Distal Tubular Cells Research Articles

Impairment of Renal Function in Hermansky-Pudlak Syndrome

Introduction: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles. The genetic types of HPS are associated with a spectrum of multisystemic clinical manifestations. Phenotypic features of HPS type 1 (HPS-1) or HPS-4, which are associated with defects in biogenesis of lysosome-related organelles complex-3 (BLOC-3), are generally more severe than those of HPS-3, HPS-5, or HPS-6, which are associated with defects in BLOC-2. A paucity of information is available about renal impairment in HPS. The objective of this study is to expand the understanding of kidney disease in HPS. Methods: Medical records and clinical data of patients with HPS evaluated at the National Institutes of Health Clinical Center from 1995 to 2020 were retrospectively reviewed. For patients with more than one visit, the most recent renal function and urinalysis tests were analyzed. Estimated glomerular filtration rate (eGFR) was calculated using standard equations (i.e., Chronic Kidney Disease Epidemiology Collaboration, Modification of Diet in Renal Disease). Kidney tissue sections from 5 patients with HPS-1 and 1 patient with HPS-6 were examined. Results: Records from 205 adults and 52 children with HPS were reviewed. Calculated eGFR of adult patients with different HPS types differed significantly, and calculated eGFR of pediatric and adult patients with BLOC-3 disorders was significantly lower than that of patients with BLOC-2 disorders. Linear regression analysis showed that renal function progressively decreases with age in patients with BLOC-3 or BLOC-2 disorders, but the rate of decline was more rapid in patients with BLOC-3 disorders compared to patients with BLOC-2 disorders. In adult patients with HPS-1, glucosuria was found in 4%, proteinuria in 12%, hematuria in 15%, high levels of urinary β2MG in 24%, and elevated urinary albumin to creatinine ratios in 9%. Histological examination of kidney tissue showed accumulation of intracellular deposits of ceroid lipofuscin in proximal renal tubular epithelial cells in patients with HPS-1. There was no evidence of fibrosis, and glomeruli, distal renal tubular epithelial cells, and interstitial regions appeared histologically normal. Conclusion: Mild impairment of renal function is a feature of HPS. Kidneys of patients with HPS-1 contain proximal renal tubular intracellular deposits and no histologic evidence of fibrosis. Consistent with other manifestations of HPS, the phenotype of renal impairment is relatively more pronounced in patients with BLOC-3 disorders than in patients with BLOC-2 disorders. Strategies to avoid nephrotoxicity or renal tubular injury and to protect renal function should be considered for patients with HPS irrespective of age.

Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

CD44 is a transmembrane glycoprotein implicated in tissue inflammation and fibrosis. We investigated its role in kidney inflammation and fibrosis in a murine model of lupus nephritis (LN), and the clinico-pathological association of serum CD44 level in patients with biopsy-proven Class III/IV ± V LN. NZB/W F1 mice were treated with control IgG or anti-CD44 monoclonal antibody for 4 weeks and disease parameters assessed. Serum CD44 level in LN patients was determined by ELISA. Control groups included healthy subjects and patients with non-renal SLE or non-lupus renal disease. CD44 expression was absent in the normal kidney, but it was expressed in proximal and distal tubular epithelial cells and infiltrating cells in renal biopsies from patients with active proliferative LN. ScRNA-Seq datasets confirmed that CD44 was predominantly expressed in tubular cells and all immune cells identified in LN patients including tissue resident, inflammatory and phagocytic macrophages, Treg cells, effector and central memory CD4+ T cells, resident memory CD8+ T cells and naïve and activated B cells. Treatment of NZB/W F1 mice with anti-CD44 antibody preserved kidney histology and reduced proteinuria, tubulo-interstitial infiltration of CD3+, CD4+ and CD19+ immune cells, and mediators of kidney fibrosis compared to Control mice. Longitudinal studies showed that serum CD44 level increased prior to clinical renal flare by 4.5 months and the level decreased after treatment. ROC curve analysis showed that CD44 level distinguished patients with active LN from healthy subjects and patients with quiescent LN, active non-renal lupus, and non-lupus CKD (ROC AUC of 0.99, 0.96, 0.99 and 0.99 respectively). CD44 level correlated with leukocyte infiltration and interstitial inflammation scores in active LN kidney biopsies. Our findings suggest that CD44 plays a pathogenic role in renal parenchymal inflammation and fibrosis in active LN and monitoring CD44 may facilitate early diagnosis of flare.

Targeting the transmembrane cytokine co-receptor neuropilin-1 in distal tubules improves renal injury and fibrosis

Neuropilin-1 (NRP1), a co-receptor for various cytokines, including TGF-β, has been identified as a potential therapeutic target for fibrosis. However, its role and mechanism in renal fibrosis remains elusive. Here, we show that NRP1 is upregulated in distal tubular (DT) cells of patients with transplant renal insufficiency and mice with renal ischemia-reperfusion (I-R) injury. Knockout of Nrp1 reduces multiple endpoints of renal injury and fibrosis. We find that Nrp1 facilitates the binding of TNF-α to its receptor in DT cells after renal injury. This signaling results in a downregulation of lysine crotonylation of the metabolic enzyme Cox4i1, decreases cellular energetics and exacerbation of renal injury. Furthermore, by single-cell RNA-sequencing we find that Nrp1-positive DT cells secrete collagen and communicate with myofibroblasts, exacerbating acute kidney injury (AKI)-induced renal fibrosis by activating Smad3. Dual genetic deletion of Nrp1 and Tgfbr1 in DT cells better improves renal injury and fibrosis than either single knockout. Together, these results reveal that targeting of NRP1 represents a promising strategy for the treatment of AKI and subsequent chronic kidney disease.

Malathion toxicity attenuated by Lactobacillus bacteria via alteration of oxidative stress, lipid peroxidation, and inflammation: a possible mechanism against organophosphorus insecticide

Rats (11 groups) were treated with malathion (MAL) and probiotic bacteria for 6 weeks. The results obtained in kidney showed a significant increase in the malondialdehyde (≈92%), superoxide dismutase (≈41%), catalase (≈56%), IL-1β (≈45), and TNF-α (≈25%) in MAL-treated group. In contrast, testicular catalase, superoxide dismutase, and TNF-α levels decreased ≈ 33, 30, and 21%, respectively, in the MAL-treated groups. In kidney, treatment with the probiotics reduced malondialdehyde and catalase activity compared with the MAL-treated group. As well as, treatment with the L. rhamnosus, L. casei, L. plantarum, and L. acidophilus enhanced testicular catalase activity (≈21, 49, 52, and 26%, respectively) compared with the MAL-treated group. Unlike superoxide dismutase, in groups treated with L. plantarum, L. rhamnosus, inflammatory factor levels decreased in kidney compare with MAL-group. In testis, probiotic bacteria blocked MAL effect on malondialdehyde, catalase and TNF-α. Micrographs of kidney sections of the MAL revealed mild to moderate proximal and distal tubular cell necrosis. Histopathological evaluation of the L. acidophilus sample revealed less tissue toxicity in comparison to MAL group. Micrographs of testis sections of the showed no histological changes. Probiotics could effectively protect against malathion-induced damage in kidneys and testes, offering a potential solution for pesticide-related toxicity.

#417 Targeting parietal epithelial cell activation with mineralocorticoid receptor antagonism controls FSGS and crescentic glomerulonephritis

Abstract Background and Aims We have recently demonstrated that targeting specific pathways in parietal epithelial cells (PEC) can markedly alleviate experimental extracapillary glomerular injury [1]. Accumulating evidence has indicated the potential contributions of the mineralocorticoid receptor (MR) to the pathophysiology of chronic kidney disease. MR is expressed in endothelial cells, glomerular mesangial cells, podocytes, and distal tubular cells. Previous studies have shown that administering mineralocorticoid receptor antagonists has beneficial effects in various renal injury animal models, the role of the MR in extracapillary glomerulopathies is still elusive and mechanistically unclear. Methods To investigate the cell-specific role of the MR in PEC in the course of focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (GN), we generated chimeric mice specifically lacking MR (Pec Cre Nr3c2lox/lox) in PECs using an inducible Cre recombinase system. Maladaptive FSGS was induced by combining a high-salt diet and deoxycorticosterone acetate (DOCA) with unilateral nephrectomy (DOCA-UniN). Crescentic glomerulonephritis (GN) was induced using the anti-glomerular basement membrane nephrotoxic serum (NTS) model. Results At baseline, Pec Cre Nr3c2wt/wt and Pec Cre Nr3c2lox/lox mice displayed no kidney morphology and function differences. When challenged by DOCA-UniN, PEC-selective Nr3c2 targeting significantly inhibited the expression of PEC activation markers Ki67, CD44, and FSGS lesions. Importantly, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function compared to Pec Cre Nr3c2 wt/wt counterparts. Likewise, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function in the NTS-induced CGN model. Crescents were also more numerous and organised in Pec Cre Nr3c2wt/wt mice than in Pec Cre Nr3c2lox/lox animals. Next, we examined whether pharmacological MR inhibition could alleviate the severity of crescentic GN. When Pec Cre Nr3c2wt/wt mice were orally given eplerenone for 14 days after the onset of the crescentic GN, they were significantly protected from renal injury and failure (decreased proteinuria, normal BUN and reduced number of crescent). Such global action was associated with less expression of the CD44 activation marker on PECs. Thus, genetic disruption of MR in PEC and pharmacological inhibition using eplerenone reduced glomerular expression of CD44 and crescent formation. Furthermore, kidney biopsies of individuals diagnosed with crescentic glomerulonephritis displayed increased expression of MR in PEC within FSGS and crescents. We next evaluated PEC migration under various conditions (DMSO, HB-EGF, Eplerenone, or HB-EGF+Eplerenone) for 24 hours. Eplerenone fully abrogated HB-EGF-induced PEC migration and Pcna, Snai1, and Vim mRNA expressions. Conclusion Altogether, these results indicate the critical role of MR in PEC activation during crescentic glomerulonephritis, along with the recently discovered CD9/EGFR/PDGFR [1] and CLDN1 pathways. This further supports the idea that the PEC phenotype switch is not a bystander event but plays a targetable critical active pathogenic role in FGSS and, more surprisingly, in crescentic GN. MR modulation using eplerenone may be a new therapeutic option for the management of such severe disease.

Influenza A virus replicates productively in primary human kidney cells and induces factors and mechanisms related to regulated cell death and renal pathology observed in virus-infected patients.

Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.

Identification of uromodulin deposition in the stroma of perinephric fibromyxoid nephrogenic adenoma by mass spectrometry.

Nephrogenic adenoma (NA) is an epithelial lesion that usually occurs in the mucosa of the urinary tract. Rare cases of deep infiltrative or perinephric lesions have also been reported. Recently, NA with characteristic fibromyxoid stroma (fibromyxoid NA) has been proposed as a distinct variant. Although shedding of distal renal tubular cells due to urinary tract rupture has been postulated as the cause of NA in general, the mechanism underlying extraurinary presentation of NA and fibromyxoid stromal change in fibromyxoid NA remains unknown. In this study, we performed mass spectrometry (MS) analysis in a case of perinephric fibromyxoid NA of an 82-year-old man who underwent right nephroureterectomy for distal ureteral cancer. The patient had no prior history of urinary tract injury or radiation. Periodic acid-Schiff staining-positive eosinophilic structureless deposits in the stroma of fibromyxoid NA were microdissected and subjected to liquid chromatography/MS. The analysis revealed the presence of a substantial amount of uromodulin (Tamm-Horsfall protein). The presence of urinary content in the stroma of perinephric fibromyxoid NA suggests that urinary tract rupture and engraftment of renal tubular epithelial cells directly cause the lesion.

Roles of the mechanosensitive ion channel Piezo1 in the renal podocyte injury of experimental hypertensive nephropathy.

Glomerular podocyte injury plays an essential role in proteinuria pathogenesis, a hallmark of chronic kidney disease, including hypertensive nephropathy. Although podocytes are susceptible to mechanical stimuli, their mechanotransduction pathways remain elusive. Piezo proteins, including Piezo1 and 2, are mechanosensing ion channels that mediate various biological phenomena. Although renal Piezo2 expression and its alteration in rodent dehydration and hypertension models have been reported, the role of Piezo1 in hypertensive nephropathy and podocyte injury is unclear. In this study, we examined Piezo1 expression and localization in the kidneys of control mice and in those of mice with hypertensive nephrosclerosis. Uninephrectomized, aldosterone-infused, salt-loaded mice developed hypertension, albuminuria, podocyte injury, and glomerulosclerosis. RNAscope in situ hybridization revealed that Piezo1 expression was enhanced in the podocytes, mesangial cells, and distal tubular cells of these mice compared to those of the uninephrectomized, vehicle-infused control group. Piezo1 upregulation in the glomeruli was accompanied by the induction of podocyte injury-related markers, plasminogen activator inhibitor-1 and serum/glucocorticoid regulated kinase 1. These changes were reversed by antihypertensive drug. Exposure of Piezo1-expressing cultured podocytes to mechanical stretch activated Rac1 and upregulated the above-mentioned markers, which was antagonized by the Piezo1 blocker grammostola mechanotoxin #4 (GsMTx4). Administration of Piezo1-specific agonist Yoda1 mimicked the effects of mechanical stretch, which was minimized by the Yoda1-specific inhibitor Dooku1 and Rac inhibitor. Rac1 was also activated in the above-mentioned hypertensive mice, and Rac inhibitor downregulated gene expression of podocyte injury-related markers in vivo. Our results suggest that Piezo1 plays a role in mechanical stress-induced podocyte injury.

Urinary PART1 and PLA2R1 Could Potentially Serve as Diagnostic Markers for Diabetic Kidney Disease Patients.

Diabetic kidney disease (DKD) is a chronic renal disease which could eventually develop into renal failure. Though albuminuria and estimated glomerular filtration rate (eGFR) are helpful for the diagnosis of DKD, the lack of specific biomarkers reduces the efficiency of therapeutic interventions. Based on bulk-seq of 56 urine samples collected at different time points (including 11 acquired from DKD patients and 11 from healthy controls), in corporation of scRNA-seq data of urine samples and snRNA-seq data of renal punctures from DKD patients (retrieved from NCBI GEO Omnibus), urine-kidney specific genes were identified by Multiple Biological Information methods. Forty urine-kidney specific genes/differentially expressed genes (DEGs) were identified to be highly related to kidney injury and proteinuria for the DKD patients. Most of these genes participate in regulating glucagon and apoptosis, among which, urinary PART1 (mainly derived from distal tubular cells) and PLA2R1 (podocyte cell surface marker) could be used together for the early diagnosis of DKD. Moreover, urinary PART1 was significantly associated with multiple clinical indicators, and remained stable over time in urine. Urinary PART1 and PLA2R1 could be shed lights on the discovery and development of non-invasive diagnostic method for DKD, especially in early stages.

High RIPK3 expression is associated with a higher risk of early kidney transplant failure

Renal ischemia-reperfusion injury (IRI) is associated with reduced allograft survival, and each additional hour of cold ischemia time increases the risk of graft failure and mortality following renal transplantation. Receptor-interacting protein kinase 3 (RIPK3) is a key effector of necroptosis, a regulated form of cell death. Here, we evaluate the first-in-human RIPK3 expression dataset following IRI in kidney transplantation. The primary analysis included 374 baseline biopsy samples obtained from renal allografts 10minutes after onset of reperfusion. RIPK3 was primarily detected in proximal tubular cells and distal tubular cells, both of which are affected by IRI. Time-to-event analysis revealed that high RIPK3 expression is associated with a significantly higher risk of one-year transplant failure and prognostic for one-year (death-censored) transplant failure independent of donor and recipient associated risk factors in multivariable analyses. The RIPK3 score also correlated with deceased donation, cold ischemia time and the extent of tubular injury.

Renal Tubular CD24 Upregulation Aggravates Folic Acid Induced Acute Kidney Injury: A Possible Role for T Regulatory Cells Inhibition in Mice.

Acute kidney injury (AKI) is characterized by cell death and inflammation. CD24 is a protein induced during tissue damage and is not expressed in mature renal tissue. We explored the role of CD24 in the pathogenesis of folic acid-induced AKI (FA-AKI) in mice. A single Intraperitoneal (IP) injection of folic acid induced AKI in WT and CD24-/- mice. Renal function tests, histological analysis, immunohistochemistry, Western blot analysis, and ELISA were performed to assess the severity of renal damage and the intensity of the inflammatory response. FA-AKI induced CD24 in the distal tubular epithelial cells. Compared to WT mice, FA-AKI CD24-/- mice exhibited an attenuated reduction in renal function and histological injury, lower serum IL-10 and interferon γ, and decreased expression of renal TNFα. In contrast, renal and systemic IL-33 upregulation were augmented. CD24-/- FA-AKI animals exhibited increased splenic margination and renal infiltration of regulatory T cells (Tregs). At day 7, FA-AKI CD24-/- mice exhibited increased expression of tubular pro-apoptotic and decreased anti-apoptotic proteins compared to WT animals. Anti-CD24 antibody administration to FA-AKI mice attenuated the decrease in renal function as well as the histological injury. Renal biopsies from patients with ATN stained strongly for CD24 in the distal tubules. In conclusion, during AKI, upregulation of CD24 promotes renal inflammation through inhibition of Treg infiltration and diversion of cell death towards necrosis rather than apoptosis. Neutralization of CD24 may prove a target for future therapies in AKI.

#3293 ESTRADIOL REPLACEMENT MITIGATED BLOOD PRESSURE ELEVATION VIA SUPPRESSION OF NCC IN ANGIOTENSIN II-INFUSED OVARIECTOMIZED FEMALE RATS

Abstract Background and Aims Female rats excrete more urine sodium than male rats with similar blood pressure (BP). However, it is not clear whether the sex difference in pressure natriuresis is conserved in menopausal rats. We assessed whether the natriuretic response and renal sodium transporter activity in ovariectomized female rats infused with angiotensin II (ANGII) could be affected by estrogen supplementation. Method Six-week-old female Sprague-Dawley rats (n=30) were ovariectomized (OVX, n =20) or sham-operated (n=10) (−4 weeks, −4W). After 2 weeks, subcutaneous estradiol was administered to half of the OVX rats (n=10) for 4 weeks (-2W∼2W). ANGII was infused via the osmotic minipump in 60% of the rats for 2 weeks (0W∼2W). Six groups (female saline: FS, female ANGII: FA, OVX saline: OS, OVX ANGII: OA, OVX estradiol saline: OES, OVX estradiol ANGII: OEA) were sacrificed after 2 weeks of the ANGII infusion (2W). We also analyzed human data from the Korean Genome and Epidemiology Study (KoGES). Results ANGII-induced BP elevation was highest in the OA group, followed by the FA and OEA groups. Urine sodium tended to increase after ANGII infusion in FA and OEA but decreased in OA. Western blot results showed that cortical phosphorylated sodium chloride cotransporter (pNCC) tended to be augmented by ANGII treatment in FA and OA, but mitigated in OEA. Cortical ANGII type 2 receptor (AT2R) expression was significantly higher in estradiol-treated OVX rats than in OVX rats, irrespective of ANGII infusion. Similarly, the enhanced pNCC in ANGII-treated distal convoluted tubular cells was reversed by estradiol treatment. In KoGES data, urine sodium was significantly lower in postmenopausal women than in premenopausal women, despite the higher BP. Conclusion Estradiol reversed BP elevation and augmented the natriuretic response in ANGII-infused OVX rats and distal tubular cells via suppression of pNCC and increase of AT2R.

THE (PRO)RENIN RECEPTOR IS INVOLVED IN KIDNEY ORGANOID DEVELOPMENT

Objective: The (pro)renin receptor [(P)RR], a receptor for prorenin and renin, is widely distributed in the body, including the kidneys. Studies in (P)RR knockout mouse models indicate that (P)RR ablation can be detrimental to renal development and function. However, the lack of human models hinders investigation into the role of (P)RR in human kidney development. The advent of human induced pluripotent stem cell (iPSC) – derived kidney organoids provides a tool to study the role of (P)RR in human kidney development. Design and method: To investigate the effects of (P)RR knockdown on kidney organoid development, human iPSC – derived kidney organoids were generated according to a previously described protocol. We silenced the (P)RR by introducing (P)RR antisense oligonucleotides (ASOs) by electroporation at the stage of iPSCs and nephron progenitor cells induction, respectively. The development of kidney organoids was monitored morphologically and through protein expression analysis of nephron markers by immunohistochemistry staining which was quantified by Image J. Results: After silencing the (P)RR at the initial stage of differentiation, the size of iPSCs-derived organoids was substantially smaller, but the size of tubular structures was bigger. We observed a decrease by 50% in both WT1 (glomerular cell marker) and PDGFR↑ (stromal cell marker) expressions in (P)RR-knockdown organoids, a 4-fold and 2-fold increase in expressions of fibrogenic cell markers ↑-SMA and COL1A1 respectively, and no change in Villin1 (proximal tubular cell marker) and Cadherin-1 (distal tubular cell marker) expression. Additionally, (P)RR knockdown at the stage of nephron progenitor cells induction decreased the expressions of Villin1 and Cadherin-1 in organoids by 50% and 60% separately, while it induced a 4-, 3-, 2- and 2-fold increase in the expressions of CD31 (endothelial cell marker), PDGFR↑, ↑-SMA and COL1A1 respectively. Conclusions: This study shows that (P)RR silencing at the initial stage of differentiation or at the stage of nephron progenitor cells induction impaired normal development of kidney organoids. Its absence caused a shift into the direction of fibrogenic cells. This study provides new insights into the understanding the role of (P)RR in human kidney development.

What UV is what you get: skin injury by ultraviolet (UV) light results in renal cell loss and damage in the presence of a CD177high neutrophil population.

Abstract Immune-mediated communication between organs has been implicated in the pathogenesis of many inflammatory and autoimmune diseases. We previously showed that skin injury by UV light triggers neutrophil migration to the kidney, stimulating expression of renal injury genes. This skin-kidney axis is highly relevant to lupus patients, for whom skin exposure to UV sunlight rays can lead to kidney disease flares. Here, we applied 10X single-cell RNA-seq on saline-perfused digested kidneys and Ly6G-enriched neutrophils of B6 mice, prior to and 2 days after a single dose of UV, to define renal cell subsets damaged by skin exposure to UV and how neutrophils mediate the injury. Dimensional reduction analysis revealed 14 kidney cell types and showed that exposure to UV led to the loss of podocytes and distal tubular cells (DTCs) and an expansion in endothelial and stromal cells. Differential gene expression analysis showed that podocytes and DTCs in UV-exposed mice upregulated genes associated with renal injury, including type I interferon genes, and downregulated genes important for cellular integrity and function. Neutrophil scRNA-seq revealed a unique activated CD177 highneutrophil population in the kidneys of UV exposed mice, defined as the endpoint of the psuedotime trajectory by diffusion mapping. Ligand-receptor pair analyses showed skin injury by UV altered neutrophil interactions with renal structural cells, with expanded binding to stromal cells. While the CD177 highneutrophil cluster was dominant in the kidney, it was also found in the lung, but not in the skin and blood. These studies introduce a novel model by which CD177 highneutrophils may mediate podocyte, DTC, and stromal kidney injury secondary to sterile skin inflammation. Supported by grants from NIH (1R21AR079661 – 01, 5P20GM130454-02), The Department of Defense (W81XWH2110889), and Dartmouth Health Start Up Funds.

Calcium oxalate crystal-induced secretome derived from proximal tubular cells, not that from distal tubular cells, induces renal fibroblast activation

BackgroundKidney stone disease (KSD) is commonly accompanied with renal fibrosis, characterized by accumulation and reorganization of extracellular matrix (ECM). During fibrogenesis, resident renal fibroblasts are activated to become myofibroblasts that actively produce ECM. However, such fibroblast–myofibroblast differentiation in KSD remained unclear. Our present study thus examined effects of secreted products (secretome) derived from proximal (HK-2) vs. distal (MDCK) renal tubular cells exposed to calcium oxalate monohydrate (COM) crystals on activation of renal fibroblasts (BHK-21).MethodsHK-2 and MDCK cells were treated with 100 µg/ml COM crystals under serum-free condition for 16 h. In parallel, the cells maintained in serum-free medium without COM treatment served as the control. Secretome derived from culture supernatant of each sample was mixed (1:1) with fresh serum-free medium and then used for BHK-21 culture for another 24 h.ResultsAnalyses revealed that COM-treated-HK-2 secretome significantly induced proliferation, caused morphological changes, increased spindle index, and upregulated fibroblast-activation markers (F-actin, α-SMA and fibronectin) in BHK-21 cells. However, COM-treated-MDCK secretome had no significant effects on these BHK-21 parameters. Moreover, level of transforming growth factor-β1 (TGF-β1), a profibrotic factor, significantly increased in the COM-treated-HK-2 secretome but not in the COM-treated-MDCK secretome.ConclusionsThese data indicate, for the first time, that proximal and distal tubular epithelial cells exposed to COM crystals send different messages to resident renal fibroblasts. Only the secretome derived from proximal tubular cells, not that from the distal cells, induces renal fibroblast activation after their exposure to COM crystals. Such differential effects are partly due to TGF-β1 secretion, which is induced by COM crystals only in proximal tubular cells.

Common gene signatures and molecular mechanisms of diabetic nephropathy and metabolic syndrome.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Multiple metabolic toxicities, redox stress, and endothelial dysfunction contribute to the development of diabetic glomerulosclerosis and DN. Metabolic syndrome (MetS) is a pathological state in which the body's ability to process carbohydrates, fats, and proteins is compromised because of metabolic disorders, resulting in redox stress and renal remodeling. However, a causal relationship between MetS and DN has not been proven. This study aimed to provide valuable information for the clinical diagnosis and treatment of MetS with DN. Here, transcriptome data of DN and MetS patients were obtained from the Gene Expression Omnibus database, and seven potential biomarkers were screened using bioinformatics analysis. In addition, the relationship between these marker genes and metabolism and immune infiltration was explored. Among the identified marker genes, the relationship between PLEKHA1 and the cellular process, oxidative phosphorylation (OXPHOS), in DN was further investigated through single-cell analysis. We found that PLEKHA1 may represent an important biomarker that perhaps initiates DN by activating B cells, proximal tubular cells, distal tubular cells, macrophages, and endothelial cells, thereby inducing OXPHOS in renal monocytes. Overall, our findings can aid in further investigation of the effects of drug treatment on single cells of patients with diabetes to validate PLEKHA1 as a therapeutic target and to inform the development of targeted therapies.

High glucose induces an activated state of partial epithelial-mesenchymal transition in human primary tubular cell cultures.

Tubulointerstitial fibrosis is observed in diabetic nephropathy. It is still debated whether tubular cells, undergoing epithelial-mesenchymal transition (EMT) in high glucose (HG) conditions, may contribute to interstitial fibrosis development. In this study, we investigated the phenotypic and molecular EMT-like changes and the alteration of inflammatory and fibrogenic secretome induced by HG in human primary tubular cell cultures. Taking advantage of this in vitro cell model composed of proximal and distal tubular cells, we showed that HG-treated tubular cells acquired a fibroblast-like morphology with increased cytoplasmic stress fibers, maintaining the expression of the epithelial markers specific of proximal and distal tubular cells. HG increased Snail1, miRNA210 and Vimentin mesenchymal markers, decreased N-cadherin expression and migration ability of primary tubular cells, while E-cadherin expression and focal adhesion distribution were not affected. Furthermore, HG treatment of tubular cells altered the inflammatory cytokine secretion creating a secretome able to enhance the proliferation and migration of fibroblasts. Our findings show that HG promotes an activated state of partial EMT in human tubular primary cells and induces a pro-inflammatory and pro-fibrogenic microenvironment, supporting the active role of tubular cells in diabetic nephropathy onset.

A Risk Model to Predict the Longitudinal Decline in Creatinine Clearance Among Patients with Precursor Plasma Cell Dyscrasia with Baseline Chronic Kidney Failure

Monoclonal Gammopathy of Unknown Significance (MGUS) is characterized as an age-dependent condition that presents with circulating monoclonal protein originating from the clonal proliferation of plasma cells in bone marrow without overt renal failure. However, these proteins have potentials to exert stress on Glomeruli, proximal or distal tubular cells through activation of TOLL-like receptors resulted from excessive endocytosis of light chain proteins, leading to subclinical renal pathologies. This phenomenon lead to forming a new clinicopathological entity named Monoclonal Gammopathy of Renal Significance (MGRS) and can be more pronounced in patients with marginal renal function due to other causes. Importantly, the establishment of MGRS diagnosis is based on renal biopsy which is avoided frequently due to its invasive nature, therefore its overall incidence remains to be defined. Here we aim to define a risk model to predict the rate of worsening renal function in patients with Chronic Kidney Disease (CKD) who have simultaneous MGUS. Methods: To define the impact of circulating monoclonal protein on natural course of CKD progression we conducted a retrospective study of CKD patients with MGUS vs. CKD patients without MGUS. Primary end point of the study was Time to End Stage Renal Disease (TTESRD) measured from the date of diagnosis of CKD to the date of diagnosis of ESRD and was censored at the date of last follow-up for those without ESRD with death as competing risk. The Fine and Gray method was used for comparisons of cumulative incidence of ESRD between groups. The effect of important factors on TTESRD was further evaluated using multivariable Fine and Gray method. Survivor distribution was estimated using Kaplan-Meier methods and the difference of OS between/among groups was examined by log-rank test. The difference of continuous variable (age) between group was examined using T-test and the association between two categorical variables was examined using Chi-square test. CKD stages is defined by Glomerular Filtration Rate (GFR) into classic 5 categories as explained before. The rate of stage progression was analyzed as secondary end point. Results: Patients without valid data (age, date of diagnosis, invalid follow-up data) were excluded for the statistical analysis. The final analysis set included 626 patients with MGUS and CKD and 15337 patients with CKD but without known MGUS. The median follow up time was 19.7 months (range: 0.03, 128). The distribution of CKD stage and other risk factors at both cohorts are illustrated in Table-1. There was significant difference of time-to-ESRD comparing the patients who had CKD alone to patients who had both CKD and MGUS (p = 0.037) in the univariate analysis. After controlling the effects of age, gender, race, CKD stage, diabetes and hypertension, there was still marginally significant difference of TTESRD, comparing the patients who had CKD alone to patients who had both CKD and MGUS (p = 0.056). Specifically, the hazard of having ESRD for patients who had CKD without MGUS was 1.64 times (95% CI: 0.99, 2.72) of the hazard of having ESRD for patients who had CKD and MGUS (see Figure-1). The CKD stage advancement according to MGUS was analyzes as the secondary end-point of the study. The CKD stage at baseline was compared to the CKD stage at year 1 and year 5 during follow-up. The progression by stage was 28.5% at year 1 for patients without MGUS vs. 29.3% for those with MGUS (p = 0.73). The progression by stage was 38.7% at year 5 for patients without MGUS vs. 39.4 % for those with MGUS (p = 0.89).There was no statistically significant difference between median OS for CKD patients with or without MGUS. Conclusion: Here we presented the data of around 16,000 CKD pts based on the MGUS diagnosis in 19 years time span and we could not detect any higher trend for CKD pts with MGUS to develop ESRD or move to higher CKD stage. These data can suggest MGRS forms a quite thin slice of the whole MGUS population. Assessing finding of this study in a larger national database is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased expression of the RNA-binding protein Musashi-2 is associated with immune infiltration and predicts better outcomes in ccRCC patients.

RNA-binding proteins (RBPs) mainly contribute to abnormalities in posttranscriptional gene regulation. The RBP Musashi-2, an evolutionarily conserved protein, has been characterized as an oncoprotein in various tumors. However, the prognostic value and potential roles of Musashi-2 in clear cell renal cell carcinoma (ccRCC) have not yet been elucidated. In this study, we found that Musashi-2 was mainly expressed in the normal distal tubular cells and collecting duct cells of the kidneys, while its expression was significantly decreased in ccRCC. And higher expression levels of Musashi-2 indicated better overall survival (OS) in ccRCC. Furthermore, immunohistochemistry demonstrated that PD-L1 expression was negatively correlated with Musashi-2 expression, and Musashi-2 was found to be remarkably correlated with multiple immune cells and immune inhibitors, including CD8+ T cells, CD4+ T cells, regulatory T (Treg) cells, PDCD1, CTLA4, Foxp3, and LAG3. Functional enrichment analysis revealed that Musashi-2 might be involved in ccRCC metabolic reprogramming and immune infiltration and further predicted the therapeutic sensitivity of ccRCC. Taken together, Musashi-2 is a prognostic biomarker for ccRCC patients that may provide novel insights into individualized treatment strategies and guide effective immunotherapy.

RETRACTED: Histological, ultrastructural and immunohistochemical studies on the ameliorative role of Cinnamon zeylanicum against high cholesterol diet-induced hypercholesterolemia in the kidney of adult male albino rats

Cholesterol is an important type of lipid as it is involved in the structure of cell membrane, synthesis of steroid hormones, bile acid and vitamin D. Many diseases affect various mammalian organs, including the kidney, as a result of high cholesterol levels (hypercholesterolemia). Cinnamon zeylanicum (C. zeylanicum) proves its efficiency as it has anti-inflammatory and antioxidant prosperities. This study aimed to investigate the possible ameliorative role of C. zeylanicum on hypercholesterolemia-induced the renal toxicity in albino rats. Forty adult male albino rats were equally divided into four groups. The first group served as the control one. The second group was supplemented with C. zeylanicum powder (15% w/w) with the standard diet. The third group was fed high cholesterol diet (HCD) to induce acute hypercholesterolemia. The fourth group was fed HCD provided with C. zeylanicum powder (15% w/w). At the end of the experiment (8th weeks), kidneys were removed and prepared for histological, immunohistochemical and ultrastructure studies. Rats-fed HCD showed degenerated glomeruli and tubular cells with vacuolated or coagulated cytoplasm and pyknotic nuclei. Moreover, the renal cortex ultrastructural examination showed degenerated podocytes, parietal and mesangial cells, as well as the proximal and distal tubular cells appeared with rarified cytoplasm, degenerated mitochondria, large fat vacuoles and complete damaged microvilli. The same group showed a significant increase in the expression of desmin and inducible nitric oxide synthase. On the other hand, animals fed HCD provided with C. zeylanicum showed an obvious improvement in the observed histological, ultrastructural and immunohistochemical changes. The architecture of the renal cortex appeared mostly similar to the control one. This study concluded that C. zeylanicum has a promising role in treating the nephron-toxicity of HCD due to its natural constituent that is responsible for its pharmaceutical effects.