Consumption of low dietary potassium, common with ultra-processed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the WNK-SPAK kinase pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high potassium “DASH-like” diets inactivate the cotransporter and whether this decreases BP. A transcriptomic screen identified Ppp1C⍺, encoding PP1A, as a potassium up-regulated gene, and its negative regulator, Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK-SPAK kinase cascade, we confirmed that PP1A dephosphorylates NCC directly in a potassium-regulated manner. Prior adaptation to a high potassium diet was required to maximally dephosphorylate NCC and lower BP in the constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a, and dephosphorylation of its cognate protein, Inhibitory Subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drives NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK-SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.
Paul Richard Grimm, Anamaria Tatomir, Lena L. Rosenbaek, Bo Young Kim, Dimin Li, Eric J. Delpire, Robert A. Fenton, Paul A. Welling
Diabetic kidney disease (DKD) can lead to end-stage kidney disease (ESKD) and mortality, however, few mechanistic biomarkers are available for high risk patients, especially those without macroalbuminuria. Urine from participants with diabetes from Chronic Renal Insufficiency Cohort (CRIC), Singapore Study of Macro-Angiopathy and Reactivity in Type 2 Diabetes (SMART2D), and the Pima Indian Study determined if urine adenine/creatinine ratio (UAdCR) could be a mechanistic biomarker for ESKD. ESKD and mortality were associated with the highest UAdCR tertile in CRIC (HR 1.57, 1.18, 2.10) and SMART2D (HR 1.77, 1.00, 3.12). ESKD was associated with the highest UAdCR tertile in patients without macroalbuminuria in CRIC (HR 2.36, 1.26, 4.39), SMART2D (HR 2.39, 1.08, 5.29), and Pima Indian study (HR 4.57, CI 1.37-13.34). Empagliflozin lowered UAdCR in non-macroalbuminuric participants. Spatial metabolomics localized adenine to kidney pathology and transcriptomics identified ribonucleoprotein biogenesis as a top pathway in proximal tubules of patients without macroalbuminuria, implicating mammalian target of rapamycin (mTOR). Adenine stimulated matrix in tubular cells via mTOR and stimulated mTOR in mouse kidneys. A specific inhibitor of adenine production was found to reduce kidney hypertrophy and kidney injury in diabetic mice. We propose that endogenous adenine may be a causative factor in DKD.
Kumar Sharma, Guanshi Zhang, Jens Hansen, Petter Bjornstad, Hak Joo Lee, Rajasree Menon, Leila Hejazi, Jian-Jun Liu, Anthony Franzone, Helen C. Looker, Byeong Yeob Choi, Roman Fernandez, Manjeri A. Venkatachalam, Luxcia Kugathasan, Vikas S. Sridhar, Loki Natarajan, Jing Zhang, Varun S. Sharma, Brian Kwan, Sushrut S. Waikar, Jonathan Himmelfarb, Katherine R. Tuttle, Bryan Kestenbaum, Tobias Fuhrer, Harold Feldman, Ian H. de Boer, Fabio C. Tucci, John Sedor, Hiddo Lambers Heerspink, Jennifer Schaub, Edgar A. Otto, Jeffrey B. Hodgin, Matthias Kretzler, Christopher R. Anderton, Theodore Alexandrov, David Cherney, Su Chi Lim, Robert G. Nelson, Jonathan Gelfond, Ravi Iyengar
Cystinosis is a lysosomal storage disease that is characterized by the accumulation of di-peptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7∆). We demonstrated that this mutant is retained in the endoplasmic reticulum (ER) and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the role of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7∆) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones, previously used for treating CFTR F508∆, and demonstrated that they could facilitate the folding and trafficking of cystinosin(7∆). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by ~70%. Conclusively, our study establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7∆).
Varsha Venkatarangan, Weichao Zhang, Xi Yang, Jess G. Thoene, Si H. Hahn, Ming Li
Background Acute tubulointerstitial nephritis (AIN) is one of the few causes of acute kidney injury with diagnosis-specific treatment options. However, due to the need to obtain a kidney biopsy for histological confirmation, AIN diagnosis can be delayed, missed, or incorrectly assumed. Here, we identify and validate urinary CXCL9, an IFN-γ-induced chemokine involved in lymphocyte chemotaxis, as a diagnostic biomarker for AIN.Methods In a prospectively enrolled cohort with pathologist-adjudicated histological diagnoses, termed the discovery cohort, we tested the association of 180 immune proteins measured by an aptamer-based assay with AIN and validated the top protein, CXCL9, using sandwich immunoassay. We externally validated these findings in 2 cohorts with biopsy-confirmed diagnoses, termed the validation cohorts, and examined mRNA expression differences in kidney tissue from patients with AIN and individuals in the control group.Results In aptamer-based assay, urinary CXCL9 was 7.6-fold higher in patients with AIN than in individuals in the control group (P = 1.23 × 10–5). Urinary CXCL9 measured by sandwich immunoassay was associated with AIN in the discovery cohort (n = 204; 15% AIN) independently of currently available clinical tests for AIN (adjusted odds ratio for highest versus lowest quartile: 6.0 [1.8–20]). Similar findings were noted in external validation cohorts, where CXCL9 had an AUC of 0.94 (0.86–1.00) for AIN diagnosis. CXCL9 mRNA expression was 3.9-fold higher in kidney tissue from patients with AIN (n = 19) compared with individuals in the control group (n = 52; P = 5.8 × 10–6).Conclusion We identified CXCL9 as a diagnostic biomarker for AIN using aptamer-based urine proteomics, confirmed this association using sandwich immunoassays in discovery and external validation cohorts, and observed higher expression of this protein in kidney biopsies from patients with AIN.Funding This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) awards K23DK117065 (DGM), K08DK113281 (KM), R01DK128087 (DGM), R01DK126815 (DGM and LGC), R01DK126477 (KNC), UH3DK114866 (CRP, DGM, and FPW), R01DK130839 (MES), and P30DK079310 (the Yale O’Brien Center). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Dennis G. Moledina, Wassim Obeid, Rex N. Smith, Ivy Rosales, Meghan E. Sise, Gilbert Moeckel, Michael Kashgarian, Michael Kuperman, Kirk N. Campbell, Sean Lefferts, Kristin Meliambro, Markus Bitzer, Mark A. Perazella, Randy L. Luciano, Jordan S. Pober, Lloyd G. Cantley, Robert B. Colvin, F. Perry Wilson, Chirag R. Parikh
Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) responding to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs of the brain (CVOs) focus on mechanosensitive membrane proteins. The present study demonstrated that an intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuronal-specific knockout (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1-cKO also blunted mannitol-indued AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.
Xin Jin, Jian Xie, Chia-Wei Yeh, Jen-Chi Chen, Chih-Jen Cheng, Cheng-Chang Lien, Chou-Long Huang
Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated α-Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.
Fabian Braun, Ahmed Abed, Dominik Sellung, Manuel Rogg, Mathias Woidy, Oysten Eikrem, Nicola Wanner, Jessica Gambardella, Sandra D. Laufer, Fabian Haas, Milagros N. Wong, Bernhard Dumoulin, Paula Rischke, Anne K. Mühlig, Wiebke Sachs, Katharina von Cossel, Kristina Schulz, Nicole Muschol, Sören W. Gersting, Ania C. Muntau, Oliver Kretz, Oliver Hahn, Markus M. Rinschen, Michael Mauer, Tillmann Bork, Florian Grahammer, Wei Liang, Thorsten Eierhoff, Winfried Römer, Arne Hansen, Catherine Meyer-Schwesinger, Guido Iaccarino, Camilla Tøndel, Hans-Peter Marti, Behzad Najafian, Victor G. Puelles, Christoph Schell, Tobias B. Huber
The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here we demonstrated that Salt Inducible Kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole tissue and single cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Lastly, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful to stimulate 1,25-vitamin D production in CKD-MBD.
Sung-Hee Yoon, Mark B. Meyer, Carlos Arevalo Rivas, Murat Tekguc, Chengcheng Zhang, Jialiang S. Wang, Christian D. Castro Andrade, Katelyn E. Strauss, Tadatoshi Sato, Nancy Benkusky, Seong Min Lee, Rebecca Berdeaux, Marc Foretz, Thomas B. Sundberg, Ramnik J. Xavier, Charles H. Adelmann, Daniel J. Brooks, Anthony Anselmo, Ruslan I. Sadreyev, Ivy A. Rosales, David E. Fisher, Navin Gupta, Ryuji Morizane, Anna Greka, J. Wesley Pike, Michael Mannstadt, Marc N. Wein
Circadian rhythmicity in renal function suggests rhythmic adaptations in renal metabolism. Todecipher the role of the circadian clock in renal metabolism, we studied diurnal changes in renal metabolic pathways using integrated transcriptomic, proteomic, and metabolomic analysisperformed on control mice and mice with inducible deletion of the circadian clock regulator Bmal1 in the renal tubule (cKOt). With this unique resource, we demonstrated that ~30% RNAs, ~20% proteins and ~20% metabolites are rhythmic in kidneys of control mice. Several key metabolic pathways including NAD+ biosynthesis, fatty acid transport, carnitine shuttle,and b-oxidation displayed impairments in kidneys of cKOt, resulting in a perturbedmitochondrial activity. Carnitine reabsorption from the primary urine was one of the mostimpacted processes with a ~50% reduction in plasma carnitine levels and a parallel systemicdecrease in tissues carnitine content. This suggests that the circadian clock in the renal tubule controls both kidney and systemic physiology.
Yohan Bignon, Leonore Wigger, Camille Ansermet, Benjamin D. Weger, Sylviane Lagarrigue, Gabriel Centeno, Fanny Durussel, Lou Götz, Mark Ibberson, Sylvain Pradervand, Manfredo Quadroni, Meltem Weger, Francesca Amati, Frédéric Gachon, Dmitri Firsov
How phosphate levels are detected in mammals is unknown. The bone-derived hormone fibroblast growth factor 23 (FGF23) lowers blood phosphate by reducing kidney phosphate reabsorption and 1,25(OH)2D production, but phosphate does not directly stimulate bone FGF23 expression. Using PET scanning and LC-MS, we show that phosphate increases kidney-specific glycolysis and synthesis of glycerol-3-phosphate (G-3-P), which then circulates to bone to trigger FGF23 production. Further, we find that glycerol-3-phosphate dehydrogenase 1 (Gpd1), a cytosolic enzyme that synthesizes G-3-P and oxidizes NADH to NAD+, is required for phosphate-stimulated G-3-P and FGF23 production and prevention of hyperphosphatemia. In proximal tubule cells, we find that phosphate availability is substrate-limiting for glycolysis and G-3-P production, and that increased glycolysis and Gpd1 activity are coupled through cytosolic NAD+ recycling. Finally, we show that the type II sodium-dependent phosphate co-transporter Npt2a, which is expressed exclusively in the proximal tubule, confers kidney specificity to phosphate-stimulated G-3-P production. Importantly, exogenous G-3-P stimulates FGF23 production when Npt2a or Gpd1 are absent, confirming that it is the key circulating factor downstream of glycolytic phosphate sensing in the kidney. Together, these findings place glycolysis at the nexus of mineral and energy metabolism and identify a kidney-bone feedback loop that controls phosphate homeostasis.
Wen Zhou, Petra Simic, Iris Y. Zhou, Peter Caravan, Xavier Vela Parada, Donghai Wen, Onica L. Washington, Maria Shvedova, Kerry A. Pierce, Clary B. Clish, Michael Mannstadt, Tatsuya Kobayashi, Marc N. Wein, Harald Jüppner, Eugene P. Rhee
Sepsis pathogenesis is complex and heterogeneous; hence, a precision medicine strategy is needed. Acute kidney injury (AKI) following sepsis portends higher mortality. Overproduction of mitochondrial reactive oxygen species (mtROS) is a potential mediator of sepsis and sepsis-induced AKI. BAM15, a chemical uncoupler, dissipates mitochondrial proton gradients without generating mtROS. We injected BAM15 into mice at 0, 6, or 12 hours after cecal ligation and puncture (CLP) treated with fluids and antibiotics. BAM15 reduced mortality, even after 12 hours, when mice were ill, and BAM15 reduced kidney damage and splenic apoptosis. Serial plasma and urinary mitochondrial DNA (mtDNA) levels increased post-CLP and decreased after BAM15 administration (at 0 or 6 hours). In vitro septic serum proportionately increased mtROS overproduction and mtDNA release from kidney tubule cells, which BAM15 prevented. BAM15 decreased neutrophil apoptosis, mtDNA release; neutrophil depletion counteracted BAM15 benefits. Further, mtDNA injection in vivo replicated inflammation and kidney injury, which was prevented by BAM15. A large dose of exogenous mtDNA reversed protection by BAM15. We conclude that BAM15 is an effective preventive and therapeutic candidate in experimental sepsis, and that BAM15 and mtDNA, a potential drug-companion diagnostic/drug efficacy pair for clinical sepsis, are mechanistically linked via mtROS.
Naoko Tsuji, Takayuki Tsuji, Tetsushi Yamashita, Naoki Hayase, Xuzhen Hu, Peter S.T. Yuen, Robert A. Star