporters expressed on the membrane of renal tubular cells involved in urate excretion. Olsalazine sodium was screened from 3167 authorized small compounds/drugs, targeting xanthine oxidoreductase. In previous study, we reported that olsalazine sodium significantly reduced the serum urate levels, and the anti-hyperuricemic activity linked with inhibiting urate formation by reducing the activity of xanthine oxidoreductase. The current research aimed to assess olsalazine sodium renal urate excretion and likely molecular mechanism. The results showed that administration of olsalazine sodium 5.0 mg/kg decreased the levels of serum urate in hyperuricemic rats, and noticeably improved the fractional excretion of urate and urate clearance, exhibiting an uricosuric action. Moreover, olsalazine sodium (2.5, 5.0, 10.0 mg/kg) reduced the level of blood urea nitrogen in rats. Further study showed that olsalazine sodium reduced the mRNA expression of urate reabsorptive transporter glucose transporter 9 (GLUT9), increased the mRNA expression of urate secretory transporters, organic anion transporter 1 (OAT1), OAT3 and type 1 sodium-dependent phosphate transporter (NPT1) as well as the protein expression of OAT3 in the kidney in hyperuricemic mice. In conclusion, olsalazine sodium exhibited a promotion of urate excretion in kidney by increasing the expression of OAT3.

Sodium-glucose co-transporters and diabetic nephropathy: Is there a link with toll-like receptors?

The incidence of diabetes mellitus (DM) has increased alarmingly over the last decades. Despite taking measures aimed at controlling hyperglycaemia and blood pressure, the rate of end-stage renal disease (ESRD) is continually growing. Upon increased amounts of advanced glycation end products (AGEs) and their correspondent receptors (RAGEs), AGE-RAGE axis is over-activated in DM, being the first step in the initiation and propagation of inflammatory cascades. Meanwhile, HMGB1, released from damaged cells in the diabetic kidneys, is the most notable ligand for the highly expressed toll-like receptors (TLRs) and RAGEs. TLRs play an indispensable role in the pathogenesis of diabetic nephropathy. Sodium-glucose co-transporter 2 (SGLT-2) inhibitors are hypoglycaemic agents acting on the renal proximal tubules to prevent glucose reabsorption and therefore increase urinary glucose excretion. Besides improving glycaemic control, these hypoglycaemic agents possess direct renoprotective properties. Here, therefore, we review the most recent findings regarding interrelationship between SGLT2 inhibitors and HMGB1-TLR4 axis.

Anti-Hyperuricemic Effects of Astaxanthin by Regulating Xanthine Oxidase, Adenosine Deaminase and Urate Transporters in Rats.

This study was designed to investigate the effects and underlying mechanisms of Astaxanthin (AST) on high-fructose-induced hyperuricemia (HUA) from the perspectives of the uric acid (UA) synthesis and excretion in rat models. Following six weeks of a 10% fructose diet, the level of serum UA effectively decreased in the AST groups as compared to the model group. The enzymatic activities of xanthine oxidase (XOD) and adenosine deaminase (ADA) were significantly inhibited, and the mRNA expression levels of XOD and ADA significantly decreased after the AST administration. These results suggested that the AST reduced UA synthesis by inhibiting the mRNA expressions and enzyme activities of XOD and ADA, thereby contributing to HUA improvement. On the hand, the relative expressions of the mRNA and protein of kidney reabsorption transport proteins (GLUT9 and URAT1) were significantly down-regulated by AST, while that of the kidney secretion proteins (OAT1, OAT3 and ABCG2) were significantly up-regulated by AST. These results indicated that the AST promoted UA excretion by regulating the urate transport proteins, and thus alleviated HUA. This study suggested that the AST could serve as an effective alternative to traditional medicinal drugs for the prevention of fructose-induced HUA.

Narrative review of the effects of antidiabetic drugs on albuminuria.

Diabetes mellitus is the most prevalent metabolic disorder worldwide. Glycemic control is the main focus of antidiabetic therapy. However, there are data suggesting that some antidiabetic drugs may have intrinsic beneficial renal effects and protect against the development and progression of albuminuria, thus minimizing the risk of diabetic nephropathy. These pharmacological agents can suppress upstream molecular pathways involved in the pathophysiology of diabetes-induced renal dysfunction such as oxidative stress, inflammatory responses, and apoptosis. In this narrative review, the pathophysiology of albuminuria in patients with diabetic nephropathy is discussed. Furthermore, the renoprotective effects of antidiabetic drugs, focusing on albuminuria, are reviewed.

Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts.

Hypertonicity increases water permeability, independently of vasopressin, in the inner medullary collecting duct (IMCD) by increasing aquaporin-2 (AQP2) membrane accumulation. We investigated whether protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) are involved in hypertonicity-regulated water permeability. Increasing perfusate osmolality from 150 to 290 mosmol/kgH2O and bath osmolality from 290 to 430 mosmol/kgH2O significantly stimulated osmotic water permeability. The PKC inhibitors chelerythrine (10 µM) and rottlerin (50 µM) significantly reversed the increase in osmotic water permeability stimulated by hypertonicity in perfused rat terminal IMCDs. Chelerythrine significantly increased phosphorylation of AQP2 at S261 but not at S256. Previous studies show that AMPK is stimulated by osmotic stress. We tested AMPK phosphorylation under hypertonic conditions. Hypertonicity significantly increased AMPK phosphorylation in inner medullary tissues. Blockade of AMPK with Compound C decreased hypertonicity-stimulated water permeability but did not alter phosphorylation of AQP2 at S256 and S261. AICAR, an AMPK stimulator, caused a transient increase in osmotic water permeability and increased phosphorylation of AQP2 at S256. When inner medullary tissue was treated with the PKC activator phorbol dibutyrate (PDBu), the AMPK activator metformin, or both, AQP2 phosphorylation at S261 was decreased with PDBu or metformin alone, but there was no additive effect on phosphorylation with PDBu and metformin together. In conclusion, hypertonicity regulates water reabsorption by activating PKC. Hypertonicity-stimulated water reabsorption by PKC may be related to the decrease in endocytosis of AQP2. AMPK activation promotes water reabsorption, but the mechanism remains to be determined. PKC and AMPK do not appear to act synergistically to regulate water reabsorption.

A model of uric acid transport in the rat proximal tubule.

The objective of the present study was to theoretically investigate the mechanisms underlying uric acid transport in the proximal tubule (PT) of rat kidneys, and their modulation by factors, including Na+, parathyroid hormone, ANG II, and Na+-glucose cotransporter-2 inhibitors. To that end, we incorporated the transport of uric acid and its conjugate anion urate in our mathematical model of water and solute transport in the rat PT. The model accounts for parallel urate reabsorption and secretion pathways on apical and basolateral membranes and their coupling to lactate and α-ketoglutarate transport. Model results agree with experimental findings at the segment level. Net reabsorption of urate by the rat PT is predicted to be ~70% of the filtered load, with a rate of urate removal from the lumen that is 50% higher than the rate of urate secretion. The model suggests that apical URAT1 deletion significantly reduces net urate reabsorption across the PT, whereas ATP-binding cassette subfamily G member 2 dysfunction affects it only slightly. Inactivation of basolateral glucose transporter-9 raises fractional urate excretion above 100%, as observed in patients with renal familial hypouricemia. Furthermore, our results suggest that reducing Na+ reabsorption across Na+/H+ exchangers or Na+-glucose cotransporters augments net urate reabsorption. The model predicts that parathyroid hormone reduces urate excretion, whereas ANG II increases it. In conclusion, we have developed the first model of uric acid transport in the rat PT; this model provides a framework to gain greater insight into the numerous solutes and coupling mechanisms that affect the renal handing of uric acid.

Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs.

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.

Nitric oxide mediates anomalous tubuloglomerular feedback in rats fed high-NaCl diet after subtotal nephrectomy.

Tubuloglomerular feedback (TGF) responses become anomalous in rats fed high-NaCl diet after subtotal nephrectomy (STN), such that stimulating TGF causes single nephron GFR (SNGFR) to increase rather than decrease. Micropuncture experiments were performed to determine whether this anomaly results from heightened nitric oxide response to distal delivery, which is a known mechanism for resetting TGF, or from connecting tubule TGF (cTGF), which is a novel amiloride-inhibitable system for offsetting TGF responses. Micropuncture was done in Wistar Froemter rats fed high-NaCl diet (HS) for 8-10 days after STN or sham nephrectomy. TGF was manipulated by orthograde microperfusion of Henle's loop with artificial tubular fluid with or without NOS inhibitor, LNMMA, or the cell-impermeant amiloride analog, benzamil. SNGFR was measured by inulin clearance in tubular fluid collections from the late proximal tubule. TGF responses were quantified as the increase in SNGFR that occurred when the perfusion rate was reduced from 50 to 8 nl/min in STN or 40 to 8 nl/min in sham animals. The baseline TGF response was anomalous in STN HS (-4 ± 3 vs 14 ± 3 nl/min, P < 0.001). TGF response was normalized by perfusing STN nephron with LNMMA (14 ± 3 nl/min, P < 0.005 for ANOVA cross term) but not with benzamil (-3 ± 4 nl/min, P = 0.4 for ANOVA cross term). Anomalous TGF occurs in STN HS due to heightened effect of tubular flow on nitric oxide signaling, which increases to the point of overriding the normal TGF response. There is no role for cTGF in this phenomenon.

SGLT2 inhibition and renal urate excretion: role of luminal glucose, GLUT9, and URAT1.

Inhibitors of the Na+-glucose cotransporter SGLT2 enhance urinary glucose and urate excretion and lower plasma urate levels. The mechanisms remain unclear, but a role for enhanced glucose in the tubular fluid, which may interact with tubular urate transporters, such as the glucose transporter GLUT9 or the urate transporter URAT1, has been proposed. Studies were performed in nondiabetic mice treated with the SGLT2 inhibitor canagliflozin and in gene-targeted mice lacking the urate transporter Glut9 in the tubule or in mice with whole body knockout of Sglt2, Sglt1, or Urat1. Renal urate handling was assessed by analysis of urate in spontaneous plasma and urine samples and normalization to creatinine concentrations or by renal clearance studies with assessment of glomerular filtration rate by FITC-sinistrin. The experiments confirmed the contribution of URAT1 and GLUT9 to renal urate reabsorption, showing a greater contribution of the latter and additive effects. Genetic and pharmacological inhibition of SGLT2 enhanced fractional renal urate excretion (FE-urate), indicating that a direct effect of the SGLT2 inhibitor on urate transporters is not absolutely necessary. Consistent with a proposed role of increased luminal glucose delivery, the absence of Sglt1, which by itself had no effect on FE-urate, enhanced the glycosuric and uricosuric effects of the SGLT2 inhibitor. The SGLT2 inhibitor enhanced renal mRNA expression of Glut9 in wild-type mice, but tubular GLUT9 seemed dispensable for the increase in FE-urate in response to canagliflozin. First evidence is presented that URAT1 is required for the acute uricosuric effect of the SGLT2 inhibitor in mice.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through induction of Tollip and negative regulation of IRAK-1.

LPS inhibits HCO3- 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-ß (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 HCO3- 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.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.