Nephrology
Abstract
Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1-/- and Epac2-/- mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the knockout mice fed with Na+ deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker, ESI-05 and Epac1&2 blocker, ESI-09 decreased ENaC activity in EpacWT mice kept on Na+ deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in EpacWT mice on Na+ deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate non-redundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC over-activation.
Authors
Victor N. Tomilin, Kyrylo Pyrshev, Anna Stavniichuk, Naghmeh Hassanzadeh Khayyat, Guohui Ren, Oleg Zaika, Sherif Khedr, Alexander Staruschenko, Fang C. Mei, Xiaodong Cheng, Oleh Pochynyuk
Abstract
We performed next generation sequencing in patients with familial steroid sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with three affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that can be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR-Cas9 mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid-responsiveness observed in SSNS patients. The p.H310Y variant also disrupts binding of clavesin-1 to alpha-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1 knockout or homozygous H310Y knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, this data identifies CLVS1 as a candidate gene for SSNS, provides insight into therapeutic effects of corticosteroids on podocyte cellular dynamics and adds to the growing evidence on the importance of endocytosis and oxidative stress regulation to podocyte function.
Authors
Brandon M. Lane, Megan Chryst-Stangl, Guanghong Wu, Mohamed Shalaby, Sherif El Desoky, Claire C. Middleton, Kinsie Huggins, Amika Sood, Alejandro Ochoa, Andrew F. Malone, Ricardo Vancini, Sara E. Miller, Gentzon Hall, So Young Kim, David N. Howell, Jameela A. Kari, Rasheed Gbadegesin
Abstract
Cisplatin is a commonly used chemotherapeutic agent to treat a wide array of cancers that is frequently associated with toxic injury to the kidney due to oxidative DNA damage and perturbations in cell cycle progression leading to cell death. In this study, we investigated whether thyroid receptor interacting protein 13 (TRIP13) plays a central role in the protection of the tubular epithelia following cisplatin treatment by circumventing DNA damage. Following cisplatin treatment, double-stranded DNA repair pathways were inhibited using selective blockers to proteins involved in either homologous recombination or non-homologous end joining. This led to increased blood markers of acute kidney injury (AKI) (creatinine and neutrophil gelatinase–associated lipocalin), tubular damage, activation of DNA damage marker (γ-H2AX), elevated appearance of G2/M blockade (phosphorylated histone H3 Ser10 and cyclin B1), and apoptosis (cleaved caspase-3). Conditional proximal tubule–expressing Trip13 mice were observed to be virtually protected from the cisplatin nephrotoxicity by restoring most of the pathological phenotypes back toward normal conditions. Our findings suggest that TRIP13 could circumvent DNA damage in the proximal tubules during cisplatin injury and that TRIP13 may constitute a new therapeutic target in protecting the kidney from nephrotoxicants and reduce outcomes leading to AKI.
Authors
Taketsugu Hama, Prashanth K.B. Nagesh, Pallabita Chowdhury, Bob M. Moore II, Murali M. Yallapu, Kevin R. Regner, Frank Park
Abstract
Alport syndrome (AS) is a genetic disorder caused by mutations in type IV collagen that leads to defective glomerular basement membrane, glomerular filtration barrier (GFB) damage, and progressive chronic kidney disease. While the genetic basis of AS is well known, the molecular and cellular mechanistic details of disease pathogenesis have been elusive, hindering the development of mechanism-based therapies. Here we performed intravital multiphoton imaging of the local kidney tissue microenvironment in a X-linked AS mouse model to directly visualize the major drivers of AS pathology. Severely distended glomerular capillaries and aneurysms were found accompanied by numerous microthrombi, increased glomerular endothelial surface layer (glycocalyx) and immune cell homing, GFB albumin leakage, glomerulosclerosis and interstitial fibrosis by 5 months of age with an intermediate phenotype at 2 months. Renal histology in mouse or patient tissues largely failed to detect capillary aberrations. Treatment of AS mice with hyaluronidase or the ACE inhibitor enalapril reduced the excess glomerular endothelial glycocalyx and blocked immune cell homing, and GFB albumin leakage. This study identified central roles of glomerular mechanical forces and endothelial and immune cell activation early in AS, which could be therapeutically targeted to reduce mechanical strain and local tissue inflammation and improve kidney function.
Authors
Georgina Gyarmati, Urvi Nikhil Shroff, Audrey Izuhara, Xiaogang Hou, Stefano Da Sacco, Sargis Sedrakyan, Kevin V. Lemley, Kerstin Amann, Laura Perin, János Peti-Peterdi
Abstract
The biosynthetic routes leading to de novo Nicotinamine Adenine Dinucleotide (NAD+) production are involved in acute kidney injury (AKI) with a critical role for Quinolinate Phosphoribosyl Transferase (QPRT), a bottleneck enzyme of de novo NAD+ biosynthesis. However, the molecular mechanisms determining reduced QPRT in AKI, and the role of impaired NAD+ biosynthesis in the progression to chronic kidney disease (CKD) are unknown. We demonstrate that a high urinary quinolinate to tryptophan ratio, an indirect indicator of impaired QPRT activity and reduced de novo NAD+ biosynthesis in the kidney, is a clinically applicable early marker of AKI after cardiopulmonary bypass, and is predictive of progression to chronic kidney disease (CKD) in kidney transplant recipients. We also provide evidence that the Endoplasmic Reticulum (ER) stress response impairs de novo NAD+ biosynthesis by repressing QPRT transcription. In conclusion, NAD+ biosynthesis impairment is an early event in AKI embedded with the ER stress response, and persistent reduction of QPRT expression is associated with AKI to CKD progression. This defines non-invasive metabolic biomarkers of kidney injury with prognostic and therapeutic implications.
Authors
Yohan Bignon, Anna Rinaldi, Zahia Nadour, Virginie Poindessous, Ivan Nemazanyy, Olivia Lenoir, Baptiste Fohlen, Pierre Weill-Raynal, Alexandre Hertig, Alexandre Karras, Pierre Galichon, Maarten Naesens, Dany Anglicheau, Pietro E. Cippà, Nicolas Pallet
Abstract
BACKGROUND. Skeletal muscle maladaptation accompanies chronic kidney disease (CKD) and negatively impacts physical function. Emphasis in CKD has historically been placed on muscle fiber intrinsic deficits, such as altered protein metabolism and atrophy. However, targeted treatment of fiber intrinsic dysfunction has produced limited improvement, whereas alterations within the fiber extrinsic environment have scarcely been examined. METHODS. We investigated alterations to the skeletal muscle interstitial environment with deep cellular phenotyping of biopsies from patients with CKD compared to age-matched control participants and performed transcriptome profiling to define the molecular underpinnings of CKD-associated muscle impairments. We further examined changes in the observed muscle maladaptation following initiation of dialysis therapy for kidney failure. RESULTS. Patients with CKD exhibited a progressive fibrotic muscle phenotype, which was associated with impaired regenerative capacity and lower vascular density. The severity of these deficits was strongly associated with the degree of kidney dysfunction. Consistent with these profound deficits, CKD was associated with broad alterations to the muscle transcriptome, including altered extracellular matrix organization, downregulated angiogenesis, and altered expression of pathways related to stem cell self-renewal. Remarkably, despite the seemingly advanced nature of this fibrotic transformation, dialysis treatment rescued these deficits, restoring a healthier muscle phenotype. Furthermore, after accounting for muscle atrophy, strength and endurance improved after dialysis initiation. CONCLUSION. These data identify a dialysis-responsive muscle fibrotic phenotype in CKD and suggest that the early dialysis window presents a unique opportunity of improved muscle regenerative capacity during which targeted interventions may achieve maximal impact. TRIAL REGISTRATION. NCT01452412 FUNDING. NIH
Authors
Camille R. Brightwell, Ameya S. Kulkarni, William Paredes, Kehao Zhang, Jaclyn B. Perkins, Knubian J. Gatlin, Matthew Custodio, Hina Farooq, Bushra Zaidi, Rima Pai, Rupinder S. Buttar, Yan Tang, Michal L. Melamed, Thomas H. Hostetter, Jeffrey E. Pessin, Meredith Hawkins, Christopher S. Fry, Matthew K. Abramowitz
Abstract
Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules amongst diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflect proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants Alpha, Beta, Gamma, Kappa, and Delta exhibit comparable levels of replication in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.
Authors
Louisa Helms, Silvia Marchiano, Ian B. Stanaway, Tien-Ying Hsiang, Benjamin A. Juliar, Shally Saini, Yan Ting Zhao, Akshita Khanna, Rajasree Menon, Fadhl Alakwaa, Carmen Mikacenic, Eric D. Morrell, Mark M. Wurfel, Matthias Kretzler, Jennifer L. Harder, Charles E. Murry, Jonathan Himmelfarb, Hannele Ruohola-Baker, Pavan K. Bhatraju, Michael Gale, Jr., Benjamin S. Freedman
Abstract
Inhibitors of the renin-angiotensin system (RAS) are widely used to treat hypertension. Using mice harboring fluorescent cell lineage tracers, single-cell RNA-seq, and long-term inhibition of RAS in both mice and humans, we found that deletion of renin or inhibition of the RAS leads to concentric thickening of the intrarenal arteries and arterioles. This severe disease is caused by the multiclonal expansion and transformation of renin cells from a classical endocrine phenotype to a matrix-secretory phenotype: the cells surround the vessel walls and induce the accumulation of adjacent smooth muscle cells and extracellular matrix, resulting in blood flow obstruction, focal ischemia, and fibrosis. Ablation of the renin cells via conditional deletion of β1integrin prevents arteriolar hypertrophy, indicating that renin cells are responsible for vascular disease. Given these findings, prospective morphological studies in humans are necessary to determine the extent of renal-vascular damage caused by the widespread use of inhibitors of RAS.
Authors
Hirofumi Watanabe, Alexandre G. Martini, Evan A. Brown, Xiuyin Liang, Silvia Medrano, Shin Goto, Ichiei Narita, Lois J. Arend, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
Abstract
The role and mechanisms for upregulating complement factor B (CFB) expression in podocyte dysfunction in diabetic kidney disease (DKD) are not fully understood. Here, analyzing Gene Expression Omnibus GSE30528 data, we identified genes enriched in mTORC1 signaling, CFB, and complement alternative pathways in podocytes from patients with DKD. In mouse models, podocyte mTOR complex 1 (mTORC1) signaling activation was induced, while blockade of mTORC1 signaling reduced CFB upregulation, alternative complement pathway activation, and podocyte injury in the glomeruli. Knocking down CFB remarkably alleviated alternative complement pathway activation and DKD in diabetic mice. In cultured podocytes, high glucose treatment activated mTORC1 signaling, stimulated STAT1 phosphorylation, and upregulated CFB expression, while blockade of mTORC1 or STAT1 signaling abolished high glucose–upregulated CFB expression. Additionally, high glucose levels downregulated protein phosphatase 2Acα (PP2Acα) expression, while PP2Acα deficiency enhanced high glucose–induced mTORC1/STAT1 activation, CFB induction, and podocyte injury. Taken together, these findings uncover a mechanism by which CFB mediates podocyte injury in DKD.
Authors
Qingmiao Lu, Qing Hou, Kai Cao, Xiaoli Sun, Yan Liang, Mengru Gu, Xian Xue, Allan Zijian Zhao, Chunsun Dai
Abstract
Proline rich 11 (PRR11), a novel tumor-related gene, has been identified in different tumors. However, the relevant biological functions of PRR11 in human clear cell renal cell carcinoma (ccRCC) have not been studied. In this study, we first identified PRR11 as a biomarker of ccRCC and predictor of poor prognosis by bioinformatics. Then, we confirmed that PRR11 silencing significantly reduced ccRCC cell proliferation and migration in vitro and in vivo. Importantly, we found that PRR11 could induce the degradation of the E2F1 protein through its interaction with E2F1, and PRR11 reduced the stability of the E2F1 protein in ccRCC cells, thereby affecting cell cycle progression. Further results indicated that the downregulation of E2F1 expression could partially reverse the changes in ccRCC cell biology caused by PRR11 deletion. In addition, we proved for the first time that PRR11 is a target gene of c-Myc. The transcription factor c-Myc may promote the expression of PRR11 in ccRCC cells by binding to the PRR11 promoter region, thereby accelerating the progression of ccRCC. In summary, we found that PRR11 could serve as a novel oncogene in ccRCC, and PRR11 could reduce the protein stability of E2F1 and could be activated by c-Myc.
Authors
Siming Chen, Zhiwen He, Tiancheng Peng, Fenfang Zhou, Gang Wang, Kaiyu Qian, Lingao Ju, Yu Xiao, Xinghuan Wang
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