Gitelman's and Bartter's Syndromes: From Genetics to the Molecular Basis of Hypertension and More.

BACKGROUND: Gitelman's and Bartter's syndromes (GS/BS) are rare genetic tubulopathies characterized by electrolyte imbalance and activation of the renin-angiotensin-aldosterone system (RAAS). These syndromes have intriguing biochemical and hormonal abnormalities that lead them to be protected from hypertension and cardiovascular and renal remodeling. SUMMARY: In this review, we explore the biochemical/molecular mechanisms induced by the activation of the RAAS and its counterregulatory arm which is particularly activated in GS/BS patients, in the context of blood pressure regulation. In addition, we report our findings in the context of the COVID-19 pandemic where we observed GS/BS subjects being protected from infection. KEY MESSAGES: The intracellular pathways induced by Ang II, starting from induction of oxidative stress and vasoconstriction, are crucial for the progression toward cardiovascular-renal remodeling and might be useful targets in order to reduce/halt the progression of Ang II/oxidative stress-induced cardiovascular-renal morbidity in several diseases.

Pathophysiology of Post Transplant Hypertension in Kidney Transplant: Focus on Calcineurin Inhibitors Induced Oxidative Stress and Renal Sodium Retention and Implications with RhoA/Rho Kinase Pathway.

Post-transplant hypertension is a common occurrence during treatment with calcineurin inhibitors (CNIs) in kidney transplant population. The pathogenesis of vasoconstriction induced by CNIs involves vascular tone alterations and kidney sodium transport regulation. Among the factors involved a key role is played by the activation of intrarenal renin-angiotensin system with enhanced release of Angiotensin II (Ang II) and increase of oxidative stress. A common pathway between oxidative stress and hypertension induced by CNIs may be identified in the involvement of the activation of RhoA/Rho kinase pathway, key for the induction of hypertension and cardiovascular-renal remodeling, of the oxidative stress mediated increased nitric oxide (NO) metabolism and increased renal sodium retention via increased activity of thiazide-sensitive sodium chloride cotransporter (NCC) in the distal tubule. We examined literature data including those coming from our group regarding the role of oxidative stress and sodium retention in CNIs induced hypertension and their involvement in cardiovascular-renal remodeling. Based on the available data, we have provided support to the activation of RhoA/Rho kinase pathway as an important effector in the pathophysiology of CNIs induced post-transplant hypertension via activation of oxidative stress and sodium retention. Clarification of how the biochemical and molecular mechanisms that regulate the processes involved in CNIs induced post transplant hypertension work and interact, would provide further insights not only into the comprehension of the pathophysiology of CNIs induced post transplant hypertension but could also have a positive impact on the clinical ground through the identification of significant targets. Their specific pharmacologic targeting might have multiple beneficial effects on the whole cardiovascular-renal function. The demonstration that in kidney transplanted patients with CNIs induced post-transplanted hypertension, the treatment of hypertension with different antihypertensive drugs inducing a comparable blood pressure reduction but different effects for example on oxidative stress and oxidative stress related proteins and/or Rho kinase and sodium retention, could be helpful for the choice of the antihypertensive treatment in these patients which takes advantage from effects of these drugs beyond blood pressure reduction.

In Patients with Chronic Kidney Disease Short Term Blood Pressure Variability is Associated with the Presence and Severity of Sleep Disorders.

BACKGROUND/AIMS: In chronic kidney disease (CKD) patients blood pressure variability (BPV) is associated with poor outcome. Sleep disturbances alter BP profiles in hypertensives but their influence on BPV in CKD patients is unknown. We screened a cohort of CKD/ESRD patients to investigate whether sleep quality impacts on BPV. METHODS: Consecutive CKD patients' sleep quality was assessed using validated questionnaires (Epworth Sleepiness Scale-ESS); International Restless legs scale-IRLS; Functional Outcomes of Sleep Questionnaire-FOSQ: Insomnia Severity Index-ISI; STOP-Bang). All patients underwent ambulatory blood pressure measurement. RESULTS: 104 out of 143 enrolled patients (78.32% stage-3 CKD; 10.49% Stage-4; 11.19% Stage-5; 6.99% ESRD-under dialysis) completed all the questionnaires. 95.8% were hypertensives, 70% were non-dippers and 27.8% had resistant hypertension. STOP-Bang>4 proved sleep disorders in 84.84% of patients. Patients with IRLS>10 had greater diastolic nocturnal standard deviation (DNSD) and a trend (p=0.05) for systolic nocturnal SD (SNSD). Patients with ISI>14 had greater SNSD and in 28.8% FOSQ showed severely impaired sleep quality. Their systolic nocturnal BPV was significantly greater. ISI was independently associated with SNSD. FOSQ and diastolic nocturnal BPV were negatively correlated at the bivariate analysis and FOSQ independently predicts systolic nocturnal BPV at multivariate regression analysis. CONCLUSIONS: In CKD patients impaired sleep quality increases BPV, might contribute to their disease progression and worsen prognosis. Searching for sleep problems in CKD patients could help planning their treatment of sleep problems contributing to CV risk reduction. Our data provide the rationale working hypothesis for the need of studies with larger number of patients aimed to demonstrate improved outcome of CKD progression and CV risk with the treatment also of sleep disorders.

Tracing angiotensin II's yin-yang effects on cardiovascular-renal pathophysiology.

Adverse changes in cardiovascular and renal systems are major contributors to overall morbidity and mortality. Human cardiovascular and renal systems exhibit a complex network of positive and negative feedback that is reflected in the control of vascular tone via angiotensin II (Ang II) based signaling. This review will examine in some depth, the multiple components and processes that control the status and reflect the health of these various cardiovascular and renal systems, such as pathways associated to monomeric G proteins, RhoA/Rho kinase system and ERK, oxidative stress and NO balance. It will specifically emphasize the "yin-yang" nature of Ang II signaling by comparing and contrasting the effects and activity of various systems, pathways and components found in hypertension to those found in Gitelman's and Bartter's syndromes (GS/BS), two rare autosomal recessive tubulopathies characterized by electrolytic imbalance, metabolic alkalosis, sodium wasting and prominent activation of the renin-angiotensin-aldosterone system. Notwithstanding the activation of the renin-angiotensin-aldosterone system, GS/BS are normo-hypotensive and protected from cardiovascular-renal remodeling and therefore can be considered the mirror image, the opposite of hypertension.

Oxidants and Cardiorenal Vascular Remodeling-Insights from Rare Genetic Tubulopathies: Bartter's and Gitelman's Syndromes.

Two human genetic tubulopathies, Bartter's (BS) and Gitelman's (GS) syndromes, have normo/hypotension and absent cardiac remodeling despite their apparent angiotensin system (RAS) activation. This seeming contradiction has led to an extensive investigation of BSGS patients, the result of which is that BSGS represents a mirror image of hypertension. BSGS's unique set of properties has then permitted their use as a human model to probe and characterize RAS system pathways and oxidative stress in cardiovascular and renal remodeling and pathophysiology. This review details the results using GSBS patients that provide a deeper understanding of Ang II signaling and its associated oxidants/oxidative stress in humans. By providing a more complete and complex picture of cardiovascular and renal remodeling pathways and processes, studies of GSBS can inform the identification and selection of new targets and therapies to treat these and other oxidant-related disorders.

Pathomechanism of oxidative stress in cardiovascularrenal remodeling and therapeutic strategies.

The high prevalence of cardiovascular disease in patients with chronic kidney disease indicates significant interactions between pathogenic pathways operating in the kidney and heart. These interactions involve all cell types (endothelial cells, smooth muscle cells, macrophages, and others), components of the vasculature, glomeruli, and heart that are susceptible to oxidative damage and structural alterations. A vicious cycle occurs whereby harmful factors such as reactive oxygen species and inflammation damage of vascular structures that themselves become sources of additional dangerous/toxic components released into the local environment. The evidence of this vicious cycle in chronic kidney disease should therefore lead to add other factors to both traditional and nontraditional risk factors. This review will examine the processes occurring during progressive kidney dysfunction with regard to vascular injury, renal remodeling, cardiac hypertrophy, and the transversal role of oxidative stress in the development of these complications.

[Reduced oxidative stress in ADPKD patients treated with tolvaptan].

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic kidney disease. It causes hypertension and progressive renal failure, both strictly linked to oxidative stress (OxSt). Treatment with tolvaptan is a consolidate option which slows renal deterioration rate, although the molecular mechanisms involved are not fully clarified. We evaluated the OxSt state in tolvaptan-treated ADPKD patients, untreated patients and healthy subjects. OxSt was assessed in 9 patients for each group as mononuclear cell protein expression, MYPT-1 phosphorylation state (Western blot) and heme oxygenase (HO-1) (ELISA). p22 phox protein expression was lower in tolvaptan treated ADPKD and controls compared to untreated patients: 0.86 ±0.15 d.u. p=0.015; 0.53 ±0.11, p<0.001; 1.42 ±0.11 respectively. The same was observed for phosphorylated MYPT-1: 0.68 ±0.09, p=0.013 and vs 0.47 ±0.13, p<0.001, 0.96 ±0.28, while HO-1 of untreated patients was significantly lower compared to treated and controls: 5.33 ±3.34 ng/mL, 2.08 ±0.79, p=0.012, 1.97 ±1.22, p=0.012. Tolvaptan-treated ADPKD patients have reduced OxSt, which might contribute to slowing down the loss of renal function.

The Effect of Green Tea as an Adjuvant to Enzyme Replacement Therapy on Oxidative Stress in Fabry Disease: A Pilot Study.

Enzymatic replacement therapy (ERT) is not very effective in halting the progression of Fabry disease (FD) toward cardiovascular (CV)-renal remodeling, particularly in case of late diagnosis. FD patients have increased oxidative stress (OS), critical for the induction of CV-renal remodeling. We investigated the effects of an adjuvant antioxidant treatment to ERT on OS and the possible advantages for related complications. OS was evaluated in 10 patients with FD before ERT, after 12 months of ERT, and after 6 months of adjuvant green tea (GT) to ERT by the following experiments: expression of p22phox; phosphorylation state of MYPT-1 and ERK 1/2 (by western blotting); and quantification of malondialdehyde (MDA) and heme oxygenase (HO)-1 levels (by ELISA). p22 phox and MYPT-1 phosphorylation decreased after ERT and significantly further decreased after GT. ERK 1/2 phosphorylation and MDA levels remained unchanged after ERT, but significantly decreased after GT. HO-1 significantly increased after ERT and further increased after GT. This study provides preliminary data highlighting the antioxidant effect exerted by ERT itself, further amplified by the adjuvant antioxidant treatment with GT. The results of this study provide evidence of the positive effect of early additive antioxidant treatment to reduce OS and prevent/alleviate cardio and cerebrovascular-renal complications related to OS.

Effects of Tolvaptan on Oxidative Stress in ADPKD: A Molecular Biological Approach.

Autosomal dominant polycystic disease (ADPKD) is the most frequent monogenic kidney disease. It causes progressive renal failure, endothelial dysfunction, and hypertension, all of which are strictly linked to oxidative stress (OxSt). Treatment with tolvaptan is known to slow the renal deterioration rate, but not all the molecular mechanisms involved in this effect are well-established. We evaluated the OxSt state in untreated ADPKD patients compared to that in tolvaptan-treated ADPKD patients and healthy subjects. OxSt was assessed in nine patients for each group in terms of mononuclear cell p22phox protein expression, NADPH oxidase key subunit, MYPT-1 phosphorylation state, marker of Rho kinase activity (Western blot) and heme oxygenase (HO)-1, induced and protective against OxSt (ELISA). p22phox protein expression was higher in untreated ADPKD patients compared to treated patients and controls: 1.42 ± 0.11 vs. 0.86 ± 0.15 d.u., p = 0.015, vs. 0.53 ± 0.11 d.u., p < 0.001, respectively. The same was observed for phosphorylated MYPT-1: 0.96 ± 0.28 vs. 0.68 ± 0.09 d.u., p = 0.013 and vs. 0.47 ± 0.13 d.u., p < 0.001, respectively, while the HO-1 expression of untreated patients was significantly lower compared to that of treated patients and controls: 5.33 ± 3.34 vs. 2.08 ± 0.79 ng/mL, p = 0.012, vs. 1.97 ± 1.22 ng/mL, p = 0.012, respectively. Tolvaptan-treated ADPKD patients have reduced OxSt levels compared to untreated patients. This effect may contribute to the slowing of renal function loss observed with tolvaptan treatment.

The Pivotal Role of Oxidative Stress in the Pathophysiology of Cardiovascular-Renal Remodeling in Kidney Disease.

The excessive activation of the renin-angiotensin system in kidney disease leads to alteration of intracellular pathways which concur altogether to the induction of cardiovascular and renal remodeling, exposing these patients since the very beginning of the renal injury to chronic kidney disease and progression to end stage renal disease, a very harmful and life threatening clinical condition. Oxidative stress plays a pivotal role in the pathophysiology of renal injury and cardiovascular-renal remodeling, the long-term consequence of its effect. This review will examine the role of oxidative stress in the most significant pathways involved in cardiovascular and renal remodeling with a focus on the detrimental effects of oxidative stress-mediated renal abnormalities on the progression of the disease and of its complications. Food for thoughts on possible therapeutic target are proposed on the basis of experimental evidences.

The Dietary Approach to the Treatment of the Rare Genetic Tubulopathies Gitelman's and Bartter's Syndromes.

Gitelman's (GS) and Bartter's (BS) syndromes are rare, inherited autosomal recessive tubulopathies characterized by hypokalemia, metabolic alkalosis, renal sodium, chloride, and potassium and magnesium-wasting. While the treatment based on potassium, sodium, chloride, and magnesium supplementation in addition to other pharmacologic options are widely established, recommendations about the dietary approach to GS and BS still remain generic. In this review we focus on the dietary strategies to increase sodium, potassium, and magnesium intake in GS and BS patients. Potassium and magnesium-rich foods and supplements are considered together with those that may reduce through different mechanisms the potassium and magnesium plasma level. Magnesium supplementation is often poorly tolerated, causing abdominal pain and diarrhea in most patients. New formulations using liposome and, in particular, sucrosomial technology have been recently proposed for magnesium supplementation in order to increase magnesium supplement tolerability and intestinal absorption. The dietary approach to GS and BS may be very important in the therapeutic approach to these syndromes. Due to the relevance of the dietary approach to these syndromes, a nutritional counseling should always be recommended and the nutritionist should join nephrologists in the follow-up of GS and BS patient care.

ACE2 and SARS-CoV-2 Infection Risk: Insights From Patients With Two Rare Genetic Tubulopathies, Gitelman's and Bartter's Syndromes.

COVID-19 is spreading globally with the angiotensin converting enzyme (ACE)-2 serving as the entry point of SARS-CoV-2 virus. This raised concerns how ACE2 and the Renin-Angiotensin (Ang)-System (RAS) are to be dealt with given their roles in hypertension and their involvement in COVID-19's morbidity and mortality. Specifically, increased ACE2 expression in response to treatment with ACE inhibitors (ACEi) and Ang II receptor blockers (ARBs) might theoretically increase COVID-19 risk by increasing SARS-CoV-2 binding sites. However, ACE2 is part of the protective counter-regulatory ACE2-Ang1-7-MasR axis, which opposes the classical ACE-AngII-AT1R regulatory axis. We used Gitelman's and Bartter's syndromes (GS/BS) patients, rare genetic tubulopathies that have endogenously increased levels of ACE2, to explore these issues. Specifically, 128 genetically confirmed GS/BS patients, living in Lombardia, Emilia Romagna and Veneto, the Northern Italy hot spots for COVID-19, were surveyed via telephone survey regarding COVID-19. The survey found no COVID-19 infection and absence of COVID-19 symptoms in any patient. Comparison analysis with the prevalence of COVID-19 in those regions showed statistical significance (p < 0.01). The results of the study strongly suggest that increased ACE2 does not increase risk of COVID-19 and that ACEi and ARBs by blocking excessive AT1R-mediated Ang II activation might favor the increase of ACE2-derived Ang 1-7. GS/BS patients' increased ACE2 and Ang 1-7 levels and their characteristic chronic metabolic alkalosis suggest a mechanism similar to that of chloroquine/hydroxychloroquine effect on ACE2 glycosylation alteration with resulting SARS-COV-2 binding inhibition and blockage/inhibition of viral entry. Studies from our laboratory are ongoing to explore GS/BS ACE2 glycosylation and other potential beneficial effects of BS/GS. Importantly, the absence of frank COVID-19 or of COVID-19 symptoms in the BS/GS patients cohort, given no direct ascertainment of COVID-19 status, suggest that elevated ACE2 levels as found in GS/BS patients at a minimum render COVID-19 infection asymptomatic and thus that COVID-19 symptoms are driven by ACE2 levels.

ROCK (RhoA/Rho Kinase) in Cardiovascular-Renal Pathophysiology: A Review of New Advancements.

Rho-associated, coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase and found to belong to the AGC family of serine/threonine kinases. They were shown to be downstream effectors of RhoA and RhoC activation. They signal via phosphorylation of proteins such as MYPT-1, thereby regulating many key cellular functions including proliferation, motility and viability and the RhoA/ROCK signaling has been shown to be deeply involved in arterial hypertension, cardiovascular-renal remodeling, hypertensive nephropathy and posttransplant hypertension. Given the deep involvement of ROCK in cardiovascular-renal pathophysiology and the interaction of ROCK signaling with other signaling pathways, the reports of trials on the clinical beneficial effects of ROCK's pharmacologic targeting are growing. In this current review, we provide a brief survey of the current understanding of ROCK-signaling pathways, also integrating with the more novel data that overall support a relevant role of ROCK for the cardiovascular-renal physiology and pathophysiology.

Could nutritional therapy take us further in our approaches to Fabry disease?

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene that result in deficiency of enzyme α-galactosidase A activity. Clinical manifestation varies from mild to severe depending on the phenotype. The main clinical manifestations are cutaneous (angiokeratomas), neurologic (acroparesthesias), gastrointestinal (nausea, diarrhea, and abdominal pain), renal (proteinuria and kidney failure), cardiovascular (cardiomyopathy and arrhythmias), and cerebrovascular (stroke). Enzyme replacement therapy with recombinant human α-galactosidase is currently the therapeutic option for FD. Although enzyme replacement therapy has changed the natural history of disease, many clinical aspects of FD require an additional specific treatment. Nutritional approach is mostly indicated in case of nephropathy and gastrointestinal symptoms. Specific dietary interventions can modulate some pathogenetic mechanisms of the disease, such as the inflammation, oxidative stress, and autophagic disorders. However, to our knowledge, limited attention has been given to the nutritional aspects of FD. The aim of this review is to examine nutritional strategies that might interfere with several pathophysiologic aspects of FD, including inflammation and oxidative stress. A dietary approach should be part of the basic treatment in renal manifestations of FD. Dietary measures recommended for irritable bowel syndrome could be recommended for gastrointestinal symptoms. Dietary factors can modulate the inflammation, oxidative stress, and autophagy involved in FD. Polyphenols, ω-3 fatty acids, microbiota, and specific dietary patterns can interfere with inflammation/oxidative stress and autophagy mechanisms and could also contribute to the slowing of FD progression.

Rho Kinase Activity, Connexin 40, and Atrial Fibrillation: Mechanistic Insights from End-Stage Renal Disease on Dialysis Patients.

Evidence on cellular/molecular mechanisms leading to atrial fibrillation (AF) are scanty. Increased expression of Rho kinase (ROCK) and myosin-phosphatase-target subunit-1 (MYPT-1), ROCK activity's marker, were shown in AF patients, which correlated with connexin 40 (Cx40) expression, membrane protein of heart gap junctions, key for rapid action potential's cell-cell transfer. AF is the most frequent arrhythmia in dialysis patients who present increased MYPT-1 phosphorylation, which correlates with left ventricular (LV) mass. Given ROCK's established role in cardiovascular-renal remodeling, induction of impaired cell-to-cell coupling/potential conduction promoting AF initiation/perpetuation, we evaluated in dialysis patients with AF, MYPT-1 phosphorylation, Cx40 expression, and their relationships to support their involvement in AF. Mononuclear cells' MYPT-1 phosphorylation, Cx40 expression, and the ROCK inhibitor fasudil's effect were assessed in dialysis patients with AF (DPAFs), dialysis patients with sinus rhythm (DPs), and healthy subjects (C) (western blot). M-mode echocardiography assessed LV mass and left atrial systolic volume. DPAF's phospho-MYPT-1 was increased vs. that of DPs and C (1.57 ± 0.17 d.u. vs. 0.69 ± 0.04 vs. 0.51 ± 0.05 respectively, p < 0.0001). DP's phospho-MYPT-1 was higher vs. that of C, p = 0.009. DPAF's Cx40 was higher vs. that of DPs and C (1.23 ± 0.12 vs. 0.74 ± 0.03 vs. 0.69 ± 0.03, p < 0.0001). DPAF's phospho-MYPT-1 correlated with Cx40 (p < 0.001), left atrial systolic volume (p = 0.013), and LV mass (p = 0.014). In DPAFs, fasudil reduced MYPT-1 phosphorylation (p < 0.01) and Cx40 expression (p = 0.03). These data point toward ROCK and Cx40's role in the mechanism(s) leading to AF in dialysis patients. Exploration of the ROCK pathway in AF could contribute to AF generation's mechanistic explanations and likely identify potential pharmacologic targets for translation into treatment.

Oxidative Stress and Cardiovascular-Renal Damage in Fabry Disease: Is There Room for a Pathophysiological Involvement?

Fabry disease is an X-linked lysosomal storage disease caused by mutations in the GLA gene that lead to a reduction or an absence of the enzyme α-galactosidase A, resulting in the progressive and multisystemic accumulation of globotriaosylceramide. Clinical manifestation varies from mild to severe, depending on the phenotype. The main clinical manifestations are cutaneous (angiokeratomas), neurological (acroparesthesias), gastrointestinal (nausea, diarrhea abdominal pain), renal (proteinuria and kidney failure), cardiovascular (cardiomyopathy and arrhythmias), and cerebrovascular (stroke). A diagnosis of Fabry disease can be made with an enzymatic assay showing absent or reduced α-galactosidase A in male patients, while in heterozygous female patients, molecular genetic testing is needed. Enzyme replacement therapy (ERT) with recombinant human α-galactosidase is nowadays the most-used disease-specific therapeutic option. Despite ERT, cardiocerebrovascular-renal irreversible organ injury occurs, therefore additional knowledge and a deeper understanding of further pathophysiological mechanisms leading to end organ damage in Fabry disease are needed. Recent data point toward oxidative stress, oxidative stress signaling, and inflammation as some such mechanisms. In this short review, the current knowledge on the involvement of oxidative stress in cardiovascular-renal remodeling is summarized and related to the most recent evidence of oxidative stress activation in Fabry disease, and clearly points toward the involvement of oxidative stress in the pathophysiology of the medium- to long-term cardiovascular-renal damage of Fabry disease.

Oxidative stress - chronic kidney disease - cardiovascular disease: A vicious circle.

Chronic kidney disease patient's progression to end-stage renal disease as well as their high mortality are linked to cardiovascular disease. However, the high incidence rate of cardiovascular morbidity and mortality in these patients is not fully accounted for by traditional cardiovascular risk factors such as diabetes, hypertension and obesity. Renal disease and CVD are associated with endothelial dysfunction, inflammation and oxidative stress and in this review we will examine what is known regarding their similar roles in both CVD and chronic kidney disease, specifically focusing on the interconnections between oxidative stress, inflammation and endothelial dysfunction. These interconnections are best visualized as a vicious circle wherein these entities coexist and communicate with each other, thereby exacerbating the processes underpinning these different entities with the end result of the high morbidity and mortality that characterize CKD patients. By exploring this vicious circle i.e. the mode and extent of the interrelationships as well as some of the underlying mechanisms involved, this review aims at outlining our current understanding as well as highlighting future avenues for research and potential targets for therapeutic intervention.

Gitelman's Syndrome: characterization of a novel c.1181G>A point mutation and functional classification of the known mutations.

We have investigated the mechanisms by which a novel missense point mutation (c.1181G>A) found in two sisters causes Gitelman's syndrome by impairing the sodium chloride co-transporter (NCC, encoded by SLC12A3 gene) function. The cDNA and in vitro transcribed mRNA of either wild-type or mutated SLC12A3 were transfected into HEK293 cells and injected into Xenopus laevis oocytes, respectively. The expression, maturation, trafficking, and function of the mutated and wild-type NCC were assessed by Western blotting, immunohistochemistry and 22Na+ uptake studies. By immunoblotting of lysates from HEK293 cells and oocytes expressing wild-type NCC, two NCC-related bands of approximately 130 kDa and 115 kDa, corresponding to fully and core-glycosylated NCC, respectively, were identified. In contrast, the mutant NCC only showed a single band of approximately 115 kDa, indicating impaired maturation of the protein. Moreover, oocytes injected with wild-type NCC showed thiazide-sensitive 22Na+ uptake, which was absent in those injected with the mutant NCC. The novel mutation was discussed in the context of the functionally characterized NCC mutations causing Gitelman's syndrome, which fit into five classes. In conclusion, the functional characterization of this novel Gly394Asp NCC and its localization on the NCC structure, alongside that of previously known mutations causing Gitelman's syndrome, may provide novel information on the function of the different domains of the human NCC.