Proximal Tubular Epithelium Research Articles

Mercapturate pathway metabolites of sotorasib, a covalent inhibitor of KRASG12C, are associated with renal toxicity in the Sprague Dawley rat

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and β-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.

Sodium-glucose co-transporter-2 inhibitors in heart failure and chronic kidney disease: the role of empagliflozin

The development of chronic kidney disease (CKD) is a risk factor not only for cardiovascular diseases, but also for heart failure (HF). This article is a literary review on the use of Sodium-glucose co-transporter-2 (NGLT2) inhibitors in patients with CKD and HF. The paper describes in detail the action of NGLT2 inhibitors in the light of nephro- and cardioprotection. In addition to the glucosuric effect of NGLT2 inhibitors, they have a natriuretic and diuretic effect. One of the effects of NGLT2 inhibitors is the ability to lower blood pressure. One of the key effects of NGLT2 inhibitors, explaining nephroprotection, is the influence on glomerular filtration. The ability of NGLT2 inhibitors to suppress the peroxidation in mitochondria of proximal tubular epithelium was shown. Another putative mechanism of the organ protection action of NGLT2 inhibitors is their ability to inhibit the activation of the sympathetic nervous system.The results of studies using empagliflozin in HF and CKD are presented. In particular, the EMPA-REG OUTCOME study showed that in patients with type 2 diabetes and concomitant cardiovascular diseases, empagliflozin led to a 35% decrease in hospitalization risk due to decompensated HF and decrease of cardiovascular death risk by 38% regardless of baseline renal function. According to the EMPEROR-Reduced study, empagliflozin showed a favorable safety profile.

Pharmacological Evaluation of Safoof-e-Pathar Phori- A Polyherbal Unani Formulation for Urolithiasis.

Safoof-e-Pathar phori (SPP) is an Unani poly-herbomineral formulation, which has for a long time been used as a medicine due to its antiurolithiatic activity, as per the Unani Pharmacopoeia. This powder formulation is prepared using six different plant/mineral constituents. In this study, we explored the antiurolithiatic and antioxidant potentials of SPP (at 700 and 1,000 mg/kg) in albino Wistar rats with urolithiasis induced by 0.75% ethylene glycol (EG) and 1% ammonium chloride (AC). Long-term oral toxicity studies were performed according to the Organization for Economic Co-operation and Development (OECD) guidelines for 90 days at an oral dose of 700 mg/kg of SPP. The EG urolithiatic toxicant group had significantly higher levels of urinary calcium, serum creatinine, blood urea, and tissue lipid peroxidation and significantly (p < 0.001 vs control) lower levels of urinary sodium and potassium than the normal control group. Histopathological examination revealed the presence of refractile crystals in the tubular epithelial cell and damage to proximal tubular epithelium in the toxicant group but not in the SPP treatment groups. Treatment of SPP at 700 and 1,000 mg/kg significantly (p < 0.001 vs toxicant) lowered urinary calcium, serum creatinine, blood urea, and lipid peroxidation in urolithiatic rats, 21 days after induction of urolithiasis compared to the toxicant group. A long-term oral toxicity study revealed the normal growth of animals without any significant change in hematological, hepatic, and renal parameters; there was no evidence of abnormal histology of the heart, kidney, liver, spleen, or stomach tissues. These results suggest the usefulness of SPP as an antiurolithiatic and an antioxidant agent, and long-term daily oral consumption of SPP was found to be safe in albino Wistar rats for up to 3 months. Thus, SPP may be safe for clinical use as an antiurolithiatic formulation.

Evaluation of the Effect of Captopril and Losartan on Tacrolimus-induced Nephrotoxicity in Rats.

Tacrolimus is an immunosuppressive drug. Activation of the renin-angiotensin system (RAS) and associated inflammations may exacerbate the toxic effects of tacrolimus. Given the significant role of the kidney in RAS this study aimed to evaluate the effect of captopril as an angiotensin-converting enzyme (ACE) blocker and losartan as an angiotensin II receptor blocker on tacrolimus-induced nephrotoxicity. In total, 36 adult male rats weighing 200-250 gr were completely randomized and divided into six groups (control, tacrolimus, tacrolimus and losartan, tacrolimus and captopril, losartan, and captopril) for 30 days. Afterwards, blood urea nitrogen (BUN), creatinine (Cr) and ACE2 enzyme were measured. Also, both kidneys were collected for histological examinations. The level of BUN and Cr significantly increased in tacrolimus group. The level of BUN and Cr were lower in the groups treated with a combination of tacrolimus and losartan or captopril. While ACE2 level increased in the groups receiving a combination of tacrolimus and losartan or captopril, the level of increase was insignificant, compared to the group treated with tacrolimus alone. The glomerulus diameter and the thickness of the renal proximal tubular epithelium significantly decreased in the group treated with tacrolimus alone. the mentioned variables increased in the group treated with a combination of tacrolimus and losartan or captopril, compared to the tacrolimus group. According to this study, tacrolimus increased the BUN and Cr levels while decreasing the ACE2 levels. However, tacrolimus in combination with losartan or captopril seemed to decrease the nephrotoxicity of the drug.

Claudin-2 inhibits renal clear cell carcinoma progression by inhibiting YAP-activation

BackgroundClaudin-2 expression is upregulated in multiple cancers and promotes cancer malignancy. Remarkably, the regulation of claudin-2 expression in kidney cell lines contrasts its reported regulation in other organs. However, claudin-2 role in renal clear cell carcinoma (RCC) remains unknown despite its predominant expression in the proximal tubular epithelium (PTE), the site of RCC origin.MethodsPublicly available and independent patient databases were examined for claudin-2 association with RCC. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by Mass spectroscopy, immunoprecipitation and mutational studies, and functional evaluations.ResultsWe show that the significant decrease in claudin-2 expression characterized PTE cells and Ex-vivo cultured mouse kidney subjected to dedifferentiation. Inhibition of claudin-2 was enough to induce mesenchymal plasticity and invasive mobility in these models. Further, a progressive loss of claudin-2 expression associated with the RCC progression and poor patient survival. Overexpression of claudin-2 in RCC-derived cancer cells inhibited tumorigenic abilities and xenograft tumor growth. These data supported a novel tumor-suppressive role of claudin-2 in RCC. Mechanistic insights further revealed that claudin-2 associates with YAP-protein and modulates its phosphorylation (S127) and nuclear expression. The tumor suppressive effects of claudin-2 expression were lost upon deletion of its PDZ-binding motif emphasizing the critical role of the PDZ-domain in claudin-2 interaction with YAP in regulating RCC malignancy.ConclusionsOur results demonstrate a novel kidney specific tumor suppressive role for claudin-2 protein and further demonstrate that claudin-2 co-operates with the YAP signaling in regulating the RCC malignancy.

Polysaccharides extracted from balanophora polyandra Griff (BPP) ameliorate renal Fibrosis and EMT via inhibiting the Hedgehog pathway.

Renal interstitial fibrosis (RIF) is a crucial pathological change leading to chronic kidney disease (CKD). Currently, no effective medicines have been available for treating it. In our research, we examined the effects of polysaccharides extracted from Balanophora polyandra Griff (BPPs) on kidney fibrosis and epithelial to mesenchymal transition (EMT) in vivo and in vitro, aiming to explore the underlying mechanisms. By using the mice with unilateral urethral obstruction (UUO) as experimental subjects, we examined the medicinal values of BPPs on alleviating RIF. The effects of BPPs were evaluated by examining the histological staining and relative mRNA and protein expression levels of the related genes. The possible underlying mechanisms were further explored with human normal renal proximal tubular epithelia (HK‐2 cells) as in vitro model. In UUO mice, BPP treatment could significantly alleviate interstitial fibrosis through reducing the components (Collagens I, III and IV) of extracellular matrix (ECM), and reducing the activation of fibroblasts producing these components, as revealed by inhibiting the hallmarks (fibronectin and α‐SMA) of fibroblast activation. Furthermore, BPP administration increased the expression levels of matrix metalloproteinases (MMPs) and declined those of tissue inhibitors of metalloproteinases (TIMPs). BPPs markedly ameliorated EMT in both the kidneys of UUO mice and TGF‐β1 treated HK‐2 cells. Moreover, BPP treatment decreased the expression levels of several transcriptional factors involved in regulating E‐cadherin expression, including snail, twist and ZEB1. Additionally, the Hedgehog pathway was found to be closely correlated with renal fibrosis and EMT. Altogether, our results clearly demonstrated that BPP treatment effectively inhibited the Hedgehog pathway both in renal tissues of UUO mice and TGF‐β1‐treated HK‐2 cells. Thus, BPPs ameliorated RIF and EMT in vivo and in vitro via suppressing Hedgehog signalling pathway.

Red blood cell exosome hemoglobin content increases after cardiopulmonary bypass and mediates acute kidney injury in an animal model

ObjectiveHemolysis, characterized by formation of free hemoglobin (Hb), occurs in patients undergoing cardiopulmonary bypass (CPB). However, there is no study of the dynamic changes in red blood cell (RBC)-derived exosomes (Exos) released during CPB, nor whether these particles mediate acute kidney injury (AKI). MethodsThis study is a comprehensive time–course analysis, at baseline, 30 minutes, to 24 hours post-crossclamp release (XCR) to determine (1) Exos Hb content; (2) free Hb/heme, haptoglobin, hemopexin; and (3) urinary markers of AKI over the same time period. In addition, we developed a model system in Sprague–Dawley rats to test for AKI after intravenous injection of Exos Hb released during CPB. ResultsIn 30 patients undergoing CPB, there is a significant increase in plasma Hb-positive Exos but not microvesicles 30 minutes post-XCR versus other time points, with a simultaneous decrease in the haptoglobin/Hb ratio. These changes presage a significant increase in urine neutrophil gelatinase–associated lipocalin and kidney injury molecule-1 at 24 hours. Intravenous injection of plasma Exos (109-10 particles obtained 30 minutes post-XCR) into rats causes AKI at 72 hours, manifested by multifocal degeneration of proximal tubular epithelium. At 21 days, there is persistent tubular injury and interstitial fibrosis. Intravenous injection of Exos from 35-day-old stored RBCs into rats results in glomerular–tubular injury, increased kidney ferritin and hemoxygenase-1 expression, and significant elevation of kidney injury molecule-1 and proteinuria at 72 hours. ConclusionsThese combined studies raise the potential for RBC-derived Exos, released during CPB, to target the kidney and mediate AKI.

Electron microscopic findings can support multiple etiologies of nephrotoxicity in renal tubules

ABSTRACT Electron microscopy (EM) has been mainly used for identifying ultrastructural abnormalities such as fusion of foot processes and immune complex deposits in glomeruli. However, electron microscopic findings in renal tubules can provide either diagnostic evidence (unique finding) or supportive evidence (additional finding) for final diagnosis. Here we present multiple situations that EM can be used for drawing conclusions of various drug-associated nephrotoxicity. Multiple cases with drug-induced nephrotoxicity are reviewed, including clinical history, EM findings, and serum creatinine (sCr) levels, prior to renal biopsy and during follow-up. Two cases with nephrotoxicity by aminoglycoside antibiotics showed acute tubular injury with EM findings of myeloid bodies, characterized by laminated dense materials in lysosomes in both proximal and distal tubular epithelium (diagnostic evidence). Five cases of vancomycin associated nephrotoxicity presented with acute tubular injury and vancomycin casts in distal tubules, characterized by central laminated casts in the lumina of distal tubules (supportive evidence). Vedolizumab, a humanized monoclonal antibody used in treating Crohn’s disease, can cause T-cell dominant acute interstitial nephritis, with EM revealing lymphocytic infiltration into tubules as tubulitis (supportive evidence). Four of Seven cases (5/8) cases had renal functional recovery upon follow-up check for sCr. EM findings of characteristic changes in renal tubules can be particularly useful as either diagnostic or supportive evidence, in correlation with clinical history and etiologies of nephrotoxicity. Therefore, EM should not only focus on glomerular changes, but renal tubular changes as well.

Urogenital System Damaging Manifestations of 3 Human Infected Coronaviruses.

At present, there are no comparative studies on the damage of the 3 coronaviruses infections to the urinary system. Therefore, we reviewed the correlations and differences between invading mechanisms and resultant urogenital system damage by the 3 kinds of coronaviruses in an effort to help doctors in the prevention and treatment of these damages, which will be of significance in improving prognosis and quality of life after rehabilitation. We searched PubMed® for English language articles published since 2003 with the key words "Coronavirus disease 2019 (COVID-19)," "severe acute respiratory syndrome-2 (SARS-CoV-2)," "severe acute respiratory syndrome (SARS)-CoV" and "Middle East respiratory syndrome (MERS)-CoV "with "semen" and "kidney." We also used relevant data from websites including the World Health Organization and Centers for Disease Control and Prevention. Recent studies have revealed that both SARS-CoV and SARS-CoV-2 invade target cells through a membrane-bound angiotensin-converting enzyme 2, an important component of the renin-angiotensin system, which maintains human homeostasis, whereas MERS utilizes host cells' receptor dipeptidyl peptidase 4 for entry. While pneumonia is the most prominent symptom in patients infected by coronaviruses due to the transmission through respiratory droplets, the urogenital system can also suffer from infection with coronaviruses, resulting in renal failure, testicular atrophy etc. The impairments of the genitourinary system would be different with the 3 coronavirus infections but they still have a strong correlation. The pathogenesis and clinical symptoms of SARS-CoV, MERS-CoV and SARS-CoV-2 coronaviruses in the genitourinary system are comparable. All 3 coronaviruses have been found to affect kidney and testicular function. Therefore, both urologists and nephrologists should pay attention to the damage caused by coronavirus infection to the genitourinary system, especially monitoring renal and semen function.

Glucose, Fructose, and Urate Transporters in the Choroid Plexus Epithelium.

The choroid plexus plays a central role in the regulation of the microenvironment of the central nervous system by secreting the majority of the cerebrospinal fluid and controlling its composition, despite that it only represents approximately 1% of the total brain weight. In addition to a variety of transporter and channel proteins for solutes and water, the choroid plexus epithelial cells are equipped with glucose, fructose, and urate transporters that are used as energy sources or antioxidative neuroprotective substrates. This review focuses on the recent advances in the understanding of the transporters of the SLC2A and SLC5A families (GLUT1, SGLT2, GLUT5, GLUT8, and GLUT9), as well as on the urate-transporting URAT1 and BCRP/ABCG2, which are expressed in choroid plexus epithelial cells. The glucose, fructose, and urate transporters repertoire in the choroid plexus epithelium share similar features with the renal proximal tubular epithelium, although some of these transporters exhibit inversely polarized submembrane localization. Since choroid plexus epithelial cells have high energy demands for proper functioning, a decline in the expression and function of these transporters can contribute to the process of age-associated brain impairment and pathophysiology of neurodegenerative diseases.

Detection of Proximal Tubule Involvement by BK Polyomavirus in Kidney Transplant Recipients With Urinary Sediment Double-Immunostaining

BackgroundThe extent and depth of BK polyomavirus (BKPyV) infection in renal allograft correlate with prognosis. This study was designed to evaluate the value of urinary sediment double-immunostaining for predicting BKPyV infection in proximal tubular epithelium.Materials and methodsA total of 76 urine sediment cell blocks, as well as the corresponding transplanted kidney tissues with BK polyomavirus associated-nephropathy (BKPyVAN), were evaluated by automatic double-immunostaining with anti-58-kDa Golgi protein (58K, a proximal renal tubular marker) + anti-SV40-T and anti-homogentisate 1, 2-dioxygenase (HGD, a renal tubular marker) + anti-SV40-T.ResultsImmunohistochemical staining demonstrated that 58K was expressed in proximal tubular epithelium but not in distal tubular epithelium or transitional epithelium. Of the 76 patients, 28 (36.8%) had urinary 58K(+)/SV40-T(+) cells and HGD(+)/SV40-T(+) cells, 41 (53.9%) had only HGD(+)/SV40-T(+) cells, one (1.3%) had only 58K(+)/SV40-T(+) cells, and six (7.9%) had only 58K(−)/HGD(−)/SV40-T(+) cells. The presence of urinary 58K(+)/SV40-T(+) cells was correlated with BKPyV infection in proximal tubular epithelium (P < 0.001, r = 0.806). The mean extent of SV40-T staining was significantly more extensive in patients with urinary 58K(+)/SV40-T(+) cells than those without urinary 58K(+)/SV40-T(+) cells (21.4 vs. 12.0%, P < 0.001). The positive predictive value, negative predictive value, sensitivity, and specificity of urinary 58K(+)/SV40-T(+) cells for predicting BKPyV infection in proximal tubular epithelium were 89.7% (95% CI: 71.5–97.3%), 91.5% (95% CI: 78.7–97.2%), 86.7% (95% CI: 68.4–95.6%), and 93.5% (95% CI: 81.1–98.3%), respectively.ConclusionUrinary sediment double-immunostaining with anti-58K and anti-SV40-T is valuable for predicting the extent and depth of BKPyV infection in renal allograft.

Сhanges in the functional state of the epithelium of the proximal renal tubules in patients with the initial stage of chronic heart failure during simvastatin therapy.

To assess the change in the functional state of the proximal renal tubule epithelium in patients with dyslipidemia on the background of obesity, by determining the concentration in the urine of the examined level of cystatin C and the degree of activity of the renal organ-specific enzymes neutral α-glucosidase (NAG) and L-alaninaminopeptidase (laap) during simvastatin therapy at a daily dose of 20 mg for 6 months. The study involved 88 people who were divided into three groups: control, comparison and main. The control group is a group of practically healthy individuals: 30 people, average age 20.67 ± 0.18 years, body mass index (BMI) 21.36 ± 0.4 kg/m2. Comparison group (obese): 27 people, average age 22.38 ± 0.76 years, BMI 31.48 ± 0.56 kg / m2. Patients of the main group were divided into 2 subgroups. The first main subgroup of persons with chronic heart failure stage I (CHF I) without type 2 diabetes mellitus (DM 2)) - 15 observed: average age 56.8 ± 1.8 years, BMI 30.28 ± 1.11 kg / m2. The second main subgroup (CHF I with DM 2) - 16 observed: average age 48.25 ± 2.45 years, BMI 30.37 ± 1.11 kg/m2. The study found that simvastatin therapy does not affect glomerular filtration rate in patients with asymptomatic heart dysfunction. There was an increased level of cystatin C in the urine of the comparison group compared to the control group, the concentration of cystatin C in the main subgroups was statistically significantly higher than the control group. On the background of simvastatin therapy for 6 months, the level of this analyte is statistically significantly increased. The activity of LAAP and NAG during simvastatin therapy during the follow-up period in the CHF I subgroup without DM2 significantly decreased. In the subgroup of CHF I + DM2, a decrease in the concentration of LAAP and an increase in the activity of NAG was revealed, which may indicate that the brush border epithelium dystrophy occurred during simvastatin therapy. Simvastatin therapy for 6 months in patients with the initial stage of heart failure at a daily dosage of 20 mg does not impair glomerular function in the form of reduced glomerular filtration rate (GFR). Cystatin C levels are higher in obese individuals without heart failure and significantly higher in those with asymptomatic heart failure. When treating dyslipidemia with simvastatin at a dose of 20 mg / day, there is a decrease in the activity of NAG and laap in patients with CHF I without DM2. In the result of lipid-lowering therapy with simvastatin in a daily dosage of 20 mg in patients with CHF I+D2M there is increased activity of NAG while reducing the concentration of the LAAP, which may be due to degeneration of the proximal tubular epithelium, amid additional load on a partially renal route of metabolism of simvastatin.

Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy.

Since the discovery of the causative agent for the novel severe acute respiratory syndrome (SARS)–like pneumonia syndrome pandemic that started in China in 2019 (1,2), a coronavirus named SARS coronavirus 2 (SARS-CoV-2), electron microscopy images have populated the medical literature (2) and media outlets alike displaying the characteristic 60–140 nm round particles surrounded by a “corona” of 9–12 nm distinctive spikes (2). Although many of these images were obtained after “ in vitro ” infection of cultured cells with SARS-CoV-2 (2) and are thus likely a true representation of viral particles, we have observed morphologically indistinguishable inclusions within podocytes and tubular epithelial cells both in patients negative for coronavirus disease 2019 (COVID-19) as well as in renal biopsies from the pre–COVID-19 era. Although direct infection of the kidney is theoretically possible, given the presence of angiotensin-converting enzyme 2 (the receptor used by SARS-CoV-2 to gain access to cells) within proximal tubular epithelium (3) and podocytes (4), the virus has not been detected by real-time RT-PCR in urine samples from patients with COVID-19 (5⇓–7). Additionally, for the virus to have access to kidney parenchyma, viremia should occur, and this has only been detected in a minority of patients (6⇓–8). We would, therefore, like to issue a note of caution for inferring viral tissue infection by morphology alone using electron microscopy images from tissues obtained from biopsies or autopsy material in patients with COVID-19. Moreover, caution should be used when interpreting immunohistochemical results, especially within proximal tubules, which are prone to nonspecific staining by a variety of antibodies due to their intense reabsorptive capacity. Additionally, more specific techniques such as immunoelectron microscopy using specific viral antigens (9), or in situ hybridization for viral RNA, are likely necessary to undoubtedly confirm tissue infection in …

PROTECTIVE EFFECT OF NIGELLA SATIVA OIL ON CARBOPLATIN INDUCED NEPHROTOXICITY IN RATS

OBJECTIVE: Carboplatin is a commonly used chemotherapeutic agent. Kidneys are an important organ affected by the adverse effects of chemotherapeutic agents. This study aimed to investigate the protective effect of Nigella sativa oil (NSO) against kidney damage due to carboplatin exposure.MATERIAL AND METHODS: All animals (n=24 female wistar-albino rats) were divided into four groups; 4 ml/kg saline was intraperitoneally (i.p.) administered on day one and two in the first group. 4 ml/kg NSO on day one and 4 ml/kg saline on day two was i.p. administered in the second group.4 ml/kg saline on day one and 80 mg/kg carboplatin on day two was i.p. administered in the third group.4 ml/kg NSO on day oneand 80 mg/kg carboplatin on day two was i.p. administered inthe fourth group. Rats were sacrificed at the end of day two and renal tissues were fixed in neutral formalin. Histopathologicchanges and apoptotic index (AI) were evaluated.RESULTS: While an increase was observed in the apoptotic index of carboplatin+saline group compared to the control group, no significant differences were found in the carboplatin+saline and carboplatin+NSO group. In the histopathological evaluation, degeneration in the proximal and distal tubular epithelium and glomerular capillary glomus bodies, congestion in the vascular formations between the tubules, increase in collagen fiber density in the tunica adventitia of intraglomerular, preglomerular, intertubular and vascular formations, and sporadic basal disintegration due to Periodic Acid Schiff (PAS) reaction were observed in the carboplatin+saline group. In the carboplatin+NSO group, degenerative changes in some areas of tubular structures continued while it was observed that glomerular structures were more regular. It was observed that sclerotic change was fewer in the carboplatin+NSO group than in the carboplatin+saline group. It drew attention that basal membranes were more regular in the carboplatin-nigella sativa oil group as a result of PAS reaction.CONCLUSIONS: NSO, is used as a spice, may have a protective effect on carboplatin induced nephrotoxicity.

Renoprotective Effects of Nobiletin on High Glucose-Induced Loss of Proximal Tubular Microvilli

Abstract Objectives Kidney proximal tubular epithelium has microvillar brush borders, which is critical to renal reabsorption. Hyperglycemia induce change the loss of brush border and cellular polarity, tight junction disruption. Nobiletin is a polymethoxyflavone present in citrus fruit and peels, and has anti-inflammatory effects. This study investigated the renoprotective effects of nobiletin on proximal tubule microvillar ultrastructure and reabsorption under diabetic condition. Methods Human renal proximal tubular epithelial cells (RPTEC) were incubated in a media exposed to 33 mM glucose in the absence and presence of 1–20 μM nobiletin up to 6 days. Antibodies F-actin, villin, cubulin, megalin, SGLT2 and GLUT2 were used for Western blot analysis. The in vivo animal model employed db/db mice orally administrated with 10 mg/kg of nobiletin. Extracts of tissues were subjected to Western blotting or immunohistochemical staining. Results High glucose declined expression of F-actin and villin required for the assembly of proximal tubule microvilli. In addition, the expression of glucose uptake proteins of GLUT2 and SGLT2 was prompted in hyperglycemia. However, nontoxic nobiletin enhanced the expression of F-actin reduced by high glucose, while the elevated expression of the GLUT2 and SGLT2 was attenuated by nobiletin. In the in vivo study, oral administration of 10 mg/kg nobiletin inhibited loss of tubular microvilli through restoring expression of F-actin and villin in diabetic kidneys. Furthermore, nobiletin reduced expressions of GLUT2 and the albumin uptake receptors of cubulin and megalin in db/db mouse kidneys. Conclusions These results demonstrated that nobiletin curtailed loss of the proximal tubular microvillar proteins responsible for tubular reabsorption. Nobiletin may be a potent renoprotective agent counteracting diabetes-associated proximal tubular dysfunctions leading to kidney failure. Funding Sources This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (2017R1A6A3A04011473).

MG53 protects against contrast-induced acute kidney injury by reducing cell membrane damage and apoptosis.

Mitsugumin 53 (MG53) is a tripartite motif family protein that has been reported to attenuate injury via membrane repair in different organs. Contrast-induced acute kidney injury (CI-AKI) is a common complication caused by the administration of iodinated contrast media (CM). While the cytotoxicity induced by CM leading to tubular cell death may be initiated by cell membrane damage, we wondered whether MG53 alleviates CI-AKI. This study was designed to investigate the effect of MG53 on CI-AKI and the underlying mechanism. A rat model of CI-AKI was established, and CI-AKI induced the translocation of MG53 from serum to injury sites on the renal proximal tubular (RPT) epithelia, as illustrated by immunoblot analysis and immunohistochemical staining. Moreover, pretreatment of rats with recombinant human MG53 protein (rhMG53, 2 mg/mL) alleviated iopromide-induced injury in the kidney, which was determined by measuring serum creatinine, blood urea nitrogen and renal histological changes. In vitro studies demonstrated that exposure of RPT cells to iopromide (20, 40, and 80 mg/mL) caused cell membrane injury and cell death, which were attenuated by rhMG53 (10 and 50 μg/mL). Mechanistically, MG53 translocated to the injury site on RPT cells and bound to phosphatidylserine to protect RPT cells from iopromide-induced injury. In conclusion, MG53 protects against CI-AKI through cell membrane repair and reducing cell apoptosis; therefore, rhMG53 might be a potential effective means to treat or prevent CI-AKI.

Identification of an individualized autophagy prognostic index in clear cell renal cell carcinoma patients.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. ccRCC arises from the proximal tubular epithelium and is associated with high mortality. Autophagy may either promote or suppress tumor cell survival at different stages of cancer development. It is essential to investigate the association between autophagy-related genes (ARGs) and prognosis in ccRCC patients. We used datasets obtained from The Cancer Genome Atlas (TCGA) database to identify the expression level of ARGs in ccRCC patients. Functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using Metascape database. Hub genes were identified by Cytoscape software. We constructed a Cox proportional hazard regression model to identify hub genes that are significantly associated with overall survival (OS) in ccRCC patients. Subsequently, a prognostic index (PI) was calculated and ccRCC patients were stratified into high-risk and low-risk groups based on a median PI value. Our study detected several altered ARGs in ccRCC, which could be a useful prognostic tool in ccRCC patients.

Tubule-derived exosomes play a central role in fibroblast activation and kidney fibrosis

Extracellular vesicles such as exosomes are involved in mediating cell-cell communication by shuttling an assortment of proteins and genetic information. Here, we tested whether renal tubule-derived exosomes play a central role in mediating kidney fibrosis. The production of exosomes was found to be increased in the early stage of unilateral ureteral obstruction, ischemia reperfusion injury or 5/6 nephrectomy models of kidney disease. Exosome production occurred primarily in renal proximal tubular epithelium and was accompanied by induction of sonic hedgehog (Shh). Invitro, upon stimulation with transforming growth factor-β1, kidney proximal tubular cells (HKC-8) increased exosome production. Purified exosomes from these cells were able to induce renal interstitial fibroblast (NRK-49F) activation. Conversely, pharmacologic inhibition of exosome secretion with dimethyl amiloride, depletion of exosome from the conditioned media or knockdown of Shh expression abolished the ability of transforming growth factor-β1-treated HKC-8 cells to induce NRK-49F activation. Invivo, injections of tubular cell-derived exosomes aggravated kidney injury and fibrosis, which was negated by an Shh signaling inhibitor. Blockade of exosome secretion invivo ameliorated renal fibrosis after either ischemic or obstructive injury. Furthermore, knockdown of Rab27a, a protein that is essential for exosome formation, also preserved kidney function and attenuated renal fibrotic lesions in mice. Thus, our results suggest that tubule-derived exosomes play an essential role in renal fibrogenesis through shuttling Shh ligand. Hence, strategies targeting exosomes could be a new avenue in developing therapeutics against renal fibrosis.

Fibroblast growth factor 23 and phosphate homeostasis.

The current review highlights recent advances in the area of renal tubular phosphate transport and its regulation by fibroblast growth factor 23 (FGF23), a potent regulator of phosphate homeostasis. Recent studies demonstrate that FGF23 binds to both membrane and soluble form of α-klotho to activate FGF receptor signaling pathways. Parathyroid hormone and FGF23 equivalently decrease sodium-dependent phosphate cotransport but the effect is not additive, suggesting a shared but not synergistic mechanism of action. Crosstalk occurs downstream of parathyroid hormone-receptor and FGF23-receptor signaling and converge at the level of the scaffolding protein, sodium-hydrogen exchanger regulatory factor-1. A novel mechanism for phosphate efflux through the basolateral membrane of renal proximal tubular epithelia via an atypical G-protein coupled receptor, Xenotropic and polytropic retrovirus receptor 1 (XPR1), was recently identified. Conditional deletion of Xpr1 gene in renal proximal tubules in mice leads to hypophosphatemic rickets and Fanconi syndrome establishing an important role for XPR1 in phosphate homeostasis. A novel anti-FGF23 antibody, burosumab, was recently approved to treat X-linked hypophosphatemia, a human disorder of FGF23 excess. Significant advances in understanding the cellular and molecular aspects of renal tubular phosphate transport and its regulation by FGF23 has led to the discovery of novel therapeutics to treat human disorders of phosphate homeostasis.

Abstract 1767: Rho-GTPase inhibits claudin-2 expression to promote proximal tubular epithelial cell plasticity and renal cell carcinoma

Abstract The clear-cell renal cell carcinoma (ccRCC) is the most prevalent and malignant type of kidney cancer, for which there are no effective treatment. Moreover, the incidence of ccRCC has been increasing steadily. Disruption of epithelial cell polarity, assisted by tight junction (TJ) deregulation, facilitates rewiring of oncogene and tumor suppressor signaling pathways. In this regard, proximal tubular epithelium (PTE), where ccRCC originates is the sole nephron segment that expresses claudin-2 (hereon Cldn2), a TJ- protein. However, role of Cldn2 in ccRCC remains unknown despite well-recognized role of Cldn2 in regulating other cancers. We here report a novel role for Cldn2 in maintaining epithelial differentiation among PTE cells as its loss induces mesenchymal traits and associates with ccRCC progression. In specific, we found a specific (among claudin proteins) loss of Cldn2 expression in PTE cells subjected to stimuli, like EGF, TGF-ß and hypoxia, known to induce EMT in PTE cells as well as in a murine model of spontaneous renal tumorigenesis. Further analysis of a large renal cancer patient cohort revealed a significant decrease in Cldn2 levels in the ccRCC, and its positive association with cancer metastasis. In vitro, genetic ablation of claudin-2 expression in HK-2 or MDCK-II cells potentiated mesenchymal traits (upregulated Vimentin and Fibronectin; p&amp;lt;0.002) and cell motility (p&amp;lt;0.0002 versus control). In contrast, forced claudin-2 expression in RCC-derived (and claudin-2 low) Caki-2 cells (P&amp;lt;0.002) promoted epithelial differentiation (downregulation of Vimentin and Fibronectin; P&amp;lt;0.0012&amp; P&amp;lt;0.00025), inhibited cell invasion (P&amp;lt;0.0001) and proliferation (P&amp;lt;0.001). Overall, our findings support a novel and tissue-specific role of claudin-2 in maintaining epithelial differentiation among renal proximal tubular epithelium and tumor suppressor. Further analysis identified a key role of Rho-GTPase in regulating renal claudin-2 expression as ROCK inhibitor (Y-27632) not only reverted EMT-associated loss of Cldn2 but also in Caci-2 cells. An ELISA assay further confirmed an early induction of Rho-GTPase activity in PTE cells subjected to the EMT. Of note, inhibiting Rho-GTPase not only improves renal fibrosis but also inhibits ccRCC. Taken together, we here report a novel role of claudin-2 in maintaining epithelial phenotype in PTE cells with potential clinical significance in acute and chronic renal diseases, and ccRCC progression. Citation Format: Balawant Kumar, Rizwan Ahmad, Pinelopi Kapitsino, Giovanna A. Giannico, Roy Zent, Raymond Clement Harris, Peter Clark, Punita Dhawan, Amar B. Singh. Rho-GTPase inhibits claudin-2 expression to promote proximal tubular epithelial cell plasticity and renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1767.