Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. METHODS AND RESULTS: We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (Sp7), osteocalcin (Bglap), and osteopontin (Spp1) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, miR-16-5p, miR-17~92 cluster-originated miR-17-5p/miR-20a-5p, and miR-106b-5p. Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in VEGFA transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or VEGFR2-targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of miR-16-5p, miR-17-5p, 20a-5p, and miR-106b-5p for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. CONCLUSIONS: The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.

LRBA is essential for urinary concentration and body water homeostasis.

Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.

Absence of ULK1 decreases AMPK activity in the kidney, leading to chronic kidney disease progression.

AMP-activated protein kinase (AMPK) inactivation in chronic kidney disease (CKD) leads to energy status deterioration in the kidney, constituting the vicious cycle of CKD exacerbation. Unc-51-like kinase 1 (ULK1) is considered a downstream molecule of AMPK; however, it was recently reported that the activity of AMPK could be regulated by ULK1 conversely. We demonstrated that AMPK and ULK1 activities were decreased in the kidneys of CKD mice. However, whether and how ULK1 is involved in the underlying mechanism of CKD exacerbation remains unknown. In this study, we investigated the ULK1 involvement in CKD, using ULK1 knockout mice. The CKD model of Ulk1-/- mice exhibited significantly exacerbated renal function and worsening renal fibrosis. In the kidneys of the CKD model of Ulk1-/- mice, reduced AMPK and its downstream ß-oxidation could be observed, leading to an energy deficit of increased AMP/ATP ratio. In addition, AMPK signaling in the kidney was reduced in control Ulk1-/- mice with normal renal function compared to control wild-type mice, suggesting that ULK1 deficiency suppressed AMPK activity in the kidney. This study is the first to present ULK1 as a novel therapeutic target for CKD treatment, which regulates AMPK activity in the kidney.

A case of unexpected diagnosis of fibronectin glomerulopathy with histological features of membranoproliferative glomerulonephritis.

Fibronectin (FN) glomerulopathy (FNG), a rare autosomal hereditary renal disease, is characterized by proteinuria resulting from the massive accumulation of FN in the glomeruli. It typically affects individuals aged 10-50 years. In this report, we describe the case of a 57-year-old man who was diagnosed with FNG through genetic analysis and histological examination that revealed membranoproliferative glomerulonephritis. Despite treatment with prednisolone, the therapeutic response was unsatisfactory. Prednisolone was subsequently tapered and discontinued because the patient had pulmonary thromboembolism. Subsequent comprehensive genetic testing, which was initially not conducted because the patient's parents did not have a history of kidney disease, identified a known disease-causing variant in the FN1 gene, indicating a de novo variant. FNG was further confirmed by positive staining of glomeruli with FN using an IST-4 antibody. Although corticosteroid therapy is commonly employed as the initial treatment for MPGN, its appropriateness depends on the underlying etiology. Thus, clinicians must be aware of potential rare genetic causes underlying MPGN.

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.

LRBA signalosomes activate vasopressin-induced AQP2 trafficking at recycling endosomes.

Aquaporin-2 (AQP2) water channels are proteins that are recycled between intracellular vesicles and the apical plasma membrane in renal collecting ducts. Lipopolysaccharide-responsive beige-like anchor protein (LRBA) is a protein kinase A (PKA) anchoring protein that creates compartmentalized PKA signalling responsible for AQP2 phosphorylation. In response to increased plasma osmolality, vasopressin/cyclic adenosine monophosphate (cAMP)/PKA signalling phosphorylates AQP2, promoting AQP2 trafficking into the apical plasma membrane and increasing water reabsorption from urine. However, the molecular mechanisms by which LRBA mediates vasopressin-induced AQP2 phosphorylation remain unknown. To investigate AQP2 intracellular localization and phosphorylation status in vivo, a density gradient ultracentrifugation technique was combined with an in situ proximity ligation assay, super-resolution structured illumination microscopy and immunoelectron microscopy. Most of the AQP2 was localized on the recycling endosome in the presence of tolvaptan, a vasopressin type 2 receptor (V2R) antagonist. Desmopressin, a V2R agonist, phosphorylated AQP2, translocating it from the recycling endosome to the apical plasma membrane. In contrast, LRBA was constitutively localized at the recycling endosome. Therefore, LRBA and AQP2 were well colocalized in the absence of vasopressin stimulation. The loss of LRBA/PKA signalling by Lrba knockout impaired vasopressin-induced AQP2 phosphorylation, resulting in AQP2 retention at the recycling endosome. Defective AQP2 trafficking caused low urinary concentrating ability in Lrba-/- mice. The LRBA-PKA complex created compartmentalized PKA signalling at the recycling endosome, which facilitated AQP2 phosphorylation in response to vasopressin. KEY POINTS: Membrane proteins are continuously internalized into the endosomal system via endocytosis, after which they are either recycled back to the plasma membrane or degraded at the lysosome. In T cells, lipopolysaccharide-responsive beige-like anchor protein (LRBA) binds directly to the cytotoxic T lymphocyte antigen 4 (CTLA-4), a checkpoint immune molecule, to prevent CTLA-4 lysosomal degradation and promote its vesicle recycling. LRBA has different physiological functions in renal collecting ducts. LRBA and aquaporin-2 (AQP2) water channels were colocalized on the recycling endosome in vivo in the absence of the anti-diuretic hormone vasopressin. LRBA promoted vasopressin-induced AQP2 trafficking, increasing water reabsorption from urine via AQP2. LRBA determined renal responsiveness to vasopressin at recycling endosomes. LRBA is a ubiquitously expressed anchor protein. LRBA signalosomes might regulate membrane trafficking of several constitutively recycled proteins at recycling endosomes.

Rapidly progressive IgA nephropathy with membranoproliferative glomerulonephritis-like lesions in an elderly man following the third dose of an mRNA COVID-19 vaccine: a case report.

BACKGROUND: As messenger RNA (mRNA)-based vaccines for coronavirus disease 2019 (COVID-19) have been administered to millions of individuals worldwide, cases of de novo and relapsing glomerulonephritis after mRNA COVID-19 vaccination are increasing in the literature. While most previous publications reported glomerulonephritis after the first or second dose of an mRNA vaccine, few reports of glomerulonephritis occurring after the third dose of an mRNA vaccine currently exist. CASE PRESENTATION: We report a case of rapidly progressive glomerulonephritis in a patient following the third dose of an mRNA COVID-19 vaccine. A 77-year-old Japanese man with a history of hypertension and atrial fibrillation was referred to our hospital for evaluation of anorexia, pruritus, and lower extremity edema. One year before referral, he received two mRNA vaccines (BNT162b2) for COVID-19. Three months before the visit, he received a third mRNA vaccine (mRNA-1273) for COVID-19. On admission, the patient presented severe renal failure with a serum creatinine level of 16.29 mg/dL, which had increased from 1.67 mg/dL one month earlier, prompting us to initiate hemodialysis. Urinalysis showed nephrotic-range proteinuria and hematuria. Renal biopsy revealed mild mesangial proliferation and expansion, a lobular appearance, and double contours of the glomerular basement membrane. Renal tubules had severe atrophy. Immunofluorescence microscopy showed strong mesangial staining for IgA, IgM, and C3c. Electron microscopy exhibited mesangial and subendothelial electron-dense deposits, leading to a diagnosis of IgA nephropathy with membranoproliferative glomerulonephritis-like changes. The kidney function remained unchanged after steroid therapy. CONCLUSIONS: Although the link between renal lesions and mRNA vaccines remains unclear, a robust immune response induced by mRNA vaccines may play a role in the pathogenesis of glomerulonephritis. Further studies of the immunological effects of mRNA vaccines on the kidney are warranted.

Phenotypic differences of mutation-negative cases in Gitelman syndrome clinically diagnosed in adulthood.

Gitelman syndrome (GS), an autosomal recessive kidney disorder, is characterized by hypokalemia, hypomagnesemia, hypocalciuria, and metabolic alkalosis. Generally, diagnosis is made in school-aged children but multiple cases have been diagnosed in adulthood. This study examines the phenotypic differences between genetically confirmed cases and mutation-negative cases in adults. A comprehensive screening of 168 genes, including GS-related genes, was performed for 84 independent individuals who were referred to our institute with a clinical diagnosis of GS. The cases of pseudo-Bartter syndrome (BS)/GS because of diuretic abuse or other causes, which was determined based on patients' medical records, were excluded during registration. Of these 70 eligible cases for analysis, 27 (38.6%) had genetic confirmation of GS, while 37 (52.8%) had no known variants associated with GS and were considered to be unsolved cases. Note that unsolved cases comprised older, mostly female, individuals with decreased kidney function and multiple basic features of GS. The phenotype of unsolved cases is similar to that of pseudo BS/GS cases, although these cases were excluded in advance. However, the genetic and autoimmune profiles of these unsolved cases have not yet been investigated to date. Therefore, these cases may be categorized into new disease groups.

Deletion of Alox15 improves kidney dysfunction and inhibits fibrosis by increased PGD2 in the kidney.

BACKGROUND: Lipid-metabolizing enzymes and their metabolites affect inflammation and fibrosis, but their roles in chronic kidney disease (CKD) have not been completely understood. METHODS: To clarify their role in CKD, we measured the mRNA levels of major lipid-metabolizing enzymes in 5/6 nephrectomized (Nx) kidneys of C57BL/6 J mice. Mediator lipidomics was performed to reveal lipid profiles of CKD kidneys. RESULTS: In 5/6 Nx kidneys, both mRNA and protein levels of Alox15 were higher when compared with those in sham kidneys. With respect to in situ hybridization, the mRNA level of Alox15 was higher in renal tubules of 5/6 Nx kidneys. To examine the role of Alox15 in CKD pathogenesis, we performed 5/6 Nx on Alox15-/- mice. Alox15-/- CKD mice exhibited better renal functions than wild-type mice. Interstitial fibrosis was also inhibited in Alox15-/- CKD mice. Mediator lipidomics revealed that Alox15-/- CKD mouse kidneys had significantly higher levels of PGD2 than the control. To investigate the effects of PGD2 on renal fibrosis, we administered PGD2 to TGF-ß1-stimulated NRK-52E cells and HK-2 cells, which lead to a dose-dependent suppression of type I collagen and αSMA in both cell lines. CONCLUSION: Increased PGD2 in Alox15-/- CKD mouse kidneys could inhibit fibrosis, thereby resulting in CKD improvement. Thus, Alox15 inhibition and PGD2 administration may be novel therapeutic targets for CKD.

Renal TNFα activates the WNK phosphorylation cascade and contributes to salt-sensitive hypertension in chronic kidney disease.

The inappropriate over-activation of the with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-sodium chloride cotransporter (NCC) phosphorylation cascade increases sodium reabsorption in distal kidney nephrons, resulting in salt-sensitive hypertension. Although chronic kidney disease (CKD) is a common cause of salt-sensitive hypertension, the involvement of the WNK phosphorylation cascade is unknown. Moreover, the effect of immune systems on WNK kinases has not been investigated despite the fact that immune systems are important for salt sensitivity. Here we demonstrate that the protein abundance of WNK1, but not of WNK4, was increased at the distal convoluted tubules in the aristolochic acid nephropathy mouse model of CKD. Accordingly, the phosphorylation of both SPAK and NCC was also increased. Moreover, a high-salt diet did not adequately suppress activation of the WNK1-SPAK-NCC phosphorylation cascade in this model, leading to salt-sensitive hypertension. WNK1 also was increased in adenine nephropathy, but not in subtotal nephrectomy, models of CKD. By comparing the transcripts of these three models focusing on immune systems, we hypothesized that tumor necrosis factor (TNF)-α regulates WNK1 protein expression. In fact, TNF-α increased WNK1 protein expression in cultured renal tubular cells by reducing the transcription and protein levels of NEDD4-2 E3-ligase, which degrades WNK1 protein. Furthermore, the TNF-α inhibitor etanercept reversed the reduction of NEDD4-2 expression and upregulation of the WNK1-SPAK-NCC phosphorylation cascade in distal convoluted tubules in vivo in the aristolochic acid nephropathy model. Thus, salt-sensitive hypertension is induced in CKD via activation of the renal WNK1- SPAK-NCC phosphorylation cascade by TNF-α, reflecting a link with the immune system.

WNK1-TAK1 signaling suppresses lipopolysaccharide-induced cytokine production and classical activation in macrophages.

With-no-lysine kinase (WNK) plays important roles in regulating electrolyte homeostasis, cell signaling, survival, and proliferation. It has been recently demonstrated that WNK1, a member of the WNK family, modifies the function of immune cells. Here we report that in macrophages, WNK1 has suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses via TGFß-activated kinase 1 (TAK1)-mediated activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. We found that WNK1 heterozygous (WNK1+/-) mice produced excessive proinflammatory cytokines in an experimental LPS-induced sepsis model, and peritoneal macrophages isolated from WNK1+/- mice produced higher levels of LPS-induced cytokines and NOS2 expression as canonical proinflammatory M1 macrophage markers. We confirmed that small hairpin RNA (shRNA)-mediated knockdown of WNK1 activated LPS-induced cytokine production and NOS2 expression in RAW 264.7 macrophages. Moreover, we demonstrated that WNK1 knockdown increased the nuclear translocation of NF-κB and activated the p38 and Jun N-terminal kinase (JNK) MAPK signaling pathway and that a TAK1 inhibitor diminished these effects of WNK1 knockdown. These results suggest that WNK1 acts as a physiologic immune modulator via interactions with TAK1. WNK1 may be a therapeutic target against the cytokine storm caused by sepsis.

Tacrolimus ameliorates the phenotypes of type 4 Bartter syndrome model mice through activation of sodium-potassium-2 chloride cotransporter and sodium-chloride cotransporter.

Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the ß-subunit of the ClC-K chloride (Cl-) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Na+)-potassium (K+)-2Cl- cotransporter (NKCC2) and Na+-Cl- cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum K+ level and suppressed urinary K+ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS.

Interleukin-1ß Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis.

Background Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined.Methods RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1α and MYC transcription factors, respectively. We modeled this metabolic switch in vivo, in experimental murine models of kidney injury, and in vitro in human kidney stromal cells (SCs) and human kidney organoids.Results In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. In vitro, stimulation of purified human kidney SCs and human kidney organoids with IL-1ß recapitulated the molecular events observed in vivo, inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1ß stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 in vivo or inhibition of MYC in vivo as well as in human kidney organoids in vitro abrogated fibrosis and reduced tubular injury.Conclusions Our findings define a connection between IL-1ß and metabolic switch in fibrosis initiation and progression and highlight IL-1ß and MYC as potential therapeutic targets in tubulointerstitial diseases.

Decreased KLHL3 expression is involved in the pathogenesis of pseudohypoaldosteronism type II caused by cullin 3 mutation in vivo.

BACKGROUND: Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four genes: WNK1, WNK4, Kelch-like3 (KLHL3), and cullin3 (CUL3). Recently, it was revealed that CUL3-KLHL3 E3 ligase complex ubiquitinates WNK1 and WNK4, leading to their degradation, and that a common pathogenesis of PHAII is defective WNK degradation due to CUL3-KLHL3 E3 ligase complex impairment. PHAII-causing CUL3 mutations mediate exon9 skipping, producing a CUL3 protein with a 57-amino acid deletion (Δ403-459). However, the pathogenic effects of KLHL3, an adaptor protein that links WNKs with CUL3, in PHAII caused by CUL3 mutation remain unclear. METHODS: To clarify detailed pathophysiological mechanisms underlying PHAII caused by CUL3 mutation in vivo, we generated and analyzed knock-in mice carrying the same CUL3 exon9 deletion (CUL3WT/Δex9) as that reported in PHAII patients. RESULTS: CUL3WT/Δex9 mice exhibited a PHAII-like phenotype. Interestingly, we confirmed markedly decreased KLHL3 expression in CUL3WT/Δex9 mice by confirming the true KLHL3 band in vivo. However, the expression of other KLHL family proteins, such as KLHL2, was comparable between WT and mutant mice. CONCLUSION: KLHL3 expression was decreased in CUL3WT/Δex9 mice. However, expression levels of other KLHL family proteins were comparable between the wild-type and mutant mice. These findings indicate that the decreased abundance of KLHL3 is a specific phenomenon caused by mutant CUL3 (Δexon9). Our findings would improve our understanding of the pathogenesis of PHAII caused by CUL3 mutation in vivo.

WNK4 is indispensable for the pathogenesis of pseudohypoaldosteronism type II caused by mutant KLHL3.

WNK-OSR1/SPAK-NCC signaling cascade is important for regulating salt balance and blood pressure. Activation of WNK-OSR1/SPAK-NaCl cotransporter (NCC) cascade increases sodium reabsorption in the kidney, leading to pseudohypoaldosteronism type II (PHA II) characterized by salt-sensitive hypertension and hyperkalemia. It has been previously demonstrated that the amount of phosphorylated and total NCC markedly decreased in WNK4-/- mice, indicating that WNK4 plays a major role for activation of OSR1/SPAK-NCC signaling. However, it is unclear whether absence of WNK4 can be compensated by other WNK kinases. We recently reported that KLHL3R528H/+ knock-in mice, a PHAII model, exhibited augmented activation of OSR1/SPAK-NCC signaling by increased protein levels of both WNK1 and WNK4 due to impaired protein degradation by the mutant KLHL3. In this study, we sought to determine the contribution of WNK4 to OSR1/SPAK-NCC signaling using an in vivo model which shows extremely increased WNK1 with absence of WNK4. We generated WNK4-/-KLHL3R528H/+ mice and WNK4-/-KLHL3R528H/R528H mice by crossing WNK4-/- mice with KLHL3R528H/+ mice. Thereafter, WNK-OSR1/SPAK-NCC phosphorylation signal cascade was examined in kidneys from these mice. As expected, both WNK4-/-KLHL3R528H/+ mice and WNK4-/-KLHL3R528H/R528H mice demonstrated increased WNK1 in the kidney, due to the KLHL3 mutation, and WNK4 deficiency. However, phosphorylation of SPAK and NCC at distal convoluted tubules were almost completely absent even in WNK4-/-KLHL3R528H/R528H mice. In conclusion, increased WNK1 was unable to compensate for WNK4 deficiency and phosphorylate the NCC, indicating that WNK4 is indispensable for the onset of PHAII.

Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice.

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na-Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3(R528H/+) knock-in mice. KLHL3(R528H/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R528H/+) mouse kidney, indicating that the KLHL3(R528H/+) knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R528H/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528H/+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.

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.

Effect of osteosarcopenia on longitudinal mortality risk and chronic kidney disease progression in older adults.

INTRODUCTION: Chronic kidney disease (CKD) causes a progressive loss of muscle and bone mass, which frequently overlap with and affect clinical outcomes. However, the impact of sarcopenia, low bone mineral density (BMD; osteopenia or osteoporosis), and osteosarcopenia (sarcopenia and low BMD) on CKD progression is yet to be determined. We aimed to address these issues in patients with CKD without kidney replacement therapy (KRT). METHODS: This prospective cohort study included 251 outpatients aged ≥65 years with CKD without KRT enrolled in our hospital between June 2016 and March 2017. Sarcopenia was defined according to the 2014 criteria of the Asian Working Group for Sarcopenia (AWGS), and low BMD was defined as a T-score of ≤-1.0. The patients were divided into four groups: normal (no sarcopenia/normal BMD), only low BMD (no sarcopenia/low BMD), only sarcopenia (sarcopenia/normal BMD), and osteosarcopenia (sarcopenia/low BMD). The primary outcome was a composite of all-cause deaths, initiating KRT, and admissions owing to major adverse cardiovascular and cerebrovascular events (MACEs). The secondary outcome was a kidney composite outcome that included a 30 % reduction in creatinine-based estimated glomerular filtration rate (eGFR) and initiating KRT. The outcome risk was determined using the Cox regression models adjusted for potential confounders. RESULTS: Median age (25th-75th percentile) and eGFR of the outpatients (35 % women) were 76 (69-81) years and 32.1 (20.8-41.7) ml/min/1.73 m2, respectively. During a median follow-up period of 5.2 years, there were 22 deaths, 117 30 % eGFR reductions, 48 KRTs, and 18 admissions owing to MACEs. The osteosarcopenia group rather than the only low BMD or only sarcopenia groups exhibited a higher risk of the primary (hazard ratio [HR]: 3.28, 95 % confidence interval [CI]: 1.52-7.08) and kidney composite (HR: 2.07, 95 % CI: 1.10-3.89) outcomes. Among the osteosarcopenia-related body compositions and physical functions, low handgrip strength (HGS) was strongly associated with a high risk of primary and kidney composite outcomes (HR: 2.44, 95 % CI: 1.46-4.08; HR: 1.48, 95 % CI: 0.97-2.24, respectively). The increase in HGS but not the body mass index, skeletal muscle mass index, or BMD was associated with lower risks of primary and kidney composite outcomes (HR: 0.93, 95 % CI: 0.89-0.98; HR: 0.96, 95 % CI: 0.92-0.99 per 1 kg, respectively). CONCLUSIONS: Osteosarcopenia was associated with poor survival and kidney outcomes in older patients with CKD. Low HGS, which is common in patients with osteosarcopenia and CKD, was associated with increased mortality risk and kidney function decline. These findings can help the risk prediction and pathogenesis of the kidney-bone-muscle axis and improving muscle strength can help mitigate CKD progression.

Impact of COVID-19 versus other pneumonia on in-hospital mortality and functional decline among Japanese dialysis patients: a retrospective cohort study.

Coronavirus disease 2019 (COVID-19) affects both life and health. However, the differentiation from other types of pneumonia and effect of kidney disease remains uncertain. This retrospective observational study investigated the risk of in-hospital death and functional decline in ≥ 20% of Barthel Index scores after COVID-19 compared to other forms of pneumonia among Japanese adults, both with and without end-stage kidney disease (ESKD). The study enrolled 123,378 patients aged 18 years and older from a national inpatient administrative claims database in Japan that covers the first three waves of the COVID-19 pandemic in 2020. After a 1:1:1:1 propensity score matching into non-COVID-19/non-dialysis, COVID-19/non-dialysis, non-COVID-19/dialysis, and COVID-19/dialysis groups, 2136 adults were included in the analyses. The multivariable logistic regression analyses revealed greater odds ratios (ORs) of death [5.92 (95% CI 3.62-9.96)] and functional decline [1.93 (95% CI 1.26-2.99)] only in the COVID-19/dialysis group versus the non-COVID-19/non-dialysis group. The COVID-19/dialysis group had a higher risk of death directly due to pneumonia (OR 6.02, 95% CI 3.50-10.8) or death due to other diseases (OR 3.00, 95% CI 1.11-8.48; versus the non-COVID-19/non-dialysis group). COVID-19 displayed a greater impact on physical function than other types of pneumonia particularly in ESKD.

CFAP47 is a novel causative gene implicated in X-linked polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a well-described condition in which ~80% of cases have a genetic explanation, while the genetic basis of sporadic cystic kidney disease in adults remains unclear in ~30% of cases. This study aimed to identify novel genes associated with polycystic kidney disease (PKD) in patients with sporadic cystic kidney disease in which a clear genetic change was not identified in established genes. A next-generation sequencing panel analyzed known genes related to renal cysts in 118 sporadic cases, followed by whole-genome sequencing on 47 unrelated individuals without identified candidate variants. Three male patients were found to have rare missense variants in the X-linked gene Cilia And Flagella Associated Protein 47 (CFAP47). CFAP47 was expressed in primary cilia of human renal tubules, and knockout mice exhibited vacuolation of tubular cells and tubular dilation, providing evidence that CFAP47 is a causative gene involved in cyst formation. This discovery of CFAP47 as a newly identified gene associated with PKD, displaying X-linked inheritance, emphasizes the need for further cases to understand the role of CFAP47 in PKD.