Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.

Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1ß, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1ß. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.

White adipose tissue mitochondrial bioenergetics in metabolic diseases.

White adipose tissue (WAT) is an important endocrine organ that regulates systemic energy metabolism. In metabolically unhealthy obesity, adipocytes become dysfunctional through hypertrophic mechanisms associated with a reduced endocrine function, reduced mitochondrial function, but increased inflammation, fibrosis, and extracellular remodelling. A pathologic WAT remodelling promotes systemic lipotoxicity characterized by fat accumulation in tissues such as muscle and liver, leading to systemic insulin resistance and type 2 diabetes. Several lines of evidence from human and animal studies suggest a link between unhealthy obesity and adipocyte mitochondrial dysfunction, and interventions that improve mitochondrial function may reduce the risk of obesity-associated diseases. This review discusses the importance of mitochondrial function and metabolism in human adipocyte biology and intercellular communication mechanisms within WAT. Moreover, a selected interventional approach for better adipocyte mitochondrial metabolism in humans is reviewed. A greater understanding of mitochondrial bioenergetics in WAT might provide novel therapeutic opportunities to prevent or restore dysfunctional adipose tissue in obesity-associated diseases.

Acute Kidney Injury by Ischemia/Reperfusion and Extracellular Vesicles.

Acute kidney injury (AKI) is often caused by ischemia-reperfusion injury (IRI). IRI significantly affects kidney metabolism, which elicits pro-inflammatory responses and kidney injury. The ischemia/reperfusion of the kidney is associated with transient high mitochondrial-derived reactive oxygen species (ROS) production rates. Excessive mitochondrial-derived ROS damages cellular components and, together with other pathogenic mechanisms, elicits a range of acute injury mechanisms that impair kidney function. Mitochondrial-derived ROS production also stimulates epithelial cell secretion of extracellular vesicles (EVs) containing RNAs, lipids, and proteins, suggesting that EVs are involved in AKI pathogenesis. This literature review focuses on how EV secretion is stimulated during ischemia/reperfusion and how cell-specific EVs and their molecular cargo may modify the IRI process. Moreover, critical pitfalls in the analysis of kidney epithelial-derived EVs are described. In particular, we will focus on how the release of kidney epithelial EVs is affected during tissue analyses and how this may confound data on cell-to-cell signaling. By increasing awareness of methodological pitfalls in renal EV research, the risk of false negatives can be mitigated. This will improve future EV data interpretation regarding EVs contribution to AKI pathogenesis and their potential as biomarkers or treatments for AKI.

Interleukin 17A infusion has no acute or long-term hypertensive action in conscious unrestrained male mice.

Interleukin 17A (IL-17A) is a candidate mediator of inflammation-driven hypertension, but its direct effect on blood pressure is obscure. The present study was designed to test the hypothesis that systemic IL-17A concentration-dependently increases blood pressure and amplifies ANGII-induced hypertension in mice. Blood pressure was measured by indwelling chronic femoral catheters before and during IL-17A infusion w/wo angiotensin II (ANGII, 60ng/kg/min) in male FVB/n mice. Baseline blood pressure was recorded, and three experimental series were conducted: (1) IL-17A infusion with increasing concentrations over 6 days (two series with IL-17A from two vendors, n = 11); (2) ANGII infusion with IL-17A or vehicle for 9 days (n = 11); and (3) acute bolus infusions with four different concentrations (n = 5). Plasma IL-17A and IL-6 concentrations were determined by ELISA. Mean arterial and systolic blood pressures (MAP, SBP) decreased significantly after IL-17A infusion while heart rate was unchanged. In these mice, plasma IL-17A and IL-6 concentrations increased up to 3500- and 2.4-fold, respectively, above baseline. ANGII infusion increased MAP (~ 25 mmHg) and co-infusion of IL-17A attenuated ANGII-induced hypertension by 4.0 mmHg. Here, plasma IL-17A increased 350-fold above baseline. Acute IL-17A bolus infusion did not change blood pressure or heart rate. IL-17A receptor and IL-6 mRNAs were detected in aorta, heart, and kidneys of mice after IL-17A infusion. Nonphysiologically high concentrations of IL-17A reduce baseline blood pressure and increase IL-6 formation in male FVB/n mice. It is concluded that IL-17A is less likely to drive hypertension as the sole cytokine mediator during inflammation in vivo.

Mineralocorticoid receptor blockade with spironolactone has no direct effect on plasma IL-17A and injury markers in urine from kidney transplant patients.

Kidney transplantation is associated with increased risk of cardiovascular morbidity. Interleukin (IL)-17A mediates kidney injury. Aldosterone promotes T helper 17 lymphocyte differentiation and IL-17A production through the mineralocorticoid receptor. In this exploratory, post hoc substudy, it was hypothesized that a 1-yr intervention with the mineralocorticoid receptor antagonist spironolactone lowers IL-17A and related cytokines and reduces epithelial injury in kidney transplant recipients. Plasma and urine samples were obtained from kidney transplant recipients from a double-blind randomized clinical trial testing spironolactone (n = 39) versus placebo (n = 41). Plasma concentrations of cytokines interferon-γ, IL-17A, tumor necrosis factor-α, IL-6, IL-1ß, and IL-10 were determined before and after 1-yr treatment. Urine calbindin-to-creatinine, clusterin-to-creatinine, kidney injury molecule-1-to-creatinine, osteoactivin-to-creatinine, trefoil factor 3 (TFF3)-to-creatinine, and VEGF-to-creatinine ratios were analyzed. Blood pressure and plasma aldosterone concentration at inclusion did not relate to plasma cytokines and injury markers expect for urine TFF3-to-creatinine ratios that correlated positively to blood pressure. None of the cytokines changed in plasma after spironolactone intervention. Plasma IL-17A increased in the placebo-treated group. Spironolactone induced an increase in plasma K+ (0.4 ± 0.4 mmol/L). This increase did not correlate with plasma IL-17A or urine calbindin and TFF3 changes. Ongoing treatment at inclusion with angiotensin-converting enzyme inhibitor and/or ANG II receptor blockers was not associated with changed levels of IL-17A and injury markers and had no effect on the response to spironolactone. Urinary calbindin and TFF3 decreased in the spironolactone-treated group with no difference in between-group analyses. In conclusion, irrespective of ongoing ANG II inhibition, spironolactone has no effect on plasma IL-17A and related cytokines or urinary injury markers in kidney transplant recipients.NEW & NOTEWORTHY The mineralocorticoid receptor antagonist spironolactone had no direct anti-inflammatory effects on prohypertensive interleukin-17A or distal nephron epithelial injury markers in kidney transplant recipients.

Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients.

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of ≥300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Na+-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.NEW & NOTEWORTHY The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation, and apical membrane attack in kidney transplant recipients. C3dg and C5b-9-associated C9 neoantigen associate with proximal tubular apical membranes as demonstrated in urine extracellular vesicles. The discovery suggests intratubular complement as a mediator between proteinuria and progressive kidney damage. Inhibitors of soluble and/or luminal complement activation with access to the tubular lumen may be beneficial.

Detection of DZIP1L mutations by whole-exome sequencing in consanguineous families with polycystic kidney disease.

BACKGROUND: Autosomal recessive polycystic kidney disease is a cystic kidney disease with early onset and clinically characterized by enlarged echogenic kidneys, hypertension, varying degrees of kidney dysfunction, and liver fibrosis. It is most frequently caused by sequence variants in the PKHD1 gene, encoding fibrocystin. In more rare cases, sequence variants in DZIP1L are seen, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L). So far, only four different DZIP1L variants have been reported. METHODS: Four children from three consanguineous families presenting with polycystic kidney disease were selected for targeted or untargeted exome sequencing. RESULTS: We identified two different, previously not reported homozygous DZIP1L sequence variants: c.193 T > C; p.(Cys65Arg), and c.216C > G; p.(Cys72Trp). Functional analyses of the c.216C > G; p.(Cys72Trp) variant indicated mislocalization of mutant DZIP1L. CONCLUSIONS: In line with published data, our results suggest a critical role of the N-terminal domain for proper protein function. Although patients with PKHD1-associated autosomal recessive polycystic kidney disease often have liver abnormalities, none of the present four patients showed any clinically relevant liver involvement. Our data demonstrate the power and efficiency of next-generation sequencing-based approaches. While DZIP1L-related polycystic kidney disease certainly represents a rare form of the disease, our results emphasize the importance of including DZIP1L in multigene panels and in the data analysis of whole-exome sequencing for cystic kidney diseases. A higher resolution version of the Graphical abstract is available as Supplementary information.

Renal claudin-14 expression is not required for regulating Mg2+ balance in mice.

The kidneys play a crucial role in maintaining Ca2+ and Mg2+ homeostasis by regulating these minerals' reabsorption. In the thick ascending limb of Henle's loop (TAL), Ca2+ and Mg2+ are reabsorbed through the tight junctions by a shared paracellular pathway formed by claudin-16 and claudin-19. Hypercalcemia activates the Ca2+-sensing receptor (CaSR) in the TAL, causing upregulation of pore-blocking claudin-14 (CLDN14), which reduces Ca2+ and Mg2+ reabsorption from this segment. In addition, a high-Mg2+ diet is known to increase both urinary Mg2+ and Ca2+ excretion. Since Mg2+ may also activate CaSR, we aimed to investigate whether CaSR-dependent increases in CLDN14 expression also regulate urinary Mg2+ excretion in response to hypermagnesemia. Here, we show that a Mg2+-enriched diet increased urinary Mg2+ and Ca2+ excretion in mice; however, this occurred without detectable changes in renal CLDN14 expression. The administration of a high-Mg2+ diet to Cldn14-/- mice did not cause more pronounced hypermagnesemia or significantly alter urinary Mg2+ excretion. Finally, in vitro evaluation of CaSR-driven Cldn14 promoter activity in response to increasing Mg2+ concentrations revealed that Cldn14 expression only increases at supraphysiological extracellular Mg2+ levels. Together, these results suggest that CLDN14 is not involved in regulating extracellular Mg2+ balance following high dietary Mg2+ intake.NEW & NOTEWORTHY Using transgenic models and in vitro assays, this study examined the effect of Mg2+ on regulating urinary excretion of Ca2+ and Mg2+ via activation of the Ca2+-sensing receptor-claudin 14 (CLDN14) pathway. The study suggests that CLDN14 is unlikely to play a significant role in the compensatory response to hypermagnesemia.

Localization and regulation of claudin-14 in experimental models of hypercalcemia.

Variations in the claudin-14 (CLDN14) gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in the kidney is localized exclusively to the thick ascending limbs (TALs), mainly restricted to the cortical and upper medullary portion of the kidney. However, not all cells in the TALs expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16 but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14 showed a restricted localization pattern in the apical domain of select cells of the TAL.

Mechanisms of sodium retention in nephrotic syndrome.

PURPOSE OF REVIEW: Proteinuria in nephrotic syndrome is associated with sodium retention and edema. Recent studies from mice, rats and humans have shown that the sodium retention depends on urinary serine proteases and that it can be mitigated by blockers (amiloride, triamterene) of the epithelial sodium channel ENaC. The present review outlines the mechanisms of protease-stimulated sodium retention during proteinuric diseases. RECENT FINDINGS: Inhibition of protease activity in nephrotic mice using aprotinin alleviates sodium retention. From both human and mice studies, an increased proteolytic cleavage of the γENaC subunit plays a role in ENaC activation. In animal models, urokinase-plasmin contributes but not as sole mediators of sodium retention. Across experimental models, human case reports and small intervention trials, amiloride alleviates nephrotic sodium retention and low-renin hypertension with high efficacy. SUMMARY: Although the exact mechanisms for proteolytic ENaC activation are not resolved, multiple, redundant proteases are involved. Experimental and clinical evidence indicate that aberrant proteolytic ENaC activation is a primary driver of sodium retention in nephrotic syndrome and contributes to hypertension in conditions with low-grade proteinuria. Thus, we foresee increased and personalized use of amiloride treatment of nephrotic and other proteinuric disease patients with associated sodium retention and hypertension.

The epithelial Na+ channel α- and γ-subunits are cleaved at predicted furin-cleavage sites, glycosylated and membrane associated in human kidney.

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.

Dietary Na+ intake in healthy humans changes the urine extracellular vesicle prostasin abundance while the vesicle excretion rate, NCC, and ENaC are not altered.

Low Na+ intake activates aldosterone signaling, which increases renal Na+ reabsorption through increased apical activity of the NaCl cotransporter (NCC) and the epithelial Na+ channel (ENaC). Na+ transporter proteins are excreted in urine as an integral part of cell-derived extracellular vesicles (uEVs). It was hypothesized that Na+ transport protein levels in uEVs from healthy humans reflect their physiological regulation by aldosterone. Urine and plasma samples from 10 healthy men (median age: 22.8 yr) were collected after 5 days on a low-Na+ (70 mmol/day) diet and 5 days on a high-Na+ (250 mmol/day) diet. uEVs were isolated by ultracentrifugation and analyzed by Western blot analysis for EV markers (CD9, CD63, and ALIX), transport proteins (Na+-K+-ATPase α1-subunit, NCC, ENaC α- and γ-subunits, and aquaporin 2), and the ENaC-cleaving protease prostasin. Plasma renin and aldosterone concentrations increased during the low-Na+ diet. uEV size and concentration were not different between diets by tunable resistive pulse sensing. EV markers ALIX and CD9 increased with the low-Na+ diet, whereas CD63 and aquaporin 2 excretion were unchanged. Full-length ENaC γ-subunits were generally not detectable in uEVs, whereas ENaC α-subunits, NCC, and phosphorylated NCC were consistently detected but not changed by Na+ intake. Prostasin increased with low Na+ in uEVs. uEV excretion of transporters was not correlated with blood pressure, urinary Na+ and K+ excretion, plasma renin, or aldosterone. In conclusion, apical Na+ transporter proteins and proteases were excreted in uEVs, and while the excretion rate and size of uEVs were not affected, EV markers and prostasin increased in response to the low-Na+ diet.

In human nephrectomy specimens, the kidney level of tubular transport proteins does not correlate with their abundance in urinary extracellular vesicles.

Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumption for years has been that uEVs might provide a noninvasive liquid biopsy that reflect physiological regulation of transporter protein expression in humans. We hypothesized that protein abundance in human kidney tissue and uEVs are directly related and tested this in paired collections of nephrectomy tissue and urine sample from 12 patients. Kidney tissue was fractioned into total kidney protein, crude membrane (plasma membrane and large intracellular vesicles)-enriched, and intracellular vesicle-enriched fractions as well as sections for immunolabeling. uEVs were isolated from spot urine samples. Antibodies were used to quantify six segment-specific proteins [proximal tubule-expressed Na+-phosphate cotransporters (NaPi-2a), thick ascending limb-expressed Tamm-Horsfall protein and renal outer medullary K+ channels, distal convoluted tubule-expressed NaCl cotransporters, intercalated cell-expressed V-type H+-ATPase subunit G3 (ATP6V1G3), and principal cell-expressed aquaporin 2] and three uEV markers (exosomal CD63, microvesicle marker vesicle-associated membrane protein 3, and ß-actin) in each fraction. By Western blot analysis and immunofluorescence labeling, we found significant positive correlations between the abundance of CD63, NaCl cotransporters, aquaporin 2, and ATP6V1G3, respectively, within the different kidney-derived fractions. We detected all nine proteins in uEVs, but their level did not correlate with kidney tissue protein abundance. uEV protein levels showed higher interpatient variability than kidney-derived fractions, indicating that factors, besides kidney protein abundance, contribute to the uEV protein level. Our data suggest that, in a random sample of nephrectomy patients, uEV protein level is not a predictor of kidney protein abundance.

Nephrotic syndrome is associated with increased plasma K+ concentration, intestinal K+ losses, and attenuated urinary K+ excretion: a study in rats and humans.

The present study tested the hypotheses that nephrotic syndrome (NS) leads to renal K+ loss because of augmented epithelial Na+ channel (ENaC) activity followed by downregulation of renal K+ secretory pathways by suppressed aldosterone. The hypotheses were addressed by determining K+ balance and kidney abundance of K+ and Na+ transporter proteins in puromycin aminonucleoside (PAN)-induced rat nephrosis. The effects of amiloride and angiotensin II type 1 receptor and mineralocorticoid receptor (MR) antagonists were tested. Glucocorticoid-dependent MR activation was tested by suppression of endogenous glucocorticoid with dexamethasone. Urine and plasma samples were obtained from pediatric patients with NS in acute and remission phases. PAN-induced nephrotic rats had ENaC-dependent Na+ retention and displayed lower renal K+ excretion but elevated intestinal K+ secretion that resulted in less cumulated K+ in NS. Aldosterone was suppressed at day 8. The NS-associated changes in intestinal, but not renal, K+ handling responded to suppression of corticosterone, whereas angiotensin II type 1 receptor and MR blockers and amiloride had no effect on urine K+ excretion during NS. In PAN-induced nephrosis, kidney protein abundance of the renal outer medullary K+ channel and γ-ENaC were unchanged, whereas the Na+-Cl- cotransporter was suppressed and Na+-K+-ATPase increased. Pediatric patients with acute NS displayed suppressed urine Na+-to-K+ ratios compared with remission and elevated plasma K+ concentration, whereas fractional K+ excretion did not differ. Acute NS is associated with less cumulated K+ in a rat model, whereas patients with acute NS have elevated plasma K+ and normal renal fractional K+ excretion. In NS rats, K+ balance is not coupled to ENaC activity but results from opposite changes in renal and fecal K+ excretion with a contribution from corticosteroid MR-driven colonic secretion.

Hydronephrosis is associated with elevated plasmin in urine in pediatric patients and rats and changes in NCC and γ-ENaC abundance in rat kidney.

Obstruction of urine flow at the level of the pelvo-ureteric junction (UPJO) and subsequent development of hydronephrosis is one of the most common congenital renal malformations. UPJO is associated with development of salt-sensitive hypertension, which is set by the obstructed kidney, and with a stimulated renin-angiotensin-aldosterone system (RAAS) in rodent models. This study aimed at investigating the hypothesis that 1) in pediatric patients with UPJO the RAAS is activated before surgical relief of the obstruction; 2) in rats with UPJO the RAAS activation is reflected by increased abundance of renal aldosterone-stimulated Na transporters; and 3) the injured UPJO kidney allows aberrant filtration of plasminogen, leading to proteolytic activation of the epithelial Na channel γ-subunit (γ-ENaC). Hydronephrosis resulting from UPJO in pediatric patients and rats was associated with increased urinary plasminogen-to-creatinine ratio. In pediatric patients, plasma renin, angiotensin II, urine and plasma aldosterone, and urine soluble prorenin receptor did not differ significantly before or after surgery, or compared with controls. Increased plasmin-to-plasminogen ratio was seen in UPJO rats. Intact γ-ENaC abundance was not changed in UPJO kidney, whereas low-molecular cleavage product abundance increased. The Na-Cl cotransporter displayed significantly lower abundance in the UPJO kidney compared with the nonobstructed contralateral kidney. The Na-K-ATPase α-subunit was unaltered. Treatment with an angiotensin-converting enzyme inhibitor (8 days, captopril) significantly lowered blood pressure in UPJO rats. It is concluded that the RAAS contributes to hypertension following partial obstruction of urine flow at the pelvo-ureteric junction with potential contribution from proteolytic activation of ENaC.

H+-ATPase B1 subunit localizes to thick ascending limb and distal convoluted tubule of rodent and human kidney.

The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.

Albuminuria in kidney transplant recipients is associated with increased urinary serine proteases and activation of the epithelial sodium channel.

Albuminuria predicts adverse renal outcome in kidney transplant recipients. The present study addressed the hypothesis that albuminuria is associated with increased urine serine proteases with the ability to activate the epithelial sodium channel (ENaC) and with greater extracellular volume and higher blood pressure. In a cross-sectional design, kidney transplant recipients with ( n = 18) and without ( n = 19) albuminuria were included for office blood pressure measurements, estimation of volume status by bioimpedance, and collection of spot urine and plasma samples. Urine was analyzed for serine proteases and for the ability to activate ENaC current in vitro. Urine exosome protein was immunoblotted for prostasin and γ-ENaC protein. In the present study, it was found that, compared with nonalbuminuria (8.8 mg/g creatinine), albuminuric (1,722 mg/g creatinine) kidney transplant recipients had a higher systolic and diastolic blood pressure, despite receiving significantly more antihypertensives, and a greater urinary total plasminogen, active plasmin, active urokinase-type plasminogen activator, and prostasin protein abundance, which correlated significantly with u-albumin. Fluid overload correlated with systolic blood pressure, urinary albumin/creatinine, and plasminogen/creatinine. Urine from albuminuric kidney transplant recipients evoked a greater amiloride- and aprotinin-sensitive inward current in single collecting duct cells (murine cell line M1). γENaC subunits at 50 and 75 kDa showed increased abundance in urine exosomes from albuminuric kidney transplant recipients when compared with controls. These findings show that albuminuria in kidney transplant recipients is associated with hypertension, ability of urine to proteolytically activate ENaC current, and increased abundance of γENaC. ENaC activity could contribute to hypertension and adverse outcome in posttransplant proteinuria.

Physiology and pathophysiology of the plasminogen system in the kidney.

The plasminogen system is important for fibrinolysis in addition to tissue remodeling and inflammation with significance for kidney disease. The system consists of the circulating zymogen plasminogen (Plg) and the tissue- and urokinase-type plasminogen activators, tPA and uPA, expressed in the glomeruli, endothelium and tubular epithelium, respectively, and the inhibitors α2-antiplasmin and plasminogen activator inhibitor-type1, PAI-1. Plasminogen is activated by surface receptors, some with renal expression: urokinase-type plasminogen activator receptor (uPAR), plasminogen receptor KT (Plg-RKT), and tPA, most evident in the endothelium. Plasmin may exert effects through protease-activated receptors, PARs, expressed in the kidney. Deletion of plasminogen system component genes confers no major developmental or renal phenotypes in normal mice. In glomerular injury and renal interstitial fibrosis, deletion of various components, notably Plg, uPA, PAI, and uPAR is associated with protection suggesting a disease promoting effect of plasmin, in some cases exerted through PAR1 receptor activation. Plasminogen and uPA are aberrantly filtrated across the glomerular barrier in proteinuria, and plasminogen is activated in the tubular fluid. In the tubular fluid, plasmin may activate proteolytically the epithelial sodium channel (ENaC) and inhibit the apical calcium transporter transient receptor potential cation channel subfamily V member 5 (TRPV5), which could explain impaired sodium excretion and enhanced calcium excretion in proteinuria. Amiloride, a potassium-sparing diuretic, inhibits urokinase and plasmin activation in the tubular fluid and uPAR expression in vitro, which highlights new indications for an old drug. Protease inhibitors lowered blood pressure and antagonized fibrosis in salt-sensitive Dahl rats. Current knowledge indicates that the plasminogen system aggravates renal disease by direct and indirect hypertensive effects and is a promising target to antagonize disease progression.

Urine exosomes from healthy and hypertensive pregnancies display elevated level of α-subunit and cleaved α- and γ-subunits of the epithelial sodium channel-ENaC.

Preeclampsia is characterized by hypertension, proteinuria, suppression of plasma renin-angiotensin-aldosterone, and impaired urine sodium excretion. Aberrantly filtered plasmin in urine may activate proteolytically the γ-subunit of the epithelial sodium channel (ENaC) and promote Na+ reabsorption and urine K+ loss. Plasma and urine was sampled from patients with preeclampsia, healthy pregnant controls and non-pregnant women, and from patients with nephrostomy catheters. Aldosterone concentration, urine plasminogen, and protein were determined. Exosomes were isolated by ultracentrifugation. Immunoblotting was used to detect exosome markers; γ-ENaC (two different epitopes within the inhibitory peptide tract), α-ENaC, and renal outer medullary K-channel (ROMK) and compared with human kidney cortex homogenate. Urine total plasmin(ogen) was significantly increased in preeclampsia, plasma and urine aldosterone was higher in pregnancy compared to non-pregnancy, and the urine Na/K ratio was lower in preeclampsia compared to healthy pregnancy. Exosome markers ALIX and AQP-2 were stably associated with exosomes across groups. Exosomal α-ENaC-subunit migrated at 75 kDa and dominantly at 50 kDa and was significantly elevated in pregnancy. In human kidney cortex tissue and two of four pelvis catheter urine, ~90-100 kDa full-length γ-ENaC was detected while no full-length γ-ENaC but 75, 60, and 37 kDa variants dominated in voided urine exosomes. There was no difference in γ-ENaC protein abundances between healthy pregnancy and preeclampsia. ROMK was detected inconsistently in urine exosomes. Pregnancy and preeclampsia were associated with increased abundance of furin-cleaved α-ENaC subunit while γ-subunit appeared predominantly in cleaved form independently of conditions and with a significant contribution from post-renal cleavage.