American journal of physiology. Renal physiology最新文献

{"title":"Optimized protocol for the multiomics processing of cryopreserved human kidney tissue.","authors":"Sydney E Gies, Sonja Hänzelmann, Dominik Kylies, Moritz Lassé, Simon Lagies, Fabian Hausmann, Robin Khatri, Nikolay Zolotarev, Manuela Poets, Tianran Zhang, Fatih Demir, Anja M Billing, Josephine Quaas, Elisabeth Meister, Jonas Engesser, Anne K Mühlig, Shun Lu, Shuya Liu, Silvia Chilla, Ilka Edenhofer, Jan Czogalla, Fabian Braun, Bernd Kammerer, Victor G Puelles, Stefan Bonn, Markus M Rinschen, Maja Lindenmeyer, Tobias B Huber","doi":"10.1152/ajprenal.00404.2023","DOIUrl":"10.1152/ajprenal.00404.2023","url":null,"abstract":"<p><p>Biobanking of tissue from clinically obtained kidney biopsies for later analysis with multiomic approaches, such as single-cell technologies, proteomics, metabolomics, and the different types of imaging, is an inevitable step to overcome the need of disease model systems and toward translational medicine. Hence, collection protocols that ensure integration into daily clinical routines by the usage of preservation media that do not require liquid nitrogen but instantly preserve kidney tissue for both clinical and scientific analyses are necessary. Thus, we modified a robust single-nucleus dissociation protocol for kidney tissue stored snap-frozen or in the preservation media RNAlater and CellCover. Using at first porcine kidney tissue as a surrogate for human kidney tissue, we conducted single-nucleus RNA sequencing with the widely recognized Chromium 10X Genomics platform. The resulting datasets from each storage condition were analyzed to identify any potential variations in transcriptomic profiles. Furthermore, we assessed the suitability of the preservation media for additional analysis techniques such as proteomics, metabolomics, and the preservation of tissue architecture for histopathological examination including immunofluorescence staining. In this study, we show that in daily clinical routines, the preservation medium RNAlater facilitates the collection of highly preserved human kidney biopsies and enables further analysis with cutting-edge techniques like single-nucleus RNA sequencing, proteomics, and histopathological evaluation. Only metabolome analysis is currently restricted to snap-frozen tissue. This work will contribute to build tissue biobanks with well-defined cohorts of the respective kidney disease that can be deeply molecularly characterized, opening up new horizons for the identification of unique cells, pathways and biomarkers for the prevention, early identification, and targeted therapy of kidney diseases.<b>NEW & NOTEWORTHY</b> In this study, we addressed challenges in integrating clinically obtained kidney biopsies into everyday clinical routines. Using porcine kidneys, we evaluated preservation media (RNAlater and CellCover) versus snap freezing for multi-omics processing. Our analyses highlighted RNAlater's suitability for single-nucleus RNA sequencing, proteome analysis and histopathological evaluation. Only metabolomics are currently restricted to snap-frozen biopsies. Our research established a cryopreservation protocol that facilitates tissue biobanking for advancing precision medicine in nephrology.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F822-F844"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142373703","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"COMMD5 counteracts cisplatin-induced nephrotoxicity by maintaining tubular epithelial integrity and autophagy flux.","authors":"Maiko Ogasawara-Nosoko, Hiroyuki Matsuda, Jin Ikeda, Masanori Abe, Yoshikazu Masuhiro, Morito Endo, Pavel Hamet, Johanne Tremblay","doi":"10.1152/ajprenal.00026.2024","DOIUrl":"10.1152/ajprenal.00026.2024","url":null,"abstract":"<p><p>Oxidative stress mediated by reactive oxygen species (ROS) contributes to apoptosis of tubular epithelial cells (TECs) and renal inflammation during acute kidney injury (AKI). Copper metabolism MURR1 domain-containing 5 [COMMD5/hypertension-related, calcium-regulated gene (HCaRG)] shows strong cytoprotective properties. COMMD5 is highly expressed in proximal tubules (PTs), where it controls cell differentiation. We assessed its role in cisplatin-induced AKI using transgenic mice in which COMMD5 is overexpressed in the PTs. Cisplatin caused the accumulation of damaged mitochondria and cellular waste in PTs, thus increasing the apoptosis of TECs. COMMD5 overexpression effectively protected TECs from cisplatin nephrotoxicity by decreasing intracellular ROS levels, mitochondrial dysfunction, and apoptosis through the preservation of tubular epithelial integrity, thus alleviating morphological and functional kidney damage. Excessive ROS production by hydrogen peroxide led to long-term autophagy activation through an increased burden on the autophagy/lysosome degradation system in TECs, and autophagic elimination of damaged mitochondria and cellular waste was compromised. COMMD5 attenuated oxidative injury by increasing autophagy flux, possibly due to a reduction of intracellular ROS levels through maintained tubular epithelial integrity, which decreased JNK/caspase-3-dependent apoptosis. Meanwhile, COMMD5 inhibition by siRNA reduced the resistance of TECs to cisplatin cytotoxicity, as shown by disrupted tubular epithelial integrity and cell viability. These data indicated that COMMD5 protects TECs from drug-induced oxidative stress and toxicity by maintaining tubular epithelial integrity and autophagy flux and ultimately decreases mitochondrial dysfunction and apoptosis. Increasing COMMD5 content in PTs is proposed as a new protective and therapeutic strategy against AKI.<b>NEW & NOTEWORTHY</b> Oxidative stress overload by drug treatment causes the accumulation of damaged mitochondria that could contribute to tubulopathy. However, effective preventive treatment for drug-induced acute kidney injury remains incompletely understood. Our study showed that copper metabolism MURR1 domain-containing 5 (COMMD5) reduced mitochondrial dysfunction and increased autophagy flux by alleviating reactive oxygen species production through maintaining tubular epithelial integrity when tubular epithelial cells were under oxidative stress, thus ameliorating renal function in cisplatin-treated mice. These results uncover a novel renoprotective mechanism underlying tubular epithelial integrity and autophagy flux.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F739-F757"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142303164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A brief harvesting-freezing delay significantly alters the kidney metabolome and leads to false positive and negative results.","authors":"Yahya Alsawaf, Igor Maksimovic, Jamie Zheng, Song Zhang, Ivan Vuckovic, Petras Dzeja, Slobodan Macura, Maria V Irazabal","doi":"10.1152/ajprenal.00131.2024","DOIUrl":"10.1152/ajprenal.00131.2024","url":null,"abstract":"<p><p>Abnormalities in distinct metabolic pathways have been associated with the pathogenesis and progression of many forms of kidney disease. Metabolomics analyses can be used to determine organ-specific metabolic fingerprints and, ideally, should represent the metabolic state of the organ at the exact moment the sample is harvested. However, conventional harvesting methods depend on posteuthanasia tissue harvest, which results in ischemia conditions and metabolome changes that could potentially introduce artifacts into the final studies. We recently optimized a modified clamp-freezing technique for rodent kidney harvesting and freezing, significantly reducing ischemia and freezing times and granting a closer snapshot of in vivo metabolism. In this study, we characterized and compared the metabolome of kidneys harvested using our modified approach versus traditional techniques to determine which metabolites are preferentially affected by a brief lapse of ischemia and freezing delay and which are more stable. We used Sprague-Dawley rats as a model of wild-type (WT) kidneys and PCK [polycystic kidney disease (PKD)] rats as a model of chronic kidney disease kidneys. Finally, we compared the metabolic profile of clamp-frozen and delayed WT and PKD kidneys to determine which metabolic changes are most likely observed in vivo in PKD and which could be subjected to false positive or negative results. Our data indicate that a short harvesting-freezing delay is sufficient to impart profound metabolic changes in WT and PKD kidneys, leading to false positive and negative differences when comparing these genotypes. In addition, we identified a group of metabolites that were more stable. Interestingly, while the delay had a similar effect between WT and PKD, there were notable differences. The data obtained indicate that the quick clamp-freezing technique for kidney metabolomics provides a more accurate interpretation of the in vivo metabolic changes associated with the disease state. <b>NEW & NOTEWORTHY</b> Our study shows that a brief harvesting-freezing delay associated with organ collection and freezing can significantly alter the kidney metabolic profile of both male and female wild-type and a genetic model of chronic kidney disease. Importantly, given that the effect of this delay differs among genotypes, it is not safe to assume that equally delaying harvesting-freezing in wild-type and polycystic kidney disease kidneys adequately controls this effect, ultimately leading to false positive and negative results among different renal diseases.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F697-F711"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11563588/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142115817","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Longitudinal intravital microscopy of the mouse kidney: inflammatory responses to abdominal imaging windows.","authors":"Michelle M Martinez, Julia R Walsh, Malgorzata M Kamocka, Hyowon Lee, Kenneth W Dunn","doi":"10.1152/ajprenal.00071.2024","DOIUrl":"10.1152/ajprenal.00071.2024","url":null,"abstract":"<p><p>Intravital microscopy enables direct observation of cell biology and physiology at subcellular resolution in real time in living animals. Implanted windows extend the scope of intravital microscopy to processes extending for weeks or even months, such as disease progression or tumor development. However, a question that must be addressed in such studies is whether the imaging window, like any foreign body, triggers an inflammatory response, and whether that response alters the biological process under investigation. To directly evaluate this question, we conducted large-scale intravital microscopy of the kidney of LysM-EGFP mice over time after implantation of abdominal imaging windows. These studies demonstrate that windows stimulated a variety of changes consistent with a foreign body response. Within a few days of implantation, leukocytes were recruited to the window and the region between the window and kidney where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular albumin permeability within 2-5 days of implantation. Similar results were obtained from mice implanted with windows coated with poly(l-lysine)-graft-polyethylene glycol (PLL-g-PEG), but not from immune-deficient mice. These studies demonstrate the importance of evaluating whether implanted windows induce an inflammatory response, and whether that response impacts the processes under evaluation in longitudinal intravital microscopy studies.<b>NEW & NOTEWORTHY</b> Intravital microscopy studies of LysM-EGFP mice demonstrate that abdominal imaging windows placed over the kidney stimulated a variety of changes consistent with a foreign body response. Within a day of implantation, leukocytes were recruited to the window where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular permeability to albumin.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F845-F868"},"PeriodicalIF":0.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11563595/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142334170","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of NET formation and the renoprotective effect of degraded NETs in lupus nephritis.","authors":"Yong Jin, Yutong Wang, Xu Ma, Hongbin Li, Manling Zhang","doi":"10.1152/ajprenal.00122.2024","DOIUrl":"10.1152/ajprenal.00122.2024","url":null,"abstract":"<p><p>To explore molecular biomarkers associated with the pathophysiology and therapy of lupus nephritis (LN), we conducted a joint analysis of transcriptomic data from 40 peripheral blood mononuclear cells (PBMCs) (GSE81622) and 21 kidney samples (GSE112943) from the Gene Expression Omnibus database using bioinformatics. A total of 976 and 2,427 differentially expressed genes (DEGs) were identified in PBMCs and renal tissues. Seven and two functional modules closely related to LN were identified. Further enrichment analysis revealed that the neutrophil activation pathway was highly active in both PBMCs and the kidney. Subsequently, 16 core genes closely associated with LN were verified by protein-protein interaction screening and quantitative PCR. In vitro cell models and MRL/lpr mouse models confirmed that the abnormal expression of these core genes was closely linked to neutrophil extracellular traps (NETs) generated by neutrophil activation, while degradation of NETs led to downregulation of core gene expression, thereby improving pathological symptoms of LN. Therefore, identification of patients with systemic lupus erythematosus exhibiting abnormal expression patterns for these core genes may serve as a useful indicator for kidney involvement. In addition, targeting neutrophils to modulate their activation levels and inhibit aberrant expression of these genes represents a potential therapeutic strategy for treating LN. <b>NEW & NOTEWORTHY</b> The mechanisms by which immune cells cause kidney injury in lupus nephritis are poorly understood. We integrated and analyzed the transcriptomic features of PBMCs and renal tissues from the GEO database to identify key molecular markers associated with neutrophil activation. We confirmed that neutrophil extracellular traps (NETs) formed by neutrophil activation promoted the upregulation of key genes in cell and animal models. Targeted degradation of NETs significantly ameliorated kidney injury in MRL/lpr mice.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F637-F654"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11483074/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142115830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bayesian mapping of protein kinases to vasopressin-regulated phosphorylation sites in renal collecting duct.","authors":"Venkatesh Deshpande, Euijung Park, Nipun U Jayatissa, Shaza Khan, Raymond Mejia, Chin-Rang Yang, Chung-Lin Chou, Viswanathan Raghuram, Mark A Knepper","doi":"10.1152/ajprenal.00142.2024","DOIUrl":"10.1152/ajprenal.00142.2024","url":null,"abstract":"<p><p>Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase β (CDC42BPB).<b>NEW & NOTEWORTHY</b> Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F591-F598"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11918269/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141725272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Histone deacetylase expression following cisplatin-induced acute kidney injury in male and female mice.","authors":"Huy Nguyen, Anabelle Gales, Sureena Monteiro-Pai, Ariana S Oliver, Nicholas Harris, Anna D Montgomery, Stephanie Franzén, Malgorzata Kasztan, Kelly A Hyndman","doi":"10.1152/ajprenal.00132.2024","DOIUrl":"10.1152/ajprenal.00132.2024","url":null,"abstract":"<p><p>The chemotherapeutic agent cisplatin accumulates in the kidneys, leading to acute kidney injury (AKI). Preclinical and clinical studies have demonstrated sex-dependent outcomes of cisplatin-AKI. Deranged histone deacetylase (HDAC) activity is hypothesized to promote the pathogenesis of male murine cisplatin-AKI; however, it is unknown whether there are sex differences in the kidney HDACs. We hypothesized that there would be sex-specific <i>Hdac</i> expression, localization, or enzymatic activity, which may explain sexual dimorphic responses to cisplatin-AKI. In normal human kidney RNA samples, <i>HDAC10</i> was significantly greater in the kidneys of women compared with men, whereas <i>HDAC1</i>, <i>HDAC6</i>, <i>HDAC10</i>, and <i>HDAC11</i> were differentially expressed between the kidney cortex and medulla, regardless of sex. In a murine model of cisplatin-AKI (3 days after a 15 mg/kg injection), we found few sex- or cisplatin-related differences in <i>Hdac</i> kidney transcripts among the mice. Although <i>Hdac9</i> was significantly greater in female mice compared with male mice, HDAC9 protein localization did not differ. <i>Hdac7</i> transcripts were greater in the inner medulla of cisplatin-AKI mice, regardless of sex, and this agreed with a greater HDAC7 abundance. HDAC activity within the cortex, outer medulla, and inner medulla was significantly lower in cisplatin-AKI mice but did not differ between the sexes. In agreement with these findings, a class I HDAC inhibitor did not improve kidney injury or function. In conclusion, even though cisplatin-AKI was evident and there were transcript level differences among the different kidney regions in this model, there were few sex- or cisplatin-dependent effects on kidney HDAC localization or activity.<b>NEW & NOTEWORTHY</b> Kidney histone deacetylases (HDACs) are abundant in male and female mice, and the inner medulla has the greatest HDAC activity. A low dose of cisplatin caused acute kidney injury (AKI) in these mice, but there were few changes in kidney HDACs at the RNA/protein/activity level. A class I HDAC inhibitor failed to improve AKI outcomes. Defining the HDAC isoform, cellular source, and interventional timing is necessary to determine whether HDAC inhibition is a therapeutic strategy to prevent cisplatin-AKI in both sexes.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F623-F636"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11483084/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141908615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Peripheral dopamine suppression and elevated cystatin C in early diabetic nephropathy in spontaneously diabetic rats.","authors":"Shoichiro Horita, Guy Watanabe, Shingen Misaka, Shu Taira, Mamoru Satoh, Yuko Maejima, Kenju Shimomura, Michio Shimabukuro, Junichiro James Kazama, Shuichi Shigetomi","doi":"10.1152/ajprenal.00180.2023","DOIUrl":"10.1152/ajprenal.00180.2023","url":null,"abstract":"<p><p>Intrarenal dopamine plays a protective role against the development of diabetic nephropathy during the early stages of the disease. In streptozotocin-induced diabetic mice with renal-specific catechol-<i>O</i>-methyl transferase knockout, intrarenal dopamine was found to suppress glomerular hyperfiltration, reduce oxidative stress and inflammation, and inhibit fibrosis. However, although dopamine activation in streptozotocin-induced diabetic models has been shown to provide renal protection, the role of dopamine in models of naturally induced diabetes mellitus is still unclear. In the present study, we orally administered 10 mg/kg benserazide, a peripheral decarboxylase inhibitor, to spontaneously diabetic Torii rats daily to investigate the activation of the renal dopaminergic system during the progression of diabetic nephropathy. Our findings show that peripheral dopamine decreased urinary 8-iso-prostaglandin F_2α and suppressed increases in plasma cystatin C levels. This study demonstrates that a reduction in peripheral dopamine can exacerbate renal dysfunction, even in the early stages of diabetic nephropathy characterized by glomerular hyperfiltration, thereby clarifying the pivotal role of endogenous peripheral dopamine in modulating oxidative stress and kidney performance.<b>NEW & NOTEWORTHY</b> By administering a peripheral decarboxylase inhibitor, we revealed that peripheral dopamine inhibits both the increase in urinary 8-iso-prostaglandin F_2α, an oxidative stress marker, and the increase in plasma cystatin C, an early renal dysfunction marker, even in the early stages of diabetic nephropathy characterized by glomerular hyperfiltration. By visualizing renal dopamine precursor distribution, we highlighted the role of endogenous renal dopamine in oxidative stress and renal function following the onset of glomerular hyperfiltration.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F581-F590"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141876941","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing renal transporter immunodetection: consequences of freeze-thaw during sample preparation.","authors":"Hannah L Hartman-Houstman, Donna L Ralph, Jonathan W Nelson, Lawrence G Palmer, Jessica E Faulkner, Jennifer C Sullivan, Desmond M Moronge, Alicia A McDonough","doi":"10.1152/ajprenal.00210.2024","DOIUrl":"10.1152/ajprenal.00210.2024","url":null,"abstract":"<p><p>Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na₂EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, \"+\") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25-50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. <b>NEW & NOTEWORTHY</b> Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F655-F666"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11483075/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142115816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Actin cytoskeleton and associated myosin motors within the renal epithelium.","authors":"Brook W Busselman, Ishara Ratnayake, Mark R Terasaki, Vedant P Thakkar, Arooba Ilyas, Karla L Otterpohl, Jenna L Zimmerman, Indra Chandrasekar","doi":"10.1152/ajprenal.00078.2024","DOIUrl":"10.1152/ajprenal.00078.2024","url":null,"abstract":"<p><p>This review highlights the complexity of renal epithelial cell membrane architectures and organelles through careful review of ultrastructural and physiological studies published over the past several decades. We also showcase the vital roles played by the actin cytoskeleton and actin-associated myosin motor proteins in regulating cell type-specific physiological functions within the cells of the renal epithelium. The purpose of this review is to provide a fresh conceptual framework to explain the structure-function relationships that exist between the actin cytoskeleton, organelle structure, and cargo transport within the mammalian kidney. With recent advances in technologies to visualize the actin cytoskeleton and associated proteins within intact kidneys, it has become increasingly imperative to reimagine the functional roles of these proteins in situ to provide a rationale for their unique, cell type-specific functions that are necessary to establish and maintain complex physiological processes.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F553-F565"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11483076/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141763108","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}