Updates and Perspectives on Aquaporin-2 and Water Balance Disorders.

Ensuring the proper amount of water inside the body is essential for survival. One of the key factors in the maintenance of body water balance is water reabsorption in the collecting ducts of the kidney, a process that is regulated by aquaporin-2 (AQP2). AQP2 is a channel that is exclusively selective for water molecules and impermeable to ions or other small molecules. Impairments of AQP2 result in various water balance disorders, including nephrogenic diabetes insipidus (NDI), which is a disease characterized by a massive loss of water through the kidney and consequent severe dehydration. Dysregulation of AQP2 is also a cause of water retention with hyponatremia in heart failure, hepatic cirrhosis, and syndrome of inappropriate antidiuretic hormone secretion (SIADH). Antidiuretic hormone vasopressin is an upstream regulator of AQP2. Its binding to the vasopressin V2 receptor promotes AQP2 targeting to the apical membrane and thus enables water reabsorption. Tolvaptan, a vasopressin V2 receptor antagonist, is effective and widely used for water retention with hyponatremia. However, there are no studies showing improvement in hard outcomes or long-term prognosis. A possible reason is that vasopressin receptors have many downstream effects other than AQP2 function. It is expected that the development of drugs that directly target AQP2 may result in increased treatment specificity and effectiveness for water balance disorders. This review summarizes recent progress in studies of AQP2 and drug development challenges for water balance disorders.

Phosphorylation profile of human AQP2 in urinary exosomes by LC-MS/MS phosphoproteomic analysis.

BACKGROUND: Aquaporin-2 (AQP2) is a key water channel protein which determines the water permeability of the collecting duct. Multiple phosphorylation sites are present at the C-terminal of AQP2 including S256 (serine at 256 residue), S261, S264 and S/T269, which are regulated by vasopressin (VP) to modulate AQP2 trafficking. As the dynamics of these phosphorylations have been studied mostly in rodents, little is known about the phosphorylation of human AQP2 which has unique T269 in the place of S269 of rodent AQP2. Because AQP2 is excreted in urinary exosomes, the phosphoprotein profile of human AQP2 can be easily examined through urinary exosomes without any intervention. METHODS: Human urinary exosomes digested with trypsin or glutamyl endopeptidase (Glu-C) were examined by the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) phosphoproteomic analysis. RESULTS: The most dominant phosphorylated AQP2 peptide identified was S256 phosphorylated form (pS256), followed by pS261 with less pS264 and far less pT269, which was confirmed by the western blot analyses using phosphorylated AQP2-specific antibodies. In a patient lacking circulating VP, administration of a VP analogue showed a transient increase (peak at 30-60 min) in excretion of exosomes with pS261 AQP2. CONCLUSION: These data suggest that all phosphorylation sites of human AQP2 including T269 are phosphorylated and phosphorylations at S256 and S261 may play a dominant role in the urinary exosomal excretion of AQP2.

Asymptomatic sinusitis as an origin of infection-related glomerulonephritis manifesting steroid-resistant nephrotic syndrome: A case report.

RATIONALE: Infection is a major trigger or pathogenic origin in a substantial proportion of glomerulonephritis (GN) patients, typically manifesting infection-related GN (IRGN). Various microorganisms, infection sites, and clinical and histopathological features are involved in IRGN. Once an infectious origin is identified and successfully eradicated, nephrotic syndrome or kidney dysfunction is spontaneously resolved. However, if patients are asymptomatic and the origin is undetermined, the diagnosis and treatment of GN is challenging. This case presentation reported on an IRGN case manifesting steroid-resistant nephrotic syndrome associated with asymptomatic sinusitis as a pathogenic origin. PATIENT CONCERNS: A 68-year-old male presented with severe kidney dysfunction and edema in both extremities. DIAGNOSIS: The patient was clinically diagnosed with hypocomplementemic nephrotic syndrome and kidney dysfunction and histopathologically with diffuse proliferative GN and a focal pattern of membranoproliferative GN. The findings suggested that idiopathic membranoproliferative glomerulonephritis type I was more likely than IRGN, given a critical lack of apparent infection. INTERVENTIONS: Combined intravenous methylprednisolone, oral prednisolone, and cyclosporin did not improve the patient's condition. Thus, IRGN associated with inapparent infectious origin was suspected. Repeated thorough and careful examinations including CT scan showed sinusitis in his left maxillary sinus. Moreover, reanalysis of kidney specimen revealed positive nephritis-associated plasmin receptor in glomeruli, a typical finding for IRGN, supporting a pathogenic significance of his sinusitis. Medical treatment was initiated with 200 mg oral clarithromycin daily. OUTCOMES: Oral clarithromycin gradually improved proteinuria and hypocomplementemia and resulted in nephrotic syndrome remission in parallel with opacification resolution of sinuses shown on CT. LESSONS: This case presentation showed that asymptomatic sinusitis is potentially a pathogenic IRGN origin. A gold standard therapy for idiopathic GN, corticosteroid could be damaging in uncontrolled or underdiagnosed infection. In asymptomatic patients, a thorough screening of infectious diseases, including sinusitis, together with a renal histological evaluation of glomerular nephritis-associated plasmin receptor deposition is also essential in treating a wide spectrum of GN.

Prognosis of chronic kidney disease with normal-range proteinuria: The CKD-ROUTE study.

BACKGROUND: Although lower estimated glomerular filtration rate (eGFR) and higher proteinuria are high risks for mortality and kidney outcomes, the prognosis of chronic kidney disease (CKD) in patients with normal-range proteinuria remains unclear. METHODS: In this prospective cohort study, 1138 newly visiting stage G2-G5 CKD patients were stratified into normal-range and abnormal-range proteinuria groups. Study endpoints were CKD progression (>50% eGFR loss or initiation of dialysis), cardiovascular events, and all-cause death. RESULTS: In total, 927 patients who were followed for >6 months were included in the analysis. The mean age was 67 years, and 70.2% were male. During a median follow-up of 35 months, CKD progression, cardiovascular events, and mortality were observed in 223, 110, and 55 patients, respectively. Patients with normal-range proteinuria had a significantly lower risk for CKD progression (hazard ratio, 0.20; 95% confidence interval, 0.10-0.38) than those with abnormal-proteinuria by multivariate Cox proportional hazard analysis. We also analyzed patients with normal-range proteinuria (n = 351). Nephrosclerosis was the most frequent cause of CKD among all patients with normal-range proteinuria (59.7%). During a median follow-up of 36 months, CKD progression, cardiovascular events, and mortality were observed in 10, 28, and 18 patients, respectively. The Kaplan-Meyer analysis demonstrated that the risks of CKD progression and cardiovascular events were not significantly different among CKD stages, whereas the risk of death was significantly higher in patients with advanced-stage CKD. Multivariate Cox proportional hazard analysis showed that the risk of three endpoints did not significantly differ among CKD stages. CONCLUSION: Newly visiting CKD patients with normal-range proteinuria, who tend to be overlooked during health checkups did not exhibit a decrease in kidney function even in advanced CKD stages under specialized nephrology care.

Low white blood cell count is independently associated with chronic kidney disease progression in the elderly: the CKD-ROUTE study.

BACKGROUND: Elevated white blood cell (WBC) count is a well-known predictor of chronic kidney disease (CKD) progression. However, elderly patients commonly fail to develop a high WBC count in response to several diseased states and may instead present a low WBC count. Therefore, we hypothesized that low WBC count, in addition to high WBC count, is associated with CKD progression in the elderly. METHODS: We conducted a prospective cohort study using 3-year follow-up data from the CKD Research of Outcomes in Treatment and Epidemiology study. In the present study, participants aged over 60 years with pre-dialysis CKD stages G2-G5 were eligible. Patients were stratified into three groups according to WBC count using tertiles (T). The primary outcome was a composite of end-stage renal disease and a 50% reduction in estimated glomerular filtration rate. Data were analyzed using Cox proportional hazard models with adjustments for covariates. RESULTS: We enrolled 697 patients (males, 69%). The median WBC count was 6100 cells/µl (T1, <5400, n =  222; T2, 5400-6900, n =  235; T3, ≥6900, n = 240). During a median follow-up of 868 days, the primary outcome was observed in 170 patients, whereas 54 patients died. T1 and T3 had significantly higher hazard ratios (HR) than T2 (T1, HR 1.69, 95% confidence interval 1.14-2.51; T3, HR 1.63, 95% confidence interval 1.10-2.41). Moreover, T1 had a significantly higher adjusted HR (1.54, 95% confidence interval 1.00-2.37). CONCLUSION: Low WBC count is independently associated with CKD progression in the elderly.

Combination of low body mass index and serum albumin level is associated with chronic kidney disease progression: the chronic kidney disease-research of outcomes in treatment and epidemiology (CKD-ROUTE) study.

BACKGROUND: The relationship between protein-energy wasting and chronic kidney disease (CKD) progression is unknown. In the present prospective cohort study, we evaluated the hypothesis that a combination of low body mass index (BMI) and serum albumin level is associated with rapid CKD progression. METHODS: The study cohort comprised 728 predialysis Japanese patients with CKD (stages 2-5) enrolled from 2010 to 2011. Patients were categorized into four groups according to their serum albumin levels and BMI: group 1, low serum albumin level (<4 g/dL) and low BMI (<23.5 kg/m2); group 2, high serum albumin level (≥4 g/dL) and low BMI; group 3, low serum albumin level and high BMI (≥23.5 kg/m2); and group 4, high serum albumin level and high BMI. The primary outcome was a 30 % decline in estimated glomerular filtration rate (eGFR) or start of dialysis within 2 years. The secondary outcome was an annual GFR decline (mL/min/1.73 m2/year). RESULTS: Logistic regression analysis adjusted for baseline characteristics (reference, group 4) showed that only group 1 was associated with a significant risk of CKD progression, with adjusted odds ratio of 3.51 [95 % confidence interval (CI) (1.63, 7.56)]. A multivariate linear regression analysis adjusted for baseline characteristics showed a significant difference in annual eGFR decline between groups 1 and 4 [coefficients ß (standard error) -2.62 (0.75), p = 0.001]. CONCLUSION: This study suggests that combined effects of low BMI (<23.5 kg/m2) and serum albumin level (<4 g/dL) are associated with CKD progression.

Association of serum chloride level with mortality and cardiovascular events in chronic kidney disease: the CKD-ROUTE study.

BACKGROUND: Electrolyte abnormalities, particularly dysnatremia, are independent predictors of adverse outcome in individuals with and without renal failure. However, the association of serum chloride level (Cl-) with mortality or risk of cardiovascular (CV) events in chronic kidney disease (CKD) remains unclear. METHODS: This prospective cohort study included 923 pre-dialysis CKD G2-G5 patients among the participants of the CKD Research of Outcomes in Treatment and Epidemiology (CKD-ROUTE) study, who newly visited 16 nephrology centers. The primary outcome was a composite of overall death and CV events, and the secondary outcome was overall death. Data were analyzed using the Cox hazards model with adjustment for potential confounders. RESULTS: Median Cl- was 106.0 mEq/L at enrollment [quartile (Q) 1: ≤103.9, n =  207; Q2: 104.0-105.9, n =  207; Q3: 106.0-108.0, n =  289; Q4: ≥108.1, n = 220]. During a median follow-up of 33 months, there were 98 CV events, 66 deaths, and 154 composite outcomes. The hazard ratio (HR) for the composite outcome was higher for Q1 than Q3 [HR 1.72; 95 % confidence interval (CI) 1.08-2.72; P =  0.022]. As a continuous variable in a subset of patients whose Cl- was ≤106.0 mEq/L, higher Cl- was associated with lower risk of the composite outcome (HR 0.93; 95 % CI 0.87-0.99; P = 0.023). HR for all-cause mortality was also higher for Q1 than Q3 (HR 2.48; 95 % CI 1.22-5.03; P =  0.012). CONCLUSION: Low Cl- was associated with increased mortality and risk of CV events in pre-dialysis CKD patients. Cl- may be an additional prognostic indicator in CKD.

The use of vitamin D analogs is independently associated with the favorable renal prognosis in chronic kidney disease stages 4-5: the CKD-ROUTE study.

BACKGROUND: Vitamin D analogs have generally been recommended for treatment of mineral bone disease in chronic kidney disease (CKD). However, the association between this treatment and CKD progression has not yet been established. METHODS: We designed a post hoc propensity score-matched cohort analysis derived from 3-year follow-up data of a prospective cohort. Adult participants with pre-dialysis CKD stages 4-5 who had newly been prescribed active vitamin D analogs during the observation period were eligible as matched cases. Then, matched controls were extracted from participants who had never been prescribed active vitamin D analogs. The primary outcome was a composite of end-stage renal disease or a 50 % reduction in estimated glomerular filtration rate (eGFR). A Cox proportional hazards model evaluated the association between the use of vitamin D analogs and the primary outcome. RESULTS: We enrolled 240 patients (males, 65 %). The number of matched cases and controls was 30 and 210, respectively. The primary outcome was observed in 94 patients, whereas 25 patients died. The mean ± standard deviation age and eGFR were 69 ± 12 years and 17 ± 5.7 ml/min/1.73 m2, respectively. In a Cox proportional hazard model, the use of vitamin D analogs was independently associated with a lower risk of the primary outcome (crude hazard ratio 0.41; 95 % confidence interval 0.19, 0.89; adjusted hazard ratio 0.38; 95 % confidence interval 0.17, 0.88). CONCLUSION: The use of vitamin D analogs is independently associated with the preservation of renal function in patients with pre-dialysis CKD stages 4-5.

Anaemia management and mortality risk in newly visiting patients with chronic kidney disease in Japan: The CKD-ROUTE study.

AIM: To investigate the association between iron deficiency anaemia and mortality risk and assess the changes in anaemia and iron status after primary management by a nephrologist. METHODS: In this prospective cohort study, we stratified 951 non-dialysis chronic kidney disease (CKD) G2-G5 patients newly visiting 16 nephrology centres into four groups according to the presence of anaemia with or without iron deficiency. All-cause mortality, cardiovascular (CV)-related mortality, and a change in anaemia and iron status after specialized primary care were the endpoints evaluated. RESULTS: During a median follow-up time of 19 months, the number of all-cause deaths and CV-related deaths were 56 and 26, respectively. Compared with the control group, the groups with isolated anaemia and iron deficiency anaemia had significantly higher all-cause mortalities (isolated anaemia: hazard ratio (HR), 3.37; 95% confidence intervals (CI), 1.76-6.44; iron deficiency anaemia: HR, 3.11; 95% CI, 1.21-8.01) and CV-related mortalities (isolated anaemia: HR, 3.64; 95% CI, 1.36-9.73; iron deficiency anaemia: HR, 3.86; 95% CI, 1.11-13.41). In the isolated anaemia group, erythropoietin-stimulating agent (ESA) prescriptions significantly increased to approximately 70%. However, in patients with both anaemia and iron deficiency, iron prescriptions only increased to 48.1%. CONCLUSIONS: Iron deficiency anaemia and isolated anaemia were associated with all-cause and CV-related mortality. The absence of relative increase in iron prescriptions suggests that iron deficiency should be accurately assessed and iron supplementation should be appropriately used to manage anaemia in non-dialysis patients with CKD.

Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model.

The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Dynamic regulation and dysregulation of the water channel aquaporin-2: a common cause of and promising therapeutic target for water balance disorders.

The human body is two-thirds water. The ability of ensuring the proper amount of water inside the body is essential for the survival of mammals. The key event for maintenance of body water balance is water reabsorption in the kidney collecting ducts, which is regulated by aquaporin-2 (AQP2). AQP2 is a channel that is exclusively selective for water molecules and never allows permeation of ions or other small molecules. Under normal conditions, AQP2 is restricted within the cytoplasm of the collecting duct cells. However, when the body is dehydrated and needs to retain water, AQP2 relocates to the apical membrane, allowing water reabsorption from the urinary tubule into the cell. Its impairments result in various water balance disorders including diabetes insipidus, which is a disease characterized by a massive loss of water through the kidney, leading to severe dehydration in the body. Dysregulation of AQP2 is also a common cause of water retention and hyponatremia that exacerbate the prognosis of congestive heart failure and hepatic cirrhosis. Many studies have uncovered the regulation mechanisms of AQP2 at the single-molecule level, the whole-body level, and the clinical level. In clinical practice, urinary AQP2 is a useful marker for body water balance (hydration status). Moreover, AQP2 is now attracting considerable attention as a potential therapeutic target for water balance disorders which commonly occur in many diseases.

Baseline characteristics and prevalence of cardiovascular disease in newly visiting or referred chronic kidney disease patients to nephrology centers in Japan: a prospective cohort study.

BACKGROUND: About 39,000 patients were newly prescribed renal replacement therapy in Japan in 2011, resulting in a total of more than 300,000 patients being treated with dialysis. This high prevalence of treated end stage kidney disease (ESKD) patients is an emergent problem that requires immediate attention. We launched a prospective cohort study to evaluate population specific characteristics of the progression of chronic kidney disease (CKD). In this report, we describe the baseline characteristics and risk factors for cardiovascular disease (CVD) prevalence among this cohort. METHODS: New patients from 16 nephrology centers who were older than 20 years of age and who visited or were referred for the treatment of CKD stage 2-5, but were not on dialysis therapy, were recruited in this study. At enrollment, medical history, lifestyle behaviors, functional status and current medications were recorded, and blood and urine samples were collected. Estimated glomerular filtration rate (eGFR) was calculated by a modified three-variable equation. RESULTS: We enrolled 1138 patients, 69.6% of whom were male, with a mean age of 68 years. Compared with Western cohorts, patients in this study had a lower body mass index (BMI) and higher proteinuria. The prevalence of CVD was 26.8%, which was lower than that in Western cohorts but higher than that in the general Japanese population. Multivariate analysis demonstrated the following association with CVD prevalence: hypertension (adjusted odds ratio (aOR) 3.57; 95% confidence interval (CI) 1.82-7.02); diabetes (aOR 2.45; 95% CI 1.86-3.23); hemoglobin level less than 11 g/dl (aOR 1.61; 95% CI 1.21-2.15); receiving anti-hypertensive agents (aOR 3.54; 95% CI 2.27-5.53); and statin therapy (aOR 2.73; 95% CI 2.04-3.66). The combination of decreased eGFR and increased proteinuria was also associated with a higher prevalence of CVD. CONCLUSIONS: The participants in this cohort had a lower BMI, higher proteinuria and lower prevalence of CVD compared with Western cohorts. Lower eGFR and high proteinuria were associated with CVD prevalence. Prospective follow up of these study patients will contribute to establishment of individual population-based treatment of CKD.

Type II diabetes mellitus is a risk factor for heart failure in pre-dialysis patients.

Chronic kidney disease (CKD) increases the risk of developing cardiovascular diseases such as heart failure (HF) and ischemic heart disease (IHD). The characteristics of patients with CKD complicated with HF at the time of starting hemodialysis have not yet been evaluated. We enrolled 347 patients in this study and compared gender, age, body mass index, laboratory data, causative disease, complications, and echocardiographic findings between groups with (n = 105) and without (n = 242) HF. Type II diabetic nephropathy and estimated glomerular filtration rate (eGFR; mL/min/1.73 m(2) ) were the independent factors for HF (OR: 3.004, 95% CI: 1.754 to 5.146 and OR: 1.215, 95% CI: 1.101 to 1.330, [per 1 mL/min/1.73 m(2) increase], respectively). The higher GFR appeared to be not a risk factor for HF, probably because the HF group included patients who required periodic dialysis to prevent fatal HF, even if their renal function was not extremely deteriorated. The prevalence of hypertension, IHD and values of body mass index, triglycerides, and LDL-cholesterol did not differ between these two groups. Echocardiographic data showed that left ventricular mass index was an independent risk factor for HF (OR: 1.006, 95% CI: 1.001 to 1.012, per 1 g/m(2) increase) and more than half of the patients appeared to have left ventricular diastolic dysfunction. Our findings suggest that not only CKD, but also type II DM, is a potent risk for left ventricular dysfunction, which causes HF and IHD in pre-dialysis patients with CKD.

Splenic artery ligation: A protection against hepatic ischemia/reperfusion injury in partially hepatectomized rats.

AIM: In liver resection, the temporary occlusion of the hepatoduodenal ligament (Pringle maneuver) is often used. However, the maneuver causes ischemia/reperfusion (I/R) injury in the remnant liver. Heme oxygenase (HO)-1 has a cytoprotective role against this injury. Our aim is to investigate whether splenic artery ligation induces HO-1 expression in the liver and ameliorates the hepatic I/R injury in partially hepatectomized rats. METHODS: Rats underwent splenic artery ligation by occluding the main splenic artery. Two days later, the total hepatic ischemia (Pringle maneuver) was conducted, and then a two-thirds partial hepatectomy (PH) was performed just before the start of reperfusion. HO inhibitor was twice injected s.c. at 3 and 16 h before the Pringle maneuver. HO-1 levels were determined by western blotting. Liver injury was biochemically assessed. RESULTS: In normal rats, HO-1 was highly expressed in the spleen, but not in the liver. Splenic artery ligation induced HO-1 in the livers. When rats underwent 20 and 30 min of Pringle maneuver/PH, survival rates were 28% and 8%, respectively. Splenic artery ligation significantly improved both the survival rates: 73% and 56%, respectively. Under these conditions, administration of HO-1 inhibitor at least partly negated the efficacy of splenic artery ligation. Splenic artery ligation also increased the recovery rate of the remnant liver mass and platelet counts in Pringle maneuver/PH-treated rats. CONCLUSION: Splenic artery ligation was significantly effective on the hepatic I/R injury in partially hepatectomized rats. Induction of HO-1 may be at least partly involved in the improvement of this injury.

Claudin-4 deficiency results in urothelial hyperplasia and lethal hydronephrosis.

Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4(-/-) mice and found that these mice had increased mortality due to hydronephrosis of relatively late onset. While the renal nephrons of Cldn4(-/-) mice showed a concomitant diminution of Cld8 expression at tight junction (TJ), accumulation of Cld3 at TJ was markedly enhanced in compensation and the overall TJ structure was unaffected. Nonetheless, Cldn4(-/-) mice showed slightly yet significantly increased fractional excretion of Ca(2+) and Cl(-), suggesting a role of Cld4 in the specific reabsorption of these ions via a paracellular route. Although the urine volume tended to be increased concordantly, Cldn4(-/-) mice were capable of concentrating urine normally on dehydration, with no evidence of diabetes insipidus. In the urothelium, the formation of TJs and uroplaques as well as the gross barrier function were also unaffected. However, intravenous pyelography analysis indicated retarded urine flow prior to hydronephrosis. Histological examination revealed diffuse hyperplasia and a thickening of pelvic and ureteral urothelial layers with markedly increased BrdU uptake in vivo. These results suggest that progressive hydronephrosis in Cldn4(-/-) mice arises from urinary tract obstruction due to urothelial hyperplasia, and that Cld4 plays an important role in maintaining the homeostatic integrity of normal urothelium.

Severe hyperparathyroidism in a pre-dialysis chronic kidney disease patient treated with a very low protein diet.

The present report describes a case of a 64-year-old pre-dialysis woman with chronic kidney disease (CKD) stage 5, who developed severe hyperparathyroidism. This patient had been on a very low protein diet (VLPD) to delay the progression of CKD and the need for renal replacement therapy (RRT). Her serum calcium levels were high-normal to slightly high during this time. However, her serum intact parathyroid hormone (PTH) levels increased from 400 to 1160 pg/ml rapidly over a period of 3 months. Serum 1,25-(OH)2D levels were low, and ultrasound of the neck showed three markedly enlarged parathyroid glands exceeding 2 cm. Parathyroidectomy was performed, and all glands showed nodular hyperplasia, which indicated severe secondary hyperparathyroidism leading to tertiary. Severe secondary hyperparathyroidism requiring surgical intervention is usually observed in patients with long-term RRT and is relatively rare in the pre-dialysis patient. In this case, extension of the pre-dialysis period by VLPD may have predisposed this patient to develop severe secondary hyperparathyroidism. Thus, careful monitoring of calcium, phosphorus, and PTH may be necessary in patients treated with VLPD even before renal replacement therapy. Furthermore, initiation of dialysis should not be excessively delayed by strict protein restriction dietary therapy.