Associations Between Dietary Potassium Intake From Different Food Sources and Hyperkalemia in Patients With Chronic Kidney Disease.

OBJECTIVE: Previous studies reported mixed results on associations between dietary potassium intake and hyperkalemia in patients with chronic kidney disease (CKD). This study investigated the association between potassium intake from different food sources and hyperkalemia in patients with non-dialysis-dependent CKD. METHODS: A total of 285 patients were recruited at a university hospital and 2 city hospitals in Tokyo. Dietary potassium intake was estimated by a validated diet history questionnaire. Associations of potassium intake from all foods and individual food groups with serum potassium were examined by multivariable linear regression among potassium binder nonusers. An association between tertile groups of potassium intake and hyperkalemia, defined as serum potassium ≥5.0 mEq/L, was evaluated by multivariable logistic regression. RESULTS: Among 245 potassium binder nonusers, total potassium intake was weakly associated with serum potassium (regression coefficient = 0.147, 95% confidence interval (CI): 0.018-0.277), while an association with hyperkalemia was not observed (first vs third tertile: adjusted odds ratio = 0.98, 95% CI: 0.29-3.26). As for food groups, potassium intakes from potatoes, pulses, and green/yellow vegetables were positively associated with serum potassium. Patients in the highest tertile of potassium intake from potatoes had higher odds of hyperkalemia as compared to those in the lowest tertile (adjusted odds ratio = 4.12, 95% CI: 1.19-14.34). CONCLUSION: Total potassium intake was weakly associated with serum potassium, but not with hyperkalemia. Potassium intake from potatoes was associated with hyperkalemia. These findings highlight the importance of considering food sources of potassium in the management of hyperkalemia in CKD.

[Kidney transplantation for end-stage renal disease after third allogeneic hematopoietic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia].

An 18-year-old man underwent allogenic bone marrow transplantation (BMT) for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Ph+ALL relapsed 3 months after the first BMT, and the patient underwent a second BMT. However, Ph+ALL relapsed 4 months after the second BMT, and he received a haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) from his father. Molecular complete remission was confirmed 29 days after haplo-PBSCT. However, the patient needed dialysis for end-stage renal disease due to thrombotic microangiopathy 3 years and 2 months after haplo-PBSCT. He received a kidney transplantation from his father 7 years and 10 months after haplo-PBSCT, and got off dialysis after the kidney transplantation. Immunosuppressive therapy with methylprednisolone, tacrolimus, and mycophenolate mofetil was started for kidney transplantation, but the dose of immunosuppressive agents was reduced successfully without rejection soon after kidney transplantation. The patient has maintained long-term remission since the haplo-PBSCT, and his kidney function was restored by the kidney transplantation from his father.

Sodium-calcium exchanger 1 is the key molecule for urinary potassium excretion against acute hyperkalemia.

The sodium (Na+)-chloride cotransporter (NCC) expressed in the distal convoluted tubule (DCT) is a key molecule regulating urinary Na+ and potassium (K+) excretion. We previously reported that high-K+ load rapidly dephosphorylated NCC and promoted urinary K+ excretion in mouse kidneys. This effect was inhibited by calcineurin (CaN) and calmodulin inhibitors. However, the detailed mechanism through which high-K+ signal results in CaN activation remains unknown. We used Flp-In NCC HEK293 cells and mice to evaluate NCC phosphorylation. We analyzed intracellular Ca2+ concentration ([Ca2+]in) using live cell Ca2+ imaging in HEK293 cells. We confirmed that high-K+-induced NCC dephosphorylation was not observed without CaN using Flp-In NCC HEK29 cells. Extracellular Ca2+ reduction with a Ca2+ chelator inhibited high-K+-induced increase in [Ca2+]in and NCC dephosphorylation. We focused on Na+/Ca2+ exchanger (NCX) 1, a bidirectional regulator of cytosolic Ca2+ expressed in DCT. We identified that NCX1 suppression with a specific inhibitor (SEA0400) or siRNA knockdown inhibited K+-induced increase in [Ca2+]in and NCC dephosphorylation. In a mouse study, SEA0400 treatment inhibited K+-induced NCC dephosphorylation. SEA0400 reduced urinary K+ excretion and induced hyperkalemia. Here, we identified NCX1 as a key molecule in urinary K+ excretion promoted by CaN activation and NCC dephosphorylation in response to K+ load.

Modified FOLFOX-6 Plus Bevacizumab Chemotherapy for Metastatic Colorectal Cancer in Patients Receiving Hemodialysis: A Report of Three Cases and Review of the Literature.

Fluorouracil plus oxaliplatin (L-OHP) (FOLFOX) plus bevacizumab (BV) therapy is commonly administered to patients with metastatic colorectal cancer. However, few reports have described L-OHP therapy in hemodialysis patients, and the efficacy and safety remain uncertain in this population. Here, we report three cases of hemodialysis patients with colorectal cancer who received a modified FOLFOX-6 (mFOLFOX-6, or FOLFOX plus folinic acid) plus BV regimen every 3 weeks. One patient, a 65-year-old man with chronic renal failure consequent to diabetic nephropathy, underwent hemodialysis 3 times/week. He exhibited a partial response after 7 cycles of mFOLFOX-6 plus BV, with the major adverse events of Grade 1 peripheral neuropathy and Grade 2 thrombocytopenia. He died of perforation-related septic shock. A 71-year-old man previously treated with bosutinib for chronic myelocytic leukemia received 9 cycles of mFOLFOX-6 plus BV and achieved stable disease. Chemotherapy was administered every 4 weeks, and the 5-fluorouracil dose was reduced after he developed Grade 4 neutropenia. A 71-year-old woman with chronic renal failure consequent to diabetic nephropathy underwent hemodialysis 3 times a week. She received 3 cycles of mFOLFOX-6 plus BV, but exhibited disease progression and developed Grade 4 neutropenia, which necessitated a reduced 5-fluorouracil dose. After completing FOLFOX therapy, she began second-line irinotecan/5-fluorouracil/leucovorin (FOLFIRI) plus BV therapy. In two cases, bone marrow suppression increased the difficulty of L-OHP dose escalation. We conclude that mFOLFOX-6 plus BV, with appropriate dose reduction, is acceptable for patients with chronic renal failure. Further data are needed to determine the adequate chemotherapy dose.

Nephrotic syndrome associated with ramucirumab therapy: A single-center case series and literature review.

Ramucirumab is a human immunoglobulin G1 monoclonal antibody that binds to vascular endothelial growth factor receptor 2 and is used for the treatment of metastatic or inoperable gastric, colorectal, and non-small cell lung cancers. However, ramucirumab can result in renal adverse events, including nephrotic syndrome, and the clinical course of this event is unclear. This study aimed to investigate the clinical course and pathological findings of patients with nephrotic syndrome after ramucirumab treatment.We evaluated 5 patients with malignancies (2 cases of gastric cancer and 3 cases of colorectal cancer) who developed nephrotic syndrome during treatment with ramucirumab. Two patients were diagnosed based on renal biopsy. We investigated the relationship between ramucirumab treatment and clinical courses, pathological findings, and renal outcomes.Four of 5 patients developed nephrotic syndrome after 1 or 2 doses of ramucirumab. All patients had hypertension, and 2 of 5 patients had renal dysfunction, defined as an increase in serum creatinine levels of ≥50% or ≥0.3 mg/dL. The 2 renal biopsy samples revealed a diffuse glomerular basement membrane double contour, intracapillary foam cell infiltration, and partial foot process effacement. Early drug discontinuation and antihypertensive therapy improved proteinuria, renal dysfunction, and hypertension in all patients.Nephrotic syndrome is a renal adverse event observed in cancer patients after ramucirumab treatment. We suggest that urinalysis, renal function, and blood pressure should be closely monitored in patients undergoing ramucirumab treatment, and treatment should be discontinued if renal adverse events are detected.

A case of entecavir-induced Fanconi syndrome.

Acquired Fanconi syndrome has been associated with the long-term ingestion of several nucleoside analogs used to treat chronic hepatitis B virus infection. However, the nucleoside analog entecavir has not been found to cause nephrotoxicity. We report a case of entecavir-induced Fanconi syndrome. Our patient was a 73-year-old man admitted to our hospital because of renal dysfunction. He also presented with hyperaminoaciduria, renal diabetes, phosphaturia, hypophosphatemia, hypokalemia, hypouricemia, and hyperchloremic metabolic acidosis, supporting a diagnosis of Fanconi syndrome. In this case, the cause of Fanconi syndrome was most likely entecavir, which had been administered as needed depending on his renal function for 5 years. After drug discontinuation and replacement with tenofovir alafenamide fumarate therapy once a week, the patient's kidney function recovered and electrolyte anomalies partially improved. We highlight the fact that entecavir may induce severe renal dysfunction, which can cause the development of Fanconi syndrome; therefore, close monitoring of proximal tubular function is recommended during entecavir therapy.

A suspected case of drug-induced tubulointerstitial nephritis by pilocarpine hydrochloride.

A 63-year-old man with pharyngeal cancer had been prescribed pilocarpine hydrochloride for xerostomia after concomitant chemoradiotherapy. After 6 months of taking pilocarpine hydrochloride, he was referred to our hospital due to gradually developing renal insufficiency. The patient underwent detailed urinalysis, blood chemistry analysis, immune-serology testing. A renal biopsy was also performed. He was diagnosed with chronic tubulointerstitial nephritis (TIN) caused by lymphocytic infiltration of the interstitium, tubular atrophy, and interstitial fibrotic changes. Infections, autoimmune diseases, and genetic factors were ruled out as causes of TIN; a drug-induced lymphocyte stimulation test confirmed that he had high stimulation index scores for pilocarpine hydrochloride and a normal range stimulation score for other supplements. These results indicated that the TIN could have been induced by pilocarpine hydrochloride. Drug discontinuation partly improved his renal function and tubule marker levels. To our knowledge, this is the first report of TIN following administration of pilocarpine hydrochloride. This finding could contribute to future treatment decisions for patients with TIN and those using pilocarpine hydrochloride.

Hematuria Alone or in Combination with Proteinuria Is a Harbinger of Poor Prognosis in Patients with Non-Hodgkin Lymphoma.

BACKGROUND: Screening for hematuria is essential during health checkups in the general population. However, urine examinations in patients with cancer tend to be overlooked. This study attempted to demonstrate the novel utility of urinalysis in the assessment of the prognosis of non-Hodgkin lymphoma (NHL). METHODS: A longitudinal, retrospective cohort study was conducted to examine the association between hematuria and mortality in 294 patients with NHL. Urinalysis was performed using a dipstick test. A multivariate, logistic regression model was constructed to evaluate factors associated with the presence of hematuria. Statistical association between hematuria and the time to all-cause mortality was analyzed using Kaplan-Meier analysis, followed by multivariate proportional hazards regression analysis adjusted for covariates that might be related to mortality. RESULTS: The prevalence of hematuria alone and in combination with proteinuria was 11.6 and 5.1%, respectively. C-reactive protein was a significant factor associated with the presence of hematuria (OR [95% CI] 1.17 [1.03-1.34], p = 0.0194). The cumulative mortality was significantly higher in patients with hematuria alone (51.1%), proteinuria alone (47.1%), and both (66.7%), than in those with neither (24.3%). Moreover, the presence of hematuria alone was significantly associated with all-cause mortality (hazard ratio [95% CI] 1.78 [1.10-3.50], p = 0.0455), and patients with concomitant proteinuria were at the highest risk (4.01 [1.71-8.33], p = 0.0001). CONCLUSIONS: In patients with hematuric NHL, systemic inflammation is likely to develop to such a great extent that kidney damage occurs. Therefore, the presence of hematuria, alone or especially in combination with proteinuria, predicts a poor prognosis of NHL.

Anticentromere Antibody-positive Scleroderma Renal Crisis Requiring Dialysis.

A 70-year-old man with prior Raynaud's phenomena developed hypertension and renal insufficiency. Raynaud's phenomena, finger skin thickening, interstitial lung disease, and positive anticentromere antibody findings indicated systemic sclerosis (SSc). Based on the presence of SSc, severe hypertension with rapidly progressive renal failure, and proliferative and obliterative arteriolar vasculopathy, scleroderma renal crisis (SRC) was diagnosed. Despite good blood pressure control with antihypertensive drugs, hemodialysis was initiated and could not be withdrawn owing to unimproved renal dysfunction. Although SRC in anticentromere antibody-positive limited cutaneous SSc is extremely rare, some patients may develop SRC, and their renal prognosis may be poor.

Prevalence and mortality of chronic kidney disease in lymphoma patients: A large retrospective cohort study.

In patients with lymphoma, an important issue that has been recognized is renal involvement, including glomerulonephritis, acute kidney injury, and lymphoma infiltrating the kidney. However, the prevalence and mortality of chronic kidney disease (CKD) have not been fully understood in lymphoma patients. This study aimed to evaluate the prevalence of CKD and its impact on mortality in those patients.This was a retrospective cohort study of 429 consecutive lymphoma patients who were admitted or regularly visited our hospital from January 2013 to October 2016. CKD was defined as estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m and/or proteinuria ≥ 1+ that was sustained for at least 3 months. The prevalence of CKD at enrollment was evaluated according to the modified CKD classification by Kidney Disease: Improving Global Outcomes (KDIGO) (eGFR and proteinuria category). Dipstick proteinuria was classified into 3 grades: A1 for - and ±; A2 for 1+ or 2+; and A3 for ≥3+. The eGFR (mL/min/1.73 m) was classified into 6 stages: G1 for ≥90, G2 for 60 to 89, G3a for 45 to 59, G3b for 30 to 44, G4 for 15 to 29, and G5 for <15. The cumulative mortality rate was estimated using the Kaplan-Meier method, with stratification into 2 groups based on the presence or absence of CKD. Furthermore, a multivariate Cox proportional hazards regression model was used to calculate the hazard ratio (HR) and its 95% confidence interval (CI) for all-cause mortality, after adjustments for age, sex, pathologic type, clinical stage of lymphoma, presence or absence of diabetes mellitus, hypertension, and cardiovascular disease.The mean follow-up period was 3.06 ±â€Š0.96 years, and the prevalence of CKD at study enrollment was 34.5%. The cumulative mortality rate was 20.7%, and was significantly higher in the CKD group than in the group without CKD (36.4% vs 18.0%, P = .02). Multivariate analysis found mortality to be significantly associated with CKD (HR 1.58; 95% CI, 1.01-2.46), and this association was the most robust with very high-risk CKD (HR 6.94; 95% CI, 2.50-17.33).The prevalence of CKD in lymphoma patients was high. CKD should be considered an independent risk factor for mortality among patients with lymphoma.

The renal pathological findings in Japanese HIV-infected individuals with CKD: a clinical case series from a single center.

BACKGROUND: Chronic kidney diseases (CKD) have emerged as a significant cause of morbidity and mortality in patients infected with human immunodeficiency virus (HIV). However, the detailed study of renal pathological findings currently remains unclear in these Japanese patients. METHODS: A retrospective cohort study was undertaken to investigate renal pathological findings between January 1996 and July 2016. Our study included 20 Japanese HIV-infected patients with CKD; 10 cases had undergone renal biopsies, and 10 cases had undergone autopsies, respectively. Moreover, in the 10 biopsied patients, their clinical courses as well as renal outcomes after renal biopsy were also reviewed. RESULTS: All of the patients had received combination antiretroviral therapy (cART). The 10 biopsy cases (mean age, 54 ± 14 years and duration of cART, 8 ± 5 years) included three cases of diabetic nephropathy (DMN), two of IgA nephropathy, two of cART-induced tubulointerstitial nephritis (TIN), one of minimal change disease, one case of only finding intrarenal arterioles, and one case without abnormal findings. Among those patients, their clinical courses were preferable except for in the DMN cases. In the autopsy cases (mean age, 52 ± 10 years and duration of cART, 5 ± 5 years), no distinct mesangial or membranous abnormalities were detected. Mild to moderate tubulointerstitial atrophies were observed in six cases. Intrarenal arteriosclerosis was identified in nine cases, and the proportion of global glomerulosclerosis seen was 8.4 ± 12.5%/100 glomeruli. CONCLUSION: DMN and cART-induced TIN was noted in the biopsy cases. In the autopsy cases, renal arteriosclerosis, global glomerulosclerosis, and tubulointerstitial atrophy were remarkable. Early diagnosis of kidney diseases should be crucial to introduce optimal management, including controlling rigorous comorbidities and appropriate use of cART, to prevent further progression of CKD.

Increased non-HDL-C level linked with a rapid rate of renal function decline in HIV-infected patients.

BACKGROUND: The risk of developing CKD is increased in HIV-infected patients; however, the relationship between renal function decline and lipid abnormalities currently remains unclear in these patients. METHODS: A retrospective cohort study was conducted on 661 HIV-infected patients, whose estimated glomerular filtration rates (eGFRs) were consecutively measured over 6 years. The rate of declines in eGFR per year was calculated, with decreases being evaluated using a linear mixed effect model. The distribution of decreases in eGFR ≥ 30 % from baseline during the follow-up period was compared across quartiles of non-high-density lipoprotein cholesterol (HDL-C) levels using the Cochran-Armitage test. A multivariate logistic regression model was built to examine the relationship between dyslipidemia and decreases in eGFR. RESULTS: The prevalence of CKD increased from 8.5 to 21.2 % during the follow-up. The average of 6 annual eGFR decline rates was 2.01 ± 0.09 ml/min/1.73 m2/year, which was more than 6-fold higher than that of age-matched controls. The distribution of decreases in eGFR significantly increased across the quartiles of non-HDL-C (p value for trend = 0.0359). Non-HDL-C levels greater than the median value of the cohort were identified as a significant risk factor for decreased eGFR [odds ratio (95 % confidence interval), 1.77 (1.07-3.00)]. CONCLUSION: Increased non-HDL-C levels are a risk factor for renal function decline in HIV-infected patients.

Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene.

Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four different genes: with-no-lysine kinases (WNK) 1 and 4, Kelch-like family member 3 (KLHL3), and cullin 3 (Cul3). Cul3 and KLHL3 form an E3 ligase complex that ubiquitinates and reduces the expression level of WNK4. PHAII-causing mutations in WNK4 and KLHL3 impair WNK4 ubiquitination. However, the molecular pathogenesis of PHAII caused by Cul3 mutations is unclear. In cultured cells and human leukocytes, PHAII-causing Cul3 mutations result in the skipping of exon 9, producing mutant Cul3 protein lacking 57 amino acids. However, whether this phenomenon occurs in the kidneys and is responsible for the pathogenesis of PHAII in vivo is unknown. We generated knock-in mice carrying a mutation in the C-terminus of intron 8 of Cul3, c.1207-1G>A, which corresponds to a PHAII-causing mutation in the human Cul3 gene. Heterozygous Cul3(G(-1)A/+) knock-in mice did not exhibit PHAII phenotypes, and the skipping of exon 9 was not evident in their kidneys. However, the level of Cul3 mRNA expression in the kidneys of heterozygous knock-in mice was approximately half that of wild-type mice. Furthermore, homozygous knock-in mice were nonviable. It suggested that the mutant allele behaved like a knockout allele and did not produce Cul3 mRNA lacking exon 9. A reduction in Cul3 expression alone was not sufficient to develop PHAII in the knock-in mice. Our findings highlighted the pathogenic role of mutant Cul3 protein and provided insight to explain why PHAII-causing mutations in Cul3 cause kidney-predominant PHAII phenotypes.

Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal.

The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction.

The WNK (with no lysine kinase)-SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin-RING E3 ubiquitin ligase complexes. To explore how a CUL3-KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na(+)/Cl(-) co-transporter)/NKCC1 (Na(+)/K(+)/2Cl(-) co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3-KLHL3 E3 ligase complex, but not a disease-causing CUL3-KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479-667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479-667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3-KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.

Phosphorylation of Na-Cl cotransporter by OSR1 and SPAK kinases regulates its ubiquitination.

Na-Cl cotransporter (NCC) is phosphorylated in its amino terminus based on salt intake under the regulation of the WNK-OSR1/SPAK kinase cascade. We have observed that total protein abundance of NCC and its apical membrane expression varies in the kidney based on the phosphorylation status. To clarify the mechanism, we examined NCC ubiquitination status in mice fed low, normal and high salt diets, as well as in a model mouse of pseudohypoaldosteronism type II (PHAII) where NCC phosphorylation is constitutively elevated. Low-salt diet decreased NCC ubiquitination, while high-salt diet increased NCC ubiquitination in the kidney, and this was inversely correlated with total and phosphorylated NCC abundance. In the PHAII model, the ubiquitination of NCC in kidney was also lower when compared to that in wild-type littermates. To evaluate the relationship between phosphorylation and ubiquitination of NCC, we expressed wild-type, phospho-deficient and -mimicking NCC in COS7 cells, and the ubiquitination of immunoprecipitated total and biotinylated surface NCC was evaluated. NCC ubiquitination was increased in the phospho-deficient NCC and decreased in phospho-mimicking NCC in both total and surface NCC. Thus, we demonstrated that NCC phosphorylation decreased NCC ubiquitination, which may contribute to the increase of NCC abundance mostly on plasma membranes.

Effect of heterozygous deletion of WNK1 on the WNK-OSR1/ SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels.

BACKGROUND: We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1(+/-)mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1(+/-) mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1(+/-) mice have not been performed. METHOD: We assess the effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels. RESULTS: Contrary to the previous report, the blood pressure of WNK1(+/-) mice was not decreased, even under a low-salt diet. Under a WNK4(D561A/+) background, the heterozygous deletion of the WNK1 gene did not reduce the high blood pressure either. We then evaluated the phosphorylation status of OSR1, SPAK, NCC, NKCC1, and NKCC2 in the kidney, but no significant decrease in the phosphorylation was observed in WNK1(+/-) mice or WNK1(+/-)WNK4(D561A/+) mice. In contrast, a significant decrease in NKCC1 phosphorylation in the aorta and a decreased pressure-induced myogenic response in the mesenteric arteries were observed in WNK1(+/-) mice. CONCLUSION: The contribution of WNK1 to total WNK kinase activity in the kidney may be small, but that WNK1 may play a substantial role in the regulation of blood pressure in the arteries.

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.

Acute insulin stimulation induces phosphorylation of the Na-Cl cotransporter in cultured distal mpkDCT cells and mouse kidney.

The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin.

Immunolocalization of WNK4 in mouse kidney.

Initial reports claim that WNK4 localization is mainly at intercellular junctions of distal convoluted tubules (DCT) and cortical collecting ducts (CCD) in the kidney. However, we recently clarified the major targets of WNK4 kinase to be the OSR1/SPAK kinases and the Na-Cl co-transporter (NCC), an apical membrane protein in the DCT, thus raising the question of whether the cellular localization of WNK4 is at intercellular junctions. In this study, we re-evaluate the intrarenal and intracellular immunolocalization of WNK4 in the mouse kidney using a newly generated anti-WNK4 antibody. By performing double immunofluorescence of WNK4 with several nephron-segment-specific markers, we have found that WNK4 is present in podocytes in glomeruli, the cortical thick ascending limb of Henle's loop including macula densa, and the medullary collecting ducts (MCD), in addition to the previously identified nephron segments, i.e., DCT and CCD. These results are consistent with the finding that WNK4 constitutes a kinase cascade with OSR1/SPAK and NCC in the DCT, and highlights a novel role for WNK4 in nephron segments newly identified as being WNK4-positive in this study.