Renal Epithelium Research Articles

Energy depletion by cell proliferation sensitizes the kidney epithelial cells to injury.

Acute kidney injury activates both proliferative and antiproliferative pathways, the consequences of which are not fully elucidated. If an initial proliferation of the renal epithelium is necessary for the successful repair, the persistence of proliferation markers is associated with the occurrence of chronic kidney disease. We hypothesized that proliferation in stress conditions impacts cell viability and renal outcomes. We found that proliferation is associated with cell death after various stresses in kidney cells. In vitro, the ATP/ADP ratio oscillates reproducibly throughout the cell cycle, and cell proliferation is associated with a decreased intracellular ATP/ADP ratio. In vivo, transcriptomic data from transplanted kidneys revealed that proliferation was strongly associated with a decrease in the expression of the mitochondria-encoded genes of the oxidative phosphorylation pathway, but not of the nucleus-encoded ones. These observations suggest that mitochondrial function is a limiting factor for energy production in proliferative kidney cells after injury. The association of increased proliferation and decreased mitochondrial function was indeed associated with poor renal outcomes. In summary, proliferation is an energy-demanding process impairing the cellular ability to cope with an injury, highlighting proliferative repair and metabolic recovery as indispensable and interdependent features for successful kidney repair.NEW & NOTEWORTHY ATP depletion is a hallmark of acute kidney injury. Proliferation is instrumental to kidney repair. We show that ATP levels vary during the cell cycle and that proliferation sensitizes renal epithelial cells to superimposed injuries in vitro. More proliferation and less energy production by the mitochondria are associated with adverse outcomes in injured kidney allografts. This suggests that controlling the timing of kidney repair might be beneficial to mitigate the extent of acute kidney injury.

ΔNp63α promotes cigarette smoke-induced renal cancer stem cell activity via the Sonic Hedgehog pathway.

Cigarette smoke (CS) has been generally recognized as a chief carcinogenic factor in renal cell carcinoma (RCC). The stimulative effect of CS on renal cancer stem cells (RCSCs) has been described previously. The Sonic Hedgehog (SHH) pathway plays an essential role in self-renewal, cell growth, drug resistance, metastasis, and recurrence of cancer stem cells (CSCs). Renal cancer-related gene ΔNp63α is highly expressed in renal epithelial tissues and contributes to the RCSCs characteristics of tumors. The aim of this study was to elucidate the role of ΔNp63α and the SHH pathway on the activity of RCSCs induced by CS through a series of in vivo and in vitro studies. It was shown that in renal cancer tissues, ΔNp63α and RCSCs markers in smokers are expressed higher than that in non-smokers. RCSCs were effectively enriched by tumor sphere formation assay. Besides, CS increased the expression of RCSCs markers and the capability of sphere-forming in vitro and in vivo. Moreover, the SHH pathway was activated, and the specialized inhibitor alleviated the promotion of CS on RCSCs. ΔNp63α activated the SHH pathway and promoted CS-induced enhancement of RCSCs activity. These findings indicate that ΔNp63α positively regulates the activity of CS-induced RCSCs via the SHH pathway.

Effect of Perfluorooctanoic Acid on Kidney Function in Diabetic and Non-‎‎Diabetic Male Guinea Pigs

Perfluorooctanoic acid (PFOA) is a synthetic fluor-surfactant chemical used widely in ‎products that resist oil, heat, grease, stains, and water. It is also used in producing other ‎fluoropolymers. The main sources of exposure to PFOA are water, soil, and animal-‎origin food (meat, fish, and dairy products). The aim of this study to evaluate the renal ‎function following oral gavage of sub-lethal dose of PFOA in diabetic and non-diabetic ‎guinea pigs. The experiment run for 4 weeks, total of 40 male guinea pigs, ‎‎(Cavia porcellus), were randomly selected and grouped into four equal groups. The first ‎group (G1) served as the negative control; 2nd group (G2) alloxan induced diabetic, 3rd ‎group (G3) non-diabetic was exposed to PFOA at 100 mg/kg BW orally/daily and 4th ‎Group (G4) was diabetic guinea pig exposed to PFOA at 100 mg/kg BW orally/daily. ‎Serum creatinine and histopathological alterations in the kidney tissue were evaluated. ‎Serum creatinine concentrations were significantly increased (P<0.05) in G3 and G4 ‎exposed to PFOA. High serum creatinine levels were suggesting impairment in kidney ‎function. Impaired kidney function was confirmed through histopathological changes ‎such as glomerular atrophy, severe necrosis, and degeneration of renal tubular epithelium ‎in guinea pigs that received PFOA in G3 and G4. In conclusion, the results confirmed ‎that PFOA was associated with renal damage and elevated creatinine concentrations in ‎diabetic and non-diabetic animals since PFOA itself can contribute to diabetes‎‎‎.

Progression of renal damage and tubular regeneration in pregnant and non-pregnant adult female rats inoculated with a sublethal dose of Shiga toxin 2

BackgroundShiga toxin-producing Escherichia coli is the main cause of post-diarrheal hemolytic uremic syndrome (HUS) which produces acute kidney injury mainly in children, although it can also affect adults. The kidneys are the organs most affected by Shiga toxin type 2 (Stx2) in patients with HUS. However, previous studies in pregnant rats showed that a sublethal dose of Stx2 causes severe damage in the uteroplacental unit and induces abortion, whereas produces mild to moderate renal damage. The aim of the present work was to study the progression of renal injury caused by a sublethal dose of Stx2, as well as renal recovery, in pregnant and non-pregnant rats, and to investigate whether pregnancy physiology may affect renal damage progression mediated by Stx2. MethodsRenal function and histopathology was evaluated in pregnant rats intraperitoneally injected with a sublethal dose of Stx2 (0.5 ng/g bwt) at the early stage of gestation (day 8 of gestation), and results in these rats were compared over time with those observed in non-pregnant female rats injected with the same Stx2 dose. Hence, progression of cell proliferation and dedifferentiation in renal tubular epithelia was also investigated. ResultsThe sublethal dose of Stx2 induced abortion in pregnant rats as well as a significant more extended functional and histological renal injury in non-pregnant rats than in pregnant rats. Stx2 also caused decreased ability to concentrate urine in non-pregnant rats compared to their controls. However, renal water handling in pregnant rats was not altered by Stx2, and was significantly different than in non-pregnant rats. The greatest renal injury in both pregnant and non-pregnant rats was observed at 4 days post-Stx2 injection, and coincided with a significant increase in tubular epithelial proliferation. Expression of mesenchymal marker vimentin in tubular epithelia was consistent with the level of tubular damage, being higher in non-pregnant rats than in pregnant rats. Recovery from Stx2-induced kidney injury was faster in pregnant rats than in non-pregnant rats. ConclusionsAdaptive mechanisms developed during pregnancy such as changes in water handle and renal hemodynamic may contribute to lessen the Stx2-induced renal injury, perhaps at the expense of fetal loss.

Histopathological study and molecular characterization of Infectious Bronchitis virus circulating in broilers in Basrah governorate, Iraq

The poultry industry plays an important role in the food economics and the food industry in both their layer and broiler chicken. Poultry diseases on the other way impact in the poultry industry especially diseases from virus sources since it does not have a specific treatment except for vaccines with routine prevention and control. This study was designed for the assessment of histopathological changes, molecular diagnosis and phylogenetic analyses of the Infectious Bronchitis virus circulating in broilers in Basrah province in the south of Iraq. The tissue samples were (trachea, lung and kidney). One hundred samples were studied with clinical signs and lesions related to IB. The histopathological changes caused by the virus were studied with hematoxylin and eosin stain, and the causative agent Infectious Bronchitis Virus (IBV) was identified using a polymerase chain reaction combined with explaining the phylogenetic analysis. The results of molecular diagnosis and identification showed the twelve Iraqi field strains of IBV were closely related and shared a high identity of 98.44 %- 99.69 % of each other. Variant 2(IS/1494 like) was a predominant genotype of IBV in the present study, and has high similarity with the variant 2(IS/1494) like a strain of IBV circulating in Iran and in the Kurdistan region north of Iraq. The histopathological findings were desquamation of the cilia of the mucosa layer of the trachea, infiltration of inflammatory cells, hyperplasia of goblet cells with thickening of mucosal layer in some areas infiltration of inflammatory exudate within capillaries and Para bronchus, where the microscopic results in kidney were showed interstitial hemorrhage, necrosis of the renal tubular epithelium, infiltration of inflammatory cell. The present results provided useful information on target organs damaged by IBV and the genetic epidemiology of such virus strains circulating in Basrah governorate Iraq during this year that would be helpful to develop a local vaccine.

Nucleocapsid protein accumulates in renal tubular epithelium of a post-COVID-19 patient

ABSTRACT In this study, we investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins and virus particles can be detected (i) in post-mortem kidney of fatal coronavirus disease 2019 (COVID-19) patients and (ii) in renal biopsy of patients suffering from kidney failure months after combating a SARS-CoV-2 infection. Using fluorescence microscopy, antibodies against viral proteins [nucleocapsid, membrane (M), non-structural proteins (nsp) 3, 4, and 13] and double-stranded RNA (dsRNA) were validated on SARS-CoV-2-infected Vero cells. dsRNA was detected in lipid-filled electron-lucent (white) compartments in SARS-CoV-2-producing cells. In the kidney of a patient with acute kidney failure, accumulation of SARS-CoV-2 nucleocapsid N protein was detected in tubular epithelium. In contrast, none of the other viral proteins (M, nsp3, 4, 13) nor dsRNA was detected in these regions, suggesting that no viral replication takes place in the kidney of these patients. High-resolution immunoelectron microscopy demonstrated N protein accumulation in Golgi-like structures but absence of intracellular virus particles. The presence of the N protein in Golgi-like structures in kidney of patients suggests that this protein may have an unforeseen role in post-COVID-19 renal complications. IMPORTANCE Even though the coronavirus disease 2019 (COVID-19) pandemic is slowly developing into a conventional infectious disease, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection are still not well understood. One of the problems is that many COVID-19 cases develop acute kidney injuries. Still, it is heavily debated whether SARS-CoV-2 virus enters and actively replicates in kidney tissue and if SARS-CoV-2 virus particles can be detected in kidney during or post-infection. Here, we demonstrated that nucleocapsid N protein was detected in kidney tubular epithelium of patients that already recovered form COVID-19. The presence of the abundantly produced N protein without signs of viral replication could have implications for the recurrence of kidney disease and have a continuing effect on the immune system.

MiR-17/-20a Alleviates Renal Interstitial Fibrosis by Repressing Frmd6 Expression in Renal Epithelia

miR-17/-20a Alleviates Renal Interstitial Fibrosis by Repressing Frmd6 Expression in Renal Epithelia

Mercury species analysis and tissue distribution in rats after continuous administration of Cinnabaris

Cinnabaris is a traditional Chinese medicine(TCM) commonly used for sedation and tranquilization in clinics, and its safety has always been a concern. This study intends to investigate the species and tissue distribution of mercury in rats after continuous administration of Cinnabaris. In the experiment, 30 rats were randomly divided into the control group(equivalent to 0.5% carboxy-methyl cellulose sodium), low-dose Cinnabaris group(0.2 g·kg~(-1)), high-dose Cinnabaris group(2 g·kg~(-1)), pseudogerm-free control group(equivalent to 0.5% sodium carboxymethyl cellulose), and pseudogerm-free Cinnabaris group(2 g·kg~(-1)). They were orally administered for 30 consecutive days. Ultrasound-assisted acid extraction method combined with high performance liquid chromatography and inductively coupled plasma-mass spectrometry(HPLC-ICP-MS) was adopted to determine inorganic mercury [Hg(Ⅱ)], methylmercury(MeHg), and ethylmercury(EtHg) in different tissue, plasma, urine, and feces of rats. The optimal detection conditions and extraction methods were optimized, and the linearity(R~2>0.999 3), precision(RSD<7.0%), and accuracy(spike recoveries ranged from 73.05% to 109.5%) of all the mercury species were satisfied, meeting the requirements of analysis. The results of mercury species detection showed that Hg(Ⅱ) was detected in all the tissue of the five experimental groups, and the main accumulating organs were the intestinal tract, stomach, and kidney. MeHg existed at a low concentration in most tissue, and EtHg was not detected in all groups. In addition, pathological examination results showed that hepatocyte vacuolar degeneration, loose cytoplasm, light staining, and mononuclear cell infiltration were observed in the high-dose Cinnabaris group, low-dose Cinnabaris group, and pseudogerm-free Cinnabaris group, with slightly milder lesions in the low-dose Cinnabaris group. Hydrous degeneration of renal tubular epithelium could be seen in the high-dose Cinnabaris group and pseudogerm-free Cinnabaris group, but there was no significant difference between the other groups and the control group. No abnormal changes were found in the brain tissue of rats in each group. This paper studied the different mercury species and tissue distribution in normal and pseudogerm-free rats after continuous administration of Cinnabaris for 30 days and clarified its effects on the tissue structure of the liver, kidney, and brain, which provided supporting evidence for the safety evaluation of Cinnabaris.

DKK3 as a potential novel biomarker in patients with autosomal polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, and leads to a steady loss of kidney function in adulthood. The variable course of the disease makes it necessary to identify the patients with rapid disease progression who will benefit the most from targeted therapies and interventions. Currently, magnetic resonance imaging-based volumetry of the kidney is the most commonly used tool for this purpose. Biomarkers that can be easily and quantitatively determined, which allow a prediction of the loss of kidney function, have not yet been established in clinical practice. The glycoprotein Dickkopf 3 (DKK3) which is secreted in the renal tubular epithelium upon stress and contributes to tubulointerstitial fibrosis via the Wnt signaling pathway, was recently described as a biomarker for estimating risk of kidney function loss, but has not been investigated for ADPKD. This study aimed to obtain a first insight into whether DKK3 may indeed improve outcome prediction in ADPKD in the future. In 184 ADPKD patients from the AD(H)PKD registry and 47 healthy controls, the urinary DKK3 (uDKK3) levels were determined using ELISA. Multiple linear regression was used to examine the potential of these values in outcome prediction. ADPKD patients showed significantly higher uDKK3 values compared with the controls (mean 1970 ± 5287 vs 112±134.7 pg/mg creatinine). Furthermore, there was a steady increase in uDKK3 with an increase in the Mayo class (A/B 1262 ± 2315 vs class D/E 3104±7627 pg/mg creatinine), the best-established biomarker of progression in ADPKD. uDKK3 also correlated with estimated glomerular filtration rate (eGFR). Patients with PKD1 mutations show higher uDKK3 levels compared with PKD2 patients (PKD1: 2304±5119; PKD2: 506.6±526.8 pg/mg creatinine). Univariate linear regression showed uDKK3 as a significant predictor of future eGFR slope estimation. In multiple linear regression this effect was not significant in models also containing height-adjusted total kidney volume and/or eGFR. However, adding both copeptin levels and the interaction term between copeptin and uDKK3 to the model resulted in a significant predictive value of all these three variables and the highest R2 of all models examined (∼0.5). uDKK3 shows a clear correlation with the Mayo classification in patients with ADPKD. uDKK3 levels correlated with kidney function, which could indicate that uDKK3 also predicts a disproportionate loss of renal function in this collective. Interestingly, we found an interaction between copeptin and uDKK3 in our prediction models and the best model containing both variables and their interaction term resulted in a fairly good explanation of variance in eGFR slope compared with previous models. Considering the limited number of patients in these analyses, future studies will be required to confirm the results. Nonetheless, uDKK3 appears to be an attractive candidate to improve outcome prediction of ADPKD in the future.

Kidney tubular epithelial cells control interstitial fibroblast fate by releasing TNFAIP8-encapsulated exosomes

Kidney fibrosis, characterized by the activation and expansion of the matrix-producing fibroblasts, is the common outcome of chronic kidney disease (CKD). While fibroblast proliferation is well studied in CKD, little is known about the regulation and mechanism of fibroblast depletion. Here, we show that exosomes derived from stressed/injured tubules play a pivotal role in dictating fibroblast apoptosis and fate. When human kidney tubular cells (HK-2) were stimulated with TGF-β1, they produced and released increased amounts of exosomes (TGFβ-Exo), which prevented renal interstitial fibroblasts from apoptosis. In vivo, injections of TGFβ-Exo promoted renal fibroblast survival, whereas blockade of exosome secretion accelerated fibroblast apoptosis in obstructive nephropathy. Proteomics profiling identified the tumor necrosis factor-α-induced protein 8 (TNFAIP8) as a key component enriched in TGFβ-Exo. TNFAIP8 was induced in renal tubular epithelium and enriched in the exosomes from fibrotic kidneys. Knockdown of TNFAIP8 in tubular cells abolished the ability of TGFβ-Exo to prevent fibroblast apoptosis. In vivo, gain- or loss- of TNFAIP8 prevented or aggravated renal fibroblast apoptosis after obstructive injury. Mechanistically, exosomal-TNFAIP8 promoted p53 ubiquitination leading to its degradation, thereby inhibiting fibroblasts apoptosis and inducing their proliferation. Collectively, these results indicate that tubule-derived exosomes play a critical role in controlling the size of fibroblast population during renal fibrogenesis through shuttling TNFAIP8 to block p53 signaling. Strategies to target exosomes may be effective strategies for the therapy of fibrotic CKD.

Ambra1 in exosomes secreted by HK-2 cells damaged by supersaturated oxalate induce mitophagy and autophagy-ferroptosis in normal HK-2 cells to participate in the occurrence of kidney stones

Injury to the renal tubular epithelium has emerged as a leading factor underlying the formation of kidney stones. Indeed, epithelial cell damage contributes to the adherence and aggregation of crystals, thereby accelerating the formation of renal stones. Meanwhile, exosomes play an instrumental role in cellular communication, including DNA, RNA, mRNA, etc. In this study, homogenous cells were treated with exosomes derived from damaged cells in an attempt to establish “positive feedback” of cell damage, and the desired results were achieved. To begin, a serum-free medium and supersaturated concentrations of oxalate were added to the HK-2 cell line, and then exosomes were isolated from the two groups for analysis and comparison, and the autophagy-related gene Ambra1 (autophagy and beclin-1 regulator 1) was detected. Subsequently, normal HK-2 cells were treated with exosomes, and the related indexes of autophagy, ferroptosis and mitophagy were determined. Thereafter, Ambra1 was knocked down in exosome-derived HK-2 cells, resulting in the down-regulation of Ambra1 expression in exosomes produced by HK-2 cells following oxalate intervention. Thereafter, the ability of exosomes to stimulate autophagy, mitophagy and ferroptosis was re-evaluated in HK-2 cells after Ambra1 knockdown. The results corroborated that exosomes secreted by oxalate-treated HK-2 can directly elevate autophagy, ferroptosis and mitophagy levels in normal cells, and this effect was significantly mitigated following Ambra1 knockdown within exosomes. Meanwhile, exosomes-induced autophagy and ferroptosis were alleviated after knockdown of beclin-1 in recipient HK-2 cells. These results further suggest that beclin-1 plays a critical role in the process of exosome-induced autophagy-ferroptosis.

Патоморфологічні зміни нирок, печінки та біохімічні показники сироватки крові червоновухих прісноводних черепах (Trachemys scripta elegans) за парентерального введення гентаміцину

Principles of reptile treatment are significantly different from the drug therapy for mammals and birds, which is primarily due to anatomical and physiological peculiarities of reptiles. Gentamicin, which has a nephrotoxic effect, is used for the treatment of reptile infectious diseases. For the purpose of studying the effects of gentamicin of reptiles a serum biochemical analysis was conducted determining aspartate aminotransferase and alanine aminotransferase activity, urea and uric acid concentration. Two research groups of red-eared sliders (Trachemys scripta elegans) were formed for the study; every 48 hours they received an injection of gentamicin in a dose 10 mg/kg into thoracic limb muscles. The animals of the first research group were withdrawn from the experiment on the 7th day, and the animals of the second group were withdrawn on the 14th day of the research. As a result of parenteral administration of gentamicin in turtles, there is an increase in the concentration of uric acid and urea, as well as the activity of aspartate aminotransferases and alanine aminotransferases in blood serum. In the kidneys, structural changes of the glomeruli were found: alternative changes in podocytes, as well as degenerative and necrotic changes in the renal tubule epithelium. Degenerative changes of nephrocytes were accompanied by the dilation of smooth endoplasmic reticulum tubules, mitochondrial crista destruction, accumulation of autophagolysosomes, cytosomes and lysosomes in the cytoplasm. Furthermore, signs of nephrocyte apoptosis with the formation of apoptotic bodies were detected. A liver examination revealed dystrophic and necrotic changes in hepatocytes, expansion and overflow of the vessels of the venous beds.

Oxalate‑induced renal pyroptotic injury and crystal formation mediated by NLRP3‑GSDMD signaling invitro and invivo.

Calcium oxalate kidney stone has become an urgent issue due to its high incidence and recurrence rate. Thus, it is necessary to explore for mechanisms of calcium oxalate stones formation. Previous studies demonstrated that oxalate crystals could induce the activation of nucleotide‑binding domain and leucine‑rich repeat‑containing family pyrin domain‑containing 3 (NLRP3) inflammasome and change the renal tubular epithelium adhesion. However, the type and molecular mechanism of NLRP3 inflammasome‑mediated calcium oxalate stones formation still need to be further investigated. In the present study, it was confirmed that the NLRP3‑gasdermin D (GSDMD) signaling was involved in oxalate‑induced cell injury invitro and invivo. Inhibition of reactive oxygen species production could effectively prevent the NLRP3 inflammasome formation in oxalate‑treated HK‑2cells. NLRP3 gene silence could inhibit the DNA damage and cellular membrane injury of HK‑2cells treated with oxalate. The ultrastructural changes of several organelles and particular structures, similar to typical cell pyroptosis, were observed in oxalate‑stimulated HK‑2cells. NLRP3 gene silence could antagonize the oxalate‑induced injury and ultrastructure changes. Additionally, NSA (GSDMD inhibitor) could prevent the oxalate‑induced injury of membrane integrity in HK‑2cells. Moreover, oxalate crystals were significantly decreased in GSDMD‑/‑ mice compared with wild‑type mice with glyoxylic acid. Together, NLRP3‑GSDMD pathway was involved in the oxalate‑induced pyroptotic injury in HK‑2cells. GSDMD and its cleavage form GSDMD‑N played an important role in the oxalate‑induced renal cell injury and oxalate calcium crystals formation invitro and invivo. This provided a new target for prevention and treatment of oxalate nephropathy and oxalate calcium stones.

Daily dose of metformin caused acute kidney injury with lactic acidosis: a case report

BackgroundMetformin-induced lactic acidosis with acute kidney injury is rare but well known. Here we report a case of a Japanese patient taking metformin who experienced severe acute renal failure accompanied with significantly elevated metformin plasma concentrations and signs of lactic acidosis.Case presentationA 60-year-old Japanese man with type II diabetes, who was taking metformin (500 mg three times a day) along with several other medications, visited the emergency department with dizziness, malaise, and oliguria. The initial laboratory test results showed elevated levels of serum creatinine and blood urea nitrogen, although his renal function was normal approximately 2 weeks earlier. His lactate level was raised (4.27 mmol/L), and he was diagnosed with lactic acidosis. Considering the low creatinine clearance and elevated urinary albumin/serum creatinine ratio, urinary N-acetyl-β-d-glucosaminidase level, and β2-microglobulin level, the patient was further diagnosed with AKI (in other words, acute tubular necrosis). A renal biopsy performed on day 3 after admission revealed renal tubular epithelium necrosis, supporting this diagnosis. The patient underwent intermittent hemodialysis until he was discharged on day 13. The metformin concentrations on days 3, 5, and 7 were 8.95, 2.58, and 0.16 μg/mL, respectively, which is significantly higher than the maximal steady-state concentration of metformin at the recommended dosage (approximately 1 μg/mL). The calculated pharmacokinetic parameters of metformin suggested poor renal excretion and a low distribution volume at higher metformin levels. Other possible acute kidney injury-causing factors included dehydration, alcohol consumption, and the use of an angiotensin receptor blocker or SGLT2 inhibitor.ConclusionsThis is the first reported case of acute kidney injury possibly caused by high levels of metformin with lactic acidosis in a patient treated with the recommended metformin dose. Thus, the development of metformin-induced acute kidney injury should be considered for patients with several acute kidney injury risk factors who are taking metformin.

Identification of microRNA editing sites in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor originating from the renal tubular epithelium. Although the microRNAs (miRNAs) transcriptome of ccRCC has been extensively studied, the role of miRNAs editing in ccRCC is largely unknown. By analyzing small RNA sequencing profiles of renal tissues of 154 ccRCC patients and 22 normal controls, we identified 1025 miRNA editing sites from 246 pre-miRNAs. There were 122 editing events with significantly different editing levels in ccRCC compared to normal samples, which include two A-to-I editing events in the seed regions of hsa-mir-376a-3p and hsa-mir-376c-3p, respectively, and one C-to-U editing event in the seed region of hsa-mir-29c-3p. After comparing the targets of the original and edited miRNAs, we found that hsa-mir-376a-1_49g, hsa-mir-376c_48g and hsa-mir-29c_59u had many new targets, respectively. Many of these new targets were deregulated in ccRCC, which might be related to the different editing levels of hsa-mir-376a-3p, hsa-mir-376c-3p, hsa-mir-29c-3p in ccRCC compared to normal controls. Our study sheds new light on miRNA editing events and their potential biological functions in ccRCC.

Oxidatively stressed extracellular microenvironment drives fibroblast activation and kidney fibrosis

Kidney fibrosis is associated with tubular injury, oxidative stress and activation of interstitial fibroblasts. However, whether these events are somehow connected is poorly understood. In this study, we show that glutathione peroxidase-3 (GPX3) depletion in renal tubular epithelium after kidney injury plays a central role in orchestrating an oxidatively stressed extracellular microenvironment, which drives interstitial fibroblast activation and proliferation. Through transcriptional profiling by RNA-sequencing, we found that the expression of GPX3 was down-regulated in various models of chronic kidney disease (CKD), which was correlated with induction of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase-4 (NOX4). By using decellularized extracellular matrix (ECM) scaffold, we demonstrated that GPX3-depleted extracellular microenvironment spontaneously induced NOX4 expression and reactive oxygen species (ROS) production in renal fibroblasts and triggered their activation and proliferation. Activation of NOX4 by advanced oxidation protein products (AOPPs) mimicked the loss of GPX3, increased the production of ROS, stimulated fibroblast activation and proliferation, and activated protein kinase C-α (PKCα)/mitogen-activated protein kinase (MAPK)/signal transducer and activator of transcription 3 (STAT3) signaling. Silencing NOX4 or inhibition of MAPK with small molecule inhibitors hampered fibroblast activation and proliferation. In mouse model of CKD, knockdown of NOX4 repressed renal fibroblast activation and proliferation and alleviated kidney fibrosis. These results indicate that loss of GPX3 orchestrates an oxidatively stressed extracellular microenvironment, which promotes fibroblast activation and proliferation through a cascade of signal transduction. Our studies underscore the crucial role of extracellular microenvironment in driving fibroblast activation and kidney fibrosis.

Abstract P183: Porcupine Protects The Kidney By Suppressing Tubular Inflammation And Preserving Mitochondrial Integrity

Chronic Kidney Disease (CKD) progression may be slowed or stopped with early diagnosis and intervention, so developing novel therapies is paramount. The Wnt acyl transferase porcupine (PORCN) regulates the secretion of all 19 Wnt ligand isoforms that propagate β-catenin signaling. We find that mice with deletion of PORCN from kidney epithelia (PORCN iKKO) had augmented kidney injury in the nephrotoxic serum nephritis (NTS) model of CKD, with higher BUNs (202 ± 12 vs. 101 ± 38, mg/dL; p<0.01), and renal mRNA levels for NGAL (6.1±1.14 vs. 1.0±0.35 au, p=0.0013), KIM-1(2.5±0.65 vs. 1.0±0.26 au, p=0.046), collagen I (2.4±0.31 vs. 1.0±0.38 au, p=0.025), and fibronectin (2.5±0.35 vs. 1.0±0.27 au, p=0.006) compared to wild-type (WT) controls. PORCN iKKOs also had upregulated renal protein levels for collagen I (1.5 ± 0.2 vs. 1.0 ± 0.1 au; p=0.01) and fibronectin (1.8 ± 0.3 vs. 1.0 ± 0.1 au; p<0.01), recapitulating the mRNA patterns. We also detected marked TNFα mRNA upregulation in the PORCN iKKO kidneys vs WTs during CKD (2.6±0.67 vs. 1.0±0.30 au, p=0.036). TNFα is a potent driver of renal injury and fibrosis, acting via TNFα receptor 1(TNFR1) to promote ROS generation. At day 14 of NTS, PORCN iKKO kidneys contained higher levels of the ROS metabolite, 8-isoprostane (2348 ± 718 vs. 486 ± 77, pg/mg kidney; p=0.02), and expressed higher levels of mitochondrial dysfunction markers, including Drp1 (1.26 ± 0.06 vs. 1.0 ± 0.04 au; p<0.01) and p62 (1.3 ± 0.09 vs. 1.0 ± 0.07 au; p=0.07). TNFR1 inhibition (R-7050, 12mg/kg, IP, every other day) reduced BUNs (94.13 ± 3 vs. 90.26 ± 4.1, mg/dL; p=0.46) and renal Drp1 protein levels (1.4 ± 0.17 vs. 1.0 ± 0.15 au; p=0.11) in the PORCN iKKO cohort to WT levels. In primary renal tubular cells (RTCs), TNFα treatment (50ng/ml, 12hrs) induced 8-isoprostane generation (387 ± 119 vs. 121 ± 39, pg/ml in medium; p=0.07), corroborating our in vivo studies. Moreover, treatment of the RTCs with the Drp1 inhibitor mdivi-1 (MD1, 10μm) for 12 hours attenuated TNFα-induced accumulation of fibronectin and collagen I. Our studies suggest that PORCN in renal epithelia protects against kidney damage by limiting TNFα induction, thereby diminishing Drp1-mediated mitochondrial dysfunction and ROS generation.

Abstract PR002: Injury-associated transcriptional state transitions in early renal carcinogenesis

Abstract Most epithelial cells with cancer driver mutations fail to form tumors. This has been particularly well established by genetic analysis of the renal epithelium, where cells with tumor-initiating mutations in the von Hippel-Lindau tumor suppressor (VHL) only rarely develop into clear cell renal carcinomas (ccRCC). The progression of VHLmutant cells to malignancy can take decades, offering a wide window of opportunity for preventative intervention. However, the cellular programs that facilitate the malignant transformation of VHL null ccRCC precursor clones remain elusive. Using a genetically engineered mouse model that allows conditional inactivation of Vhl and Polybromo 1 (Pbrm1), the second most commonly mutated ccRCC gene after VHL, in the renal epithelium of adult mice, we have analyzed the phenotypic evolution of the earliest stages of renal carcinoma formation over months at single cell resolution. Transcriptomic analysis by single cell RNA-sequencing (scRNA-seq) revealed that the acute activation of the hypoxia signaling response, a predicted consequence of genetic Vhl inactivation, is followed by distinct transcriptomic state transitions that precede the emergence of early carcinoma cells. These transitions are associated with constitutive activation of transcriptional programs that normally mediate acute and transient responses to renal injury, and are progressively activated at a low level in aging normal renal epithelium. Analysis of DNA accessibility by the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) identified widespread opening of chromatin regions in association with progression towards malignancy, revealing a chromatin footprint of tissue repair pathway activation. Analysis of human ccRCC samples, including early-stage cases from VHL mutation carriers, validated our observations at the transcriptomic and protein level. Furthermore, genetic perturbation experiments in transplantable ccRCC models demonstrated the functional role of the renal injury pathway in tumor re-initiation. These results are compatible with a model whereby activation of the renal epithelial injury pathway facilitates the early progression of mutant ccRCC precursor clones towards malignancy. That the same repair pathway is increasingly activated over time in normal renal epithelium points at a link between ageing and ccRCC development. An understanding of the causes of ageing-associated renal damage could provide opportunities for early intervention in ccRCC, especially in high-risk individuals. Citation Format: Shoko Hirosue, Jianfeng Ge, Leticia Castillon, Paulo Rodrigues, Dóra Bihary, Alyson Speed, Ludovic Wesolowski, Veronica Caraffini, Anne Y. Warren, Grant D. Stewart, Sarah J. Welsh, Thomas J. Mitchell, Athena Matakidou, Fatemeh S. Seyednasrollah, Shamith A. Samarajiwa, Sakari Vanharanta. Injury-associated transcriptional state transitions in early renal carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR002.

Recent Advances of Proteomics in Management of Acute Kidney Injury.

Acute Kidney Injury (AKI) is currently recognized as a life-threatening disease, leading to an exponential increase in morbidity and mortality worldwide. At present, AKI is characterized by a significant increase in serum creatinine (SCr) levels, typically followed by a sudden drop in glomerulus filtration rate (GFR). Changes in urine output are usually associated with the renal inability to excrete urea and other nitrogenous waste products, causing extracellular volume and electrolyte imbalances. Several molecular mechanisms were proposed to be affiliated with AKI development and progression, ultimately involving renal epithelium tubular cell-cycle arrest, inflammation, mitochondrial dysfunction, the inability to recover and regenerate proximal tubules, and impaired endothelial function. Diagnosis and prognosis using state-of-the-art clinical markers are often late and provide poor outcomes at disease onset. Inappropriate clinical assessment is a strong disease contributor, actively driving progression towards end stage renal disease (ESRD). Proteins, as the main functional and structural unit of the cell, provide the opportunity to monitor the disease on a molecular level. Changes in the proteomic profiles are pivotal for the expression of molecular pathways and disease pathogenesis. Introduction of highly-sensitive and innovative technology enabled the discovery of novel biomarkers for improved risk stratification, better and more cost-effective medical care for the ill patients and advanced personalized medicine. In line with those strategies, this review provides and discusses the latest findings of proteomic-based biomarkers and their prospective clinical application for AKI management.

Basigin is released in extracellular vesicles derived from the renal tubular epithelium in response to albuminuria.

Irrespective of the cause, albumin/proteinuria induces tubulointerstitial damage and accelerates the progression of kidney diseases. Our series of studies demonstrated that proteinuria, an independent prognostic factor for chronic kidney disease (CKD), is correlated with urinary basigin/CD147 (Bsg) levels. We examined the morphology and origin of Bsg in the tubular lumen through the effects of filtered glucose and protein solutes on the tubules. Diabetic kidney disease (DKD) patients (N = 50) were treated with spironolactone 25 mg for 4 weeks or by conservative treatment. The associations between urinary Bsg values and clinical indicators were examined. Primary-cultured proximal tubular epithelial cells (PTECs) from human adult kidneys were exposed to high glucose or bovine serum albumin (BSA). In patients with early phase DKD, urinary Bsg levels were closely correlated with proteinuria but not HbA1c. Full-length Bsg on extracellular vesicles (EVs) was investigated primarily in urine collected from DKD patients. EVs obtained from the urine of DKD patients included Bsg and SGLT2 proteins. Notably, spironolactone treatment concomitantly suppressed the release of Bsg-bearing EVs in correlation with decreased albuminuria. Exposure of PTECs to BSA (but not high glucose) enhanced the storage of supernatant Bsg in EVs despite the absence of exposure-specific changes in Bsg transcription. Proteinuria induces the release of Bsg-bearing EVs derived from PTECs into the tubular lumen.