DREAM is a transcriptional repressor complex that regulates cell proliferation and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we utilized an assembly defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis, but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys, but not the brain or bone marrow. Using laser capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting AApoAIV amyloidosis. AApoAIV is a recently described form whereby wildtype apoA-IV has been shown to predominate in amyloid plaques. We determined that DREAM directly regulates Apoa4 by chromatin immunoprecipitation and that the histone variant H2AZ is reduced from the Apoa4 gene body in DREAM’s absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of non-genetic amyloid subtypes.
Pirunthan Perampalam, Haider M. Hassan, Grace E. Lilly, Daniel T. Passos, Joseph Torchia, Patti K. Kiser, Andrea Bozovic, Vathany Kulasingam, Frederick A. Dick
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease, despite higher mitochondrial content. We sought to illuminate non-canonical, cell-specific roles for CoQ, independent of the electron transport chain (ETC). Here we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway, rather than ETC dysfunction. Single nucleus RNA sequencing from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ-deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
Eriene-Heidi Sidhom, Choah Kim, Maria Kost-Alimova, May Theng Ting, Keith Keller, Julian Avila-Pacheco, Andrew J.B. Watts, Katherine A. Vernon, Jamie L. Marshall, Estefanía Reyes-Bricio, Matthew Racette, Nicolas Wieder, Giulio Kleiner, Elizabeth J. Grinkevich, Fei Chen, Astrid Weins, Clary B. Clish, Jillian L. Shaw, Catarina M. Quinzii, Anna Greka
Human metabolic incorporation of non-human sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via pre-existing antibodies, likely contributing to red-meat-induced atherosclerosis acceleration. Exploring if this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. Levels of Kdn precursor mannose also increased but within normal range in ESRD patients. Mannose ingestion by healthy volunteers raised urinary mannose and Kdn. Kdn production pathways remain conserved in mammals but were diminished by a M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in two lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that potential toxicities of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals likely conserved Kdn biosynthesis and modulated it in lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates/metabolites. However, human cells can be forced to express Kdn-glycans, via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing CMP-Kdn. Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.
Kunio Kawanishi, Sudeshna Saha, Sandra Diaz, Michael Vaill, Aniruddha Sasmal, Shoib S. Siddiqui, Biswa P. Choudhury, Kumar Sharma, Xi Chen, Ian C. Schoenhofen, Chihiro Sato, Ken Kitajima, Hudson H. Freeze, Anja Münster-Kühnel, Ajit Varki
Primary membranous nephropathy (pMN) is a leading cause of the nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1 positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induce proteolysis of the two essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1-IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1-titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 was required to induce proteolysis of synaptopodin and NEPH1 by two distinct proteolytic pathways, mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrate a mechanism by which aberrantly glycosylated IgG4 activates the lectin pathway and induces podocyte injury in primary membranous nephropathy.
George Haddad, Johan M. Lorenzen, Hong Ma, Noortje de Haan, Harald Seeger, Christelle Zaghrini, Simone Brandt, Malte Kölling, Urs Wegmann, Bence Kiss, Gábor Pál, Péter Gál, Rudolf P. Wuthrich, Manfred Wuhrer, Laurence H. Beck, David J. Salant, Gérard Lambeau, Andreas D. Kistler
Introduction: Acute kidney injury and chronic kidney disease (CKD) are common in hospitalized patients. To inform clinical decision-making, more accurate information regarding risk of long-term progression to kidney failure is required. Methods: We enrolled 1538 hospitalized patients in a multicenter, prospective cohort study. Monocyte chemoattractant protein-1 (MCP-1/CCL2), uromodulin (UMOD), and YKL-40 (CHI3L1) were measured in urine samples collected during outpatient follow-up at 3 months. We followed patients for a median of 4.3 years and assessed the relationship between biomarker levels and changes in estimated glomerular filtration rate (eGFR) over time and the development of a composite kidney outcome (CKD incidence, CKD progression, or end-stage renal disease). We paired these clinical studies with investigations in mouse models of renal atrophy and renal repair to further understand the molecular basis of these markers in kidney disease progression. Results: Higher MCP-1 and YKL-40 levels were associated with greater eGFR decline and increased incidence of the composite renal outcome, whereas higher UMOD levels were associated with smaller eGFR declines and decreased incidence of the composite kidney outcome. A multimarker score increased prognostic accuracy and reclassification compared with traditional clinical variables alone. The mouse model of renal atrophy showed greater Ccl2 and Chi3l1 mRNA expression in infiltrating macrophages and neutrophils, respectively, and evidence of progressive renal fibrosis compared with the repair model. The repair model showed greater Umod expression in the loop of Henle and correspondingly less fibrosis. Conclusions: Biomarker levels at 3 months after hospitalization identify patients at risk for kidney disease progression. Funding: National Institutes of Health grants U01DK082223, U01DK082185, U01DK082192, U01DK082183, R01HL085757, R01DK098233, R01DK101507, R01DK114014, K23DK100468, R03DK111881, R01DK093771, K01DK120783, P30DK079310, P30DK114809.
Jeremy Puthumana, Heather Thiessen-Philbrook, Leyuan Xu, Steven G. Coca, Amit X. Garg, Jonathan Himmelfarb, Pavan K. Bhatraju, Talat Alp Ikizler, Edward Siew, Lorraine B. Ware, Kathleen D. Liu, Alan S. Go, James S. Kaufman, Paul L. Kimmel, Vernon M. Chinchilli, Lloyd Cantley, Chirag R. Parikh
The sodium-phosphate co-transporter NPT2a plays a key role in reabsorbing filtered phosphate in proximal renal tubules thereby critically contributing to phosphate homeostasis. Inadequate urinary phosphate excretion can lead to severe hyperphosphatemia as in tumoral calcinosis, and in chronic kidney disease (CKD). Pharmacological inhibition of NPT2a may therefore represent a novel approach for treating hyperphosphatemic conditions. The NPT2a-selective small molecule inhibitor, PF-06869206, was previously shown to reduce phosphate uptake in human proximal tubular cells in vitro. We now investigated the acute and chronic effects of the inhibitor in vivo and report that administration of PF-06869206 was well-tolerated and elicited a dose-dependent increase in fractional phosphate excretion. This phosphaturic effect lowered plasma phosphate levels in wild-type mice and in rats with CKD due to subtotal nephrectomy. PF-06869206 had no effect in Npt2a-null mice, but promoted phosphate excretion and reduced plasma phosphate in normophophatemic mice lacking Npt2c and in hyperphosphatemic mice lacking Fgf23 or Galnt3. In CKD rats, once daily administration of PF-06869206 for eight weeks induced an unabated acute phosphaturic and hypophosphatemic effect, but had no significant effect on FGF23 or PTH levels. Selective pharmacological inhibition of NPT2a thus holds promises as a novel therapeutic option for genetic and acquired hyperphosphatemic disorders.
Valerie Clerin, Hiroshi Saito, Kevin J. Filipski, An Hai Nguyen, Jeonifer Garren, Janka Kisucka, Monica Reyes, Harald Jüppner
Dysregulation of autophagy in diabetic kidney disease (DKD) has been reported, but the underlying mechanism and its pathogenic role remain elusive. We show that autophagy was inhibited in DKD models and in human diabetic kidneys. Ablation of autophagy-related gene 7 (Atg7) from kidney proximal tubules led to autophagy deficiency and worse renal hypertrophy, tubular damage, inflammation, fibrosis, and albuminuria in diabetic mice, indicating a protective role of autophagy in DKD. Autophagy impairment in DKD was associated with the downregulation of unc-51–like autophagy-activating kinase 1 (ULK1), which was mediated by the upregulation of microRNA-214 (miR-214) in diabetic kidney cells and tissues. Ablation of miR-214 from kidney proximal tubules prevented a decrease in ULK1 expression and autophagy impairment in diabetic kidneys, resulting in less renal hypertrophy and albuminuria. Furthermore, blockade of p53 attenuated miR-214 induction in DKD, leading to higher levels of ULK1 and autophagy, accompanied by an amelioration of DKD. Compared with nondiabetic samples, renal biopsies from patients with diabetes showed induction of p53 and miR-214, associated with downregulation of ULK1 and autophagy. We found a positive correlation between p53/miR-214 and renal fibrosis, but a negative correlation between ULK1/LC3 and renal fibrosis in patients with diabetes. Together, these results identify the p53/miR-214/ULK1 axis in autophagy impairment in diabetic kidneys, pinpointing possible therapeutic targets for DKD.
Zhengwei Ma, Lin Li, Man J. Livingston, Dongshan Zhang, Qingsheng Mi, Ming Zhang, Han-Fei Ding, Yuqing Huo, Changlin Mei, Zheng Dong
Gain-of-function mutations in the WNK1 and WNK4 genes are responsible for Familial Hyperkalemic Hypertension (FHHt), a rare inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the KLHL3-CUL3 E3 ubiquitin ligase complex have shed light on the importance of WNKs cellular degradation on renal ion transport. Using full exome sequencing in a four-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants at the WNK1 gene, clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early-onset and a marked hyperkalemic phenotype, but normal blood pressure values. Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex, rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR-Cas9 engineered mouse model recapitulated both the clinical and biological phenotype. Renal investigations showed increased activation of the SPAK-NCC phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Altogether, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
Helene Louis-Dit-Picard, Ilektra Kouranti, Chloe Rafael, Irmine Loisel-Ferreira, Maria Chavez-Canales, Waed Abdel Khalek, Eduardo Argaiz, Stephanie Baron, Sarah Vacle, Tiffany Migeon, Richard Coleman, Marcio Do Cruzeiro, Marguerite Hureaux, Nirubiah Thurairajasingam, Stéphane Decramer, Xavier Girerd, Kevin M. O'Shaughnessy, Paolo Mulatero, Gwenaelle Roussey, Ivan Tack, Robert J. Unwin, Rosa Vargas-Poussou, Olivier Staub, P. Richard Grimm, Paul A. Welling, Gerardo Gamba, Eric Clauser, Juliette Hadchouel, Xavier Jeunemaitre
The origin and fate of renal myofibroblasts is not clear after acute kidney injury (AKI). Here, we demonstrate that myofibroblasts were activated from quiescent pericytes (qPericytes) and the cell numbers increased after ischemia/reperfusion injury–induced AKI (IRI-AKI). Myofibroblasts underwent apoptosis during renal recovery but one-fifth of them survived in the recovered kidneys on day 28 after IRI-AKI and their cell numbers increased again after day 56. Microarray data showed the distinctive gene expression patterns of qPericytes, activated pericytes (aPericytes, myofibroblasts), and inactivated pericytes (iPericytes) isolated from kidneys before, on day 7, and on day 28 after IRI-AKI. Hypermethylation of the Acta2 repressor Ybx2 during IRI-AKI resulted in epigenetic modification of iPericytes to promote the transition to chronic kidney disease (CKD) and aggravated fibrogenesis induced by a second AKI induced by adenine. Mechanistically, transforming growth factor-β1 decreased the binding of YBX2 to the promoter of Acta2 and induced Ybx2 hypermethylation, thereby increasing α-smooth muscle actin expression in aPericytes. Demethylation by 5-azacytidine recovered the microvascular stabilizing function of aPericytes, reversed the profibrotic property of iPericytes, prevented AKI-CKD transition, and attenuated fibrogenesis induced by a second adenine-AKI. In conclusion, intervention to erase hypermethylation of pericytes after AKI provides a strategy to stop the transition to CKD.
Yu-Hsiang Chou, Szu-Yu Pan, Yu-Han Shao, Hong-Mou Shih, Shi-Yao Wei, Chun-Fu Lai, Wen-Chih Chiang, Claudia Schrimpf, Kai-Chien Yang, Liang-Chuan Lai, Yung-Ming Chen, Tzong-Shinn Chu, Shuei-Liong Lin
Utilizing the Nephrotic Syndrome Study Network Consortium and other publicly available transcriptomic datasets, we identified Retinoic acid receptor responder protein 1 (RARRES1) as a gene whose expression positively correlated with renal function decline in human glomerular disease. The glomerular expression of RARRES1, which is largely restricted to podocytes, increased in focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). Tumor necrosis factor-α (TNF-α) was a potent inducer of RARRES1 expression in cultured podocytes, and transcriptomic analysis showed the enrichment of cell death pathway genes with RARRES1 overexpression. The overexpression of RARRES1 indeed induced podocyte apoptosis in vitro. Notably, this effect was dependent on its cleavage in the extracellular domain, as the mutation of its cleavage site abolished the apoptotic effect. Mechanistically, the soluble RARRES1 is endocytosed and interacts with and inhibits RIO kinase 1 (RIOK1), resulting in p53 activation and podocyte apoptosis. In mice, podocyte-specific overexpression of RARRES1 resulted in marked glomerular injury and albuminuria, while the overexpression of RARRES1 cleavage mutant had no effect. Conversely, podocyte-specific knockdown of Rarres1 in mice ameliorated glomerular injury in the setting of Adriamycin-induced nephropathy. Together, our study demonstrates an important role and the mechanism of RARRES1 in podocyte injury in glomerular disease.
Anqun Chen, Ye Feng, Han Lai, Wenjun Ju, Zhengzhe Li, Yu Li, Andrew Wang, Quan Hong, Fang Zhong, Chengguo Wei, Jia Fu, Tian-Jun Guan, Bi-Cheng Liu, Matthias Kretzler, Kyung Lee, John Cijiang He