Human Proximal Tubule Research Articles

Epac activation ameliorates tubulointerstitial inflammation in diabetic nephropathy.

Tubulointerstitial inflammation plays an important role in the progression of diabetic nephropathy (DN), and tubular epithelial cells (TECs) are crucial promoters of the inflammatory cascade. Exchange protein activated by cAMP (Epac) has been shown to suppress the angiotensin II (Ang-II)-induced release of inflammatory cytokines in tubular cells. However, the role of Epac in TEC-mediated tubulointerstitial inflammation in DN remains unknown. We found that administering the Epac agonist 8-pCPT-2'-O-Me-cAMP (8-O-cAMP) to db/db mice inhibited tubulointerstitial inflammation characterized by macrophage infiltration and increased inflammatory cytokine release and consequently alleviated tubulointerstitial fibrosis in the kidney. Furthermore, 8-O-cAMP administration restored CCAAT/enhancer binding protein β (C/EBP-β) expression and further upregulated the expression of Suppressor of cytokine signaling 3 (SOCS3), while inhibiting p-STAT3, MCP-1, IL-6, and TNF-α expression in the kidney cortex in db/db mice. And in vitro study showed that macrophage migration and MCP-1 expression induced by high glucose (HG, 30 mM) were notably reduced by 8-O-cAMP in human renal proximal tubule epithelial (HK-2) cells. In addition, 8-O-cAMP treatment restored C/EBP-β expression in HK-2 cells and promoted C/EBP-β translocation to the nucleus, where it transcriptionally upregulated SOCS3 expression, subsequently inhibiting STAT3 phosphorylation. Under HG conditions, siRNA-mediated knockdown of C/EBP-β or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on the release of MCP-1. In contrast, SOCS3 overexpression inhibited HG-induced activation of STAT3 and MCP-1 expression in HK-2 cells. These findings indicate that Epac activation via 8-O-cAMP ameliorates tubulointerstitial inflammation in DN through the C/EBP-β/SOCS3/STAT3 pathway.

Ochratoxin A induces ERK1/2 phosphorylation-dependent apoptosis through NF-κB/ERK axis in human proximal tubule HK-2 cell line

Ochratoxin A (OTA) is a food contaminant mycotoxin with hazardous effects on human and animal health, primarily affecting the kidneys. OTA's mode of action is not well understood. OTA activates both MAPK/ERK and PI3K/Akt signaling pathways, which play role in apoptosis and cell survival, respectively. OTA is also known to induce toxicity by activating the NF-κB pathway in immune cells. However, its role in determining the cell fate upon OTA exposure in a human kidney cell line (HK-2) has not been fully explored. We made use of pharmacological inhibition of NF-κB to define its role in viability of OTA-treated HK-2 cells. We show that OTA-induced p65 NF-κB subunit translocation into the nucleus in a time-dependent manner using both Western blotting and immunofluorescence (IF). We also document the DNA-binding and reporter gene expression activities of NF-κB by electrophoretic mobility shift (EMSA) and luciferase reporter assays, respectively. Our results indicate that, following 6 h of exposure, OTA fully activates NF-κB pathway and its downstream effectors in HK-2 cells. In addition, Bay11-7085 treatment causes attenuation of the relative levels of OTA-mediated ERK1/2 phosphorylation, suggesting a cross-talk between NF-κB and the MAPK/ERK pathway. Critically, co-treatment of HK-2 cells with OTA and Bay11-7085 leads to the inhibition of OTA-induced apoptosis in a time-dependent manner. Our results support a robust association between NF-κB and the MAPK/ERK pathways in the modulation of apoptotic effects of OTA in HK-2 cells.

Klotho protects against diabetic kidney disease via AMPK- and ERK-mediated autophagy.

Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and results in serious public health problems. Although a great number of studies have been performed to elucidate the mechanisms of this disease, these mechanisms remain largely unknown. Cell and animal models were first constructed using human renal proximal tubule cells stimulated by high glucose (HG) and mice induced by streptozotocin (STZ). After Klotho overexpression, Klotho expression was assessed by RT-PCR and western blot, immunofluorescence; autophagy and AMPK/ERK proteins were confirmed using western blot or immunohistochemical assay; the autophagosomes were observed by transmission electron microscope; the pathological structure, fibrosis, polysaccharides and glycogen of kidney were evaluated by H&E staining, Masson staining and PAS staining. We first confirmed that Klotho expression and autophagic activity were reduced in DM mice and HG-induced human renal proximal tubule cells. Besides, overexpression of Klotho could significantly enhance autophagy and AMPK and ERK1/2 activities in vivo and in vitro, which also could be abolished by selective AMPK inhibitor and ERK activator. Moreover, we proved that Klotho could inhibit hyperglycemia-induced renal tubular damage. In summary, our results proved that Klotho improved renal tubular cell autophagy via the AMPK and ERK pathways and played a role in renal protection. These findings provide new insight into the mechanism of Klotho and autophagy in DKD.

Hypothesis for renin-angiotensin inhibitor mitigation of COVID-19

Preexisting hypertension is a known risk factor for severe COVID-19. Abnormal activation of RAS upregulates angiotensin II (Ang-II) and contributes to severe manifestations of COVID-19. Although RAS inhibitors (RASi) are a mainstay of antihypertensive therapy, they have been associated (in some animal studies) with an increase in angiotensin converting enzyme 2 (ACE2) receptors that facilitate cellular entry of the SARS-CoV-2 virus. Nonetheless, current medical practice does not recommend curtailing RASi to protect hypertensive patients from COVID. On the contrary, there is clinical evidence to support a beneficial effect of RASi for hypertensive patients in the midst of a COVID-19 pandemic, although the precise mechanism for this is unclear. In this paper, we hypothesize that RASi reduces the severity of COVID-19 by promoting ACE2-AT1R complex formation at the cell surface, where AT1R mediates the major vasopressor effects of Ang-II. Furthermore, we propose that the interaction between ACE2 and AT1R impedes binding of SARS-CoV-2 to ACE2, thereby allowing ACE2 to convert Ang-II to the more beneficial Ang(1–7), that has vasodilator and anti-inflammatory activity. Evidence for ACE2-AT1R complex formation during reduced Ang-II comes from receptor colocalization studies in isolated HEK293 cells, but this has not been confirmed in cells having endogenous expression of ACE2 and AT1R. Since the SARS-CoV-2 virus attacks the kidney, as well as the heart and lung, our hypothesis for the effect of RASi on COVID-19 could be tested in vitro using human proximal tubule cells (HK-2), having ACE2 and AT1 receptors. Specifically, colocalization of fluorescent labelled: SARS-CoV-2 spike protein, ACE2, and AT1R in HK-2 cells can be used to clarify the mechanism of RASi action in renal and lung epithelia, which could lead to protocols for reducing the severity of COVID-19 in both hypertensive and normotensive patients.

Three-dimensional spheroid culture induces apical-basal polarity and the original characteristics of immortalized human renal proximal tubule epithelial cells

The proximal tubules, which are part of the kidney, maintain blood homeostasis by absorbing amino acids, glucose, water, and ions such as sodium (Na), potassium, and bicarbonate. Proximal tubule dysfunction is associated with the pathogenesis of many kidney diseases. Renal proximal tubular epithelial cells (RPTECs) are responsible for the main functions of the proximal tubules. Therefore, in vitro experiments using RPTECs would greatly enhance our understanding of nephron physiology and pathobiology. It is preferable to use immortalized cell lines, such as human kidney-2 (HK-2) cells, because they are derived from humans and maintain growth indefinitely. However, tissue-specific RPTEC phenotypes, including apical-basal polarization, are frequently lost in conventional two-dimensional culture methods in part due to microenvironmental deficiencies. To overcome this limitation, we developed a three-dimensional (3D) spheroid culture method for HK-2 cells using an extracellular matrix. HK-2 spheroids in 3D culture formed a tubule-like architecture with cellular polarity and showed markedly restored Na transport function. 3D culture of HK-2 cells also increased expression of kidney development–related genes, including WNT9B. Models of human renal tubules using HK-2 spheroids will greatly improve our understanding of the physiology and pathobiology of the kidney.

Nephrotoxic antisense oligonucleotide SPC5001 induces kidney injury biomarkers in a proximal tubule-on-a-chip

Antisense oligonucleotides (ASOs) are a promising therapeutic modality. However, failure to predict acute kidney injury induced by SPC5001 ASO observed in a clinical trial suggests the need for additional preclinical models to complement the preceding animal toxicity studies. To explore the utility of in vitro systems in this space, we evaluated the induction of nephrotoxicity and kidney injury biomarkers by SPC5001 in human renal proximal tubule epithelial cells (HRPTEC), cultured in 2D, and in a recently developed kidney proximal tubule-on-a-chip. 2D HRPTEC cultures were exposed to the nephrotoxic ASO SPC5001 or the safe control ASO 556089 (0.16-40 µM) for up to 72 h, targeting PCSK9 and MALAT1, respectively. Both ASOs induced a concentration-dependent downregulation of their respective mRNA targets but cytotoxicity (determined by LDH activity) was not observed at any concentration. Next, chip-cultured HRPTEC were exposed to SPC5001 (0.5 and 5 µM) and 556089 (1 and 10 µM) for 48 h to confirm downregulation of their respective target transcripts, with 74.1 ± 5.2% for SPC5001 (5 µM) and 79.4 ± 0.8% for 556089 (10 µM). During extended exposure for up to 20 consecutive days, only SPC5001 induced cytotoxicity (at the higher concentration; 5 µM), as evaluated by LDH in the perfusate medium. Moreover, perfusate levels of biomarkers KIM-1, NGAL, clusterin, osteopontin and VEGF increased 2.5 ± 0.2-fold, 3.9 ± 0.9-fold, 2.3 ± 0.6-fold, 3.9 ± 1.7-fold and 1.9 ± 0.4-fold respectively, in response to SPC5001, generating distinct time-dependent profiles. In conclusion, target downregulation, cytotoxicity and kidney injury biomarkers were induced by the clinically nephrotoxic ASO SPC5001, demonstrating the translational potential of this kidney on-a-chip.

Bringing Microphysiological Systems to Practical Use: Evaluation of transporter‐mediated DDI and Renal Clearance

Active tubular secretion of drugs from the blood to urine is mediated by multiple drug transporters expressed on both basolateral and apical membrane of the proximal tubule. Human proximal tubule microphysiological system (MPS) has been proposed as a more physiologically relevant in vitro system to assess nephrotoxicity and tubular reabsorption.[1,2] However, drug-transporter studies in MPSs are currently sparse and quantitative translation of MPS data to in vivo is not yet established. This study aimed to assess the feasibility of NORTIS MPS to study quantitatively OAT1-mediated transport of pyridoxic acid (PDA) in the absence and presence of 1mM probenecid (prototypical OAT inhibitor). Conditionally immortalized proximal tubule epithelial cells overexpressing OAT1 (ciPTEC-OAT1) were seeded in the NORTIS MPS and were cultured for at least 5 days to allow cell maturation and tight tubule formation prior to transport experiments. PDA secretion rate in the apical channel effluent was linear up to 8h and plateaued between 8-24h. Inhibition of PDA secretion rate by probenecid ranged between 14 – 46% over 2-8h time period. PDA CLu,int,invitro obtained from the initial linear uptake phase was 0.07 and 0.09 µL/min/mm2 in the absence and presence of probenecid, respectively. PDA CLu,int,invitro was quantitatively scaled via total human proximal tubule surface area to CLu,int,invivo which was subsequently used to predict PDA CLR. Translation of MPS transporter data resulted in 5-fold under-prediction of PDA CLR obtained from clinical data (48.2 vs. 215 mL/min). The findings of this study demonstrate a first attempt of quantitative translation of MPS transporter data. The study also highlighted that the extent of inhibition by probenecid was not fully captured in the MPS-ciPTEC-OAT1. Further investigations of the feasibility to derive additional scaling factors for in vitro-in vivo extrapolation of transporter data from MPS are ongoing. [1] Weber, E. J., Chapron, A., Chapron, B. D., Voellinger, J. L., Lidberg, K. A., Yeung, C. K., … & Shen, D. D. (2016). Development of a microphysiological model of human kidney proximal tubule function. Kidney international, 90(3), 627-637. [2] Sakolish, C., Chen, Z., Dalaijamts, C., Mitra, K., Liu, Y., Fulton, T., … & Chiu, W. A. (2020). Predicting tubular reabsorption with a human kidney proximal tubule tissue-on-a-chip and physiologically-based modeling. Toxicology in Vitro, 63, 104752.

G Protein‐Coupled Receptor Kinase 4 (GRK4) Variant 65L‐Mediated Impairment of Dopamine D1 Receptor Function causes Hypertension

Salt sensitivity is associated with impaired dopamine receptor function, which is due, in part, to its desensitization by constitutively active GRK4γ variants. While GRK4 is essential for the normal desensitization and resensitization of dopamine receptors, certain GRK4 polymorphisms desensitize and internalize the dopamine D1 (D1R), thus impairing its natriuretic function. Activating variants of the human GRK4 (65R>L, 142A>V, and 486A>V) are involved in the desensitization of the D1R and are associated with hypertension in several ethnic groups. Furthermore, transgenic mice expressing the GRK4 65L are hypertensive when fed a high salt diet. We have reported that global transgenic mice expressing hGRK4 65L have salt-sensitive hypertension. To avoid the effect of protein overexpression in these transgenic mice, we generated mice globally expressing hGRK4 65L by crispr-cas9-mediated genome editing in C57Bl/6 mice (hGRK4 65L GE). Systolic blood pressure (SBP) in these mice under pentobarbital anesthesia was higher (126±2 vs 103±1 mmHg, n=3/group, P<0.05) than in their littermates not expressing the human gene (GE WT). Renal D1R expression was slightly but not significantly lower (100 ± 2 vs 85 ± 4%, n=3/group) but D1R phosphorylation was higher in hGRK4 65L GE than GE WT (112±2 vs 100±1%, n=3/group, P<0.05). Renal ENaC and NHE3 expressions were similar in both genotypes. To determine the role of the kidney in the salt sensitivity of these mice, we generated mice with kidney-specific (KS) expression of the GRK4 65L (KS hGRK4 65L) by the bilateral ureteral infusion of adeno-associated virus (AAV) vectors carrying hGRK4 65L in GRK4 knockout mice. Mice infused with hGRK4 wild type served as controls (KS hGRK4 WT). SBP (under anesthesia) before AAV was similar in both groups. SBP post AAV increased in KS hGRK4 65L (93±1 vs 117±4 mm Hg, P<0.05, n=4) but not in KS hGRK4 WT (96±2 vs 105±6, n=5). Renal cortical expression of D1R (63±1 vs 100±4 % n=5, P<0.01) was lower but D1R phosphorylation was higher (170 ± 10 vs 100 ±15%; n=5, P<0.02) in KS hGRK4 65L than KS hGRK4 WT. In addition, the renal expression of ENaC (1.4±0.14 vs 1.0±0.11, P<0.05) was increased KS hGRK4 65L but NHE3 was similar in both groups. We also studied human renal proximal tubule cells (hRPTCs) endogenously carrying GRK4 wild type or GRK4 65L. In hRPTCs with GRK4 65L, relative to GRK4 WT, D1R expression (599±80 vs 100±8 % n=6/group; P<0.05), as well as D1R phosphorylation (204 ± 22 vs 100±24%, n=6/group; P<0.05), were increased. Our results show across mouse models and hRPTCs that the presence of the GRK4 65L results in increased D1R phosphorylation, which impairs D1R function for normal renal sodium handling and increases blood pressure in GRK4 65L mice. These results show the important role of renal D1R in the regulation of blood pressure.

The Effects of Dopamine D2 Receptor Specific Deletion in the Renal Proximal Tubule of Mice are Sex Dependent

We have reported that the dopamine D2 receptor (D2R) has a major effect in preventing inflammation and injury in the mouse kidney and in human renal proximal tubule cells (hRPTCs). Lack of or decreased expression of D2R in the mouse kidney or in hRPTCs carrying genetic variants of the receptor that decrease its expression results in an increase in inflammatory factors and injury markers. Because dopamine is synthesized in the RPT and regulated by D2R, we developed a model to determine the effects of D2R in the RPT by generating Drd2fl/fl, PSGLT2::Cre+ mice (D2RPT-/-) that lack D2R only in the RPT and Drd2 fl/fl, PSGLT2::Cre- (D2RPT+/+) mice that do not have the deletion. We hypothesized that the D2R plays a role in the protection of females from renal injury, relative to males. We studied male and female mice on normal salt (NS; 0.4% NaCl) and high salt (HS; 4% NaCl) diets. On NS diet, male D2RPT-/- had higher D2R protein expression in the renal cortex than male D2RPT+/+ (0.85 ± 0.06 vs 0.49 ± 0.11, D2R/GAPDH; n=5; P<0.05) and female D2RPT-/- had higher D2R expression than female D2RPT+/+ (2.95 ± 0.69 vs 1.72 ± 0.35, D2R/GAPDH; n=5; P<0.05). HS increased D2R expression in both male groups (D2RPT+/+ 0.49 ± 0.11 vs 1.04 ± 0.08; n=3-5; P<0.05; D2RPT-/- 0.85 ± 0.06 vs 1.28 ± 0.19, D2R/GAPDH; n=3-5, P<0.05) but decreased its expression in both female groups (D2RPT+/+ 1.72 ± 0.35 vs 0.91 ± 0.05; n=4-5; P<0.05; n=5; D2RPT-/- 2.95 ± 0.69 vs 0.95 ± 0.04, D2R/GAPDH; n=4-5, P<0.05). The transcription factor Nurr1 is involved in the regulation of D2R expression. Nurr1 expression on NS was higher in females of both groups (D2RPT+/+ 1.57± 0.29; D2RPT-/-1.96 ± 0.20, Nurr1/GAPDH; n=4-5; P<0.05) than in males (D2RPT+/+ 0.24 ± 0.05; D2RPT-/-0.38 ± 0.17, Nurr1/GAPDH). HS increased Nurr1 expression in males (D2RPT+/+0.24± 0.05 vs 1.36 ± 0.22; D2RPT-/-0.38± 0.17 vs 0.89 ± 0.19, Nurr1/GAPDH; n=4-5; P<0.05), tended to decrease it in female D2RPT+/+ (1.57± 0.29 vs 1.26 ± 0.19, Nurr1/GAPDH) but decreased significantly in D2RPT-/- (1.96 ± 0.20 vs 0.76± 0.11 ratio Nurr1/GAPDH; n=4-5; P<0.05). The mRNA expression of the injury marker Kim-1 on NS was lower in female than in male mice and was increased in both males and females with D2RPT-/- (1.69 ± 0.09 and 0.36 ± 0.02 fold-change) compared with D2R PT+/+ (1.02 ± 0.03 and 0.15 ± 0.01 fold-change). On HS, there was a decrease in females with D2RPT-/- (0.36 ± 0.02 vs 0.13 ± 0.01 fold-change; n=4-5; p<0.05) and an increase in males with D2RPT-/- (1.69 ± 0.09 vs 3.34 ± 0.56 fold-change; n=4-5; p<0.05). These results show that renal D2R expression is higher in female mice then males and HS diet resulting in increased inflammation and injury elicits an increase in the expression of the receptor in males but a decrease in females. Nurr1 expression is related to D2R expression on NS but has a different response to HS in males and females. The expression of Kim-1 was lower in females than in males regardless of diet and genotype suggesting that increased basal levels of the D2R results in a protective effect. Our findings indicate that the D2R is part of the protective response to renal injury in females.

Changes in Energy Metabolism and Cell Damage in RPTEC‐TERT1 Cells Exposed to Hypoxia or Ischemia after Shear Stress Conditioning

Study Objective Renal proximal tubules are exposed in vivo to physiological levels of shear stress. We aim to determine how preconditioning human proximal tubule cells with shear stress affects their sensibility to hypoxia or ischemia. Hypothesis Physiological levels of shear stress would induce further differentiation and metabolic specialization resulting in changes in their response to low oxygen or nutrient conditions. Methods Immortalized human proximal tubule cells (RPTEC-TERT1 from Evercyte) were grown to confluence in Ibidi µSlides and then exposed for three days to two flow regimes providing negligible (0.01 dyn/cm2) or physiological shear stress (0.3 dyn/cm2). Hypoxia or ischemic conditions were applied for 16h, by keeping cells under 1%O2 alone or together with glucose deprivation. Results RPTEC-TERT1 cells were first adapted to a culturing regime under 5 mM glucose culture medium, instead of 17 mM glucose present in the medium recommended by this cell line vendor. Proliferation rates and expression of phenotypic markers were unchanged. RPTEC-TERT1 cells switched from proliferative-glycolytic metabolism to non-proliferative-oxidative metabolism upon reaching confluency, approximately in 6-7 days. Seven days confluent, not-perfused RPTEC-TERT1 cells exposed to hypoxia or ischemia for 16 hours exhibited 58% and 75% mortality, respectively. In cells perfused for 3 days, preconditioning with shear stress reduced their sensitivity to hypoxia (mortality = 51% no-SS vs. 35% SS) and ischemia (mortality = 83% no-SS vs. 75% SS). Shear stress conditioning increased expression of genes related to energy metabolism and cells stress (AMPK alpha-2, HIF-1alpha, KIM-1) Conclusions Preconditioning of RPTEC-TERT1 by exposing them to physiological shear-stress modifies expression of genes related to mechanisms used to cope with cellular stress and this may explain the reduction in cell mortality when exposed to hypoxia or oxygen-glucose deprivation.

Therapeutic concentrations of calcineurin inhibitors do not deregulate glutathione redox balance in human renal proximal tubule cells.

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.

Live Imaging of Calciprotein Particle Clearance and Receptor Mediated Uptake: Role of Calciprotein Monomers.

BackgroundThe liver-derived plasma protein fetuin A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes calcium phosphate mineral initially as ion clusters to form calciprotein monomers (CPM), and then as larger multimeric consolidations containing amorphous calcium phosphate (primary CPP, CPP 1) or more crystalline phases (secondary CPP, CPP 2). CPM and CPP mediate excess mineral stabilization, transport and clearance from circulation.MethodsWe injected i.v. synthetic fluorescent CPM and studied their clearance by live two-photon microscopy. We analyzed organ sections by fluorescence microscopy to assess CPM distribution. We studied cellular clearance and cytotoxicity by flow cytometry and live/dead staining, respectively, in cultured macrophages, liver sinusoidal endothelial cells (LSEC), and human proximal tubule epithelial HK-2 cells. Inflammasome activation was scored in macrophages. Fetuin A monomer and CPM charge were analyzed by ion exchange chromatography.ResultsLive mice cleared CPP in the liver as published previously. In contrast, CPM were filtered by kidney glomeruli into the Bowman space and the proximal tubules, suggesting tubular excretion of CPM-bound calcium phosphate and reabsorption of fetuin A. Fetuin-A monomer clearance was negligible in liver and low in kidney. Anion exchange chromatography revealed that fetuin A monomer was negatively charged, whereas CPM appeared neutral, suggesting electrochemical selectivity of CPM versus fetuin A. CPM were non-toxic in any of the investigated cell types, whereas CPP 1 were cytotoxic. Unlike CPP, CPM also did not activate the inflammasome.ConclusionsFetuin-A prevents calcium phosphate precipitation by forming CPM, which transform into CPP. Unlike CPP, CPM do not trigger inflammation. CPM are readily cleared in the kidneys, suggesting CPM as a physiological transporter of excess calcium and phosphate. Upon prolonged circulation, e.g., in chronic kidney disease, CPM will coalesce and form CPP, which cannot be cleared by the kidney, but will be endocytosed by liver sinusoidal endothelial cells and macrophages. Large amounts of CPP trigger inflammation. Chronic CPM and CPP clearance deficiency thus cause calcification by CPP deposition in blood vessels and soft tissues, as well as inflammation.

Influence of vitamin D treatment on functional expression of drug disposition pathways in human kidney proximal tubule cells during simulated uremia

Effects of cholecalciferol (VitD3) and calcitriol (1,25-VitD3), on the expression and function of major vitamin D metabolizing enzymes (cytochrome P450 [CYP]2R1, CYP24A1) and select drug transport pathways (ABCB1/P-gp, SLCO4C1/OATP4C1) were evaluated in human kidney proximal tubule epithelial cells (hPTECs) under normal and uraemic serum conditions. hPTECs were incubated with 10% normal or uraemic serum for 24 h followed by treatment with 2% ethanol vehicle, or 100 and 240 nM doses of VitD3, or 1,25-VitD3 for 6 days. The effects of treatment on mRNA and protein expression and functional activity of select CYP enzymes and transporters were assessed Under uraemic serum, treatment with 1,25-VitD3 resulted in increased mRNA but decreased protein expression of CYP2R1. Activity of CYP2R1 was not influenced by serum or VitD analogues. CYP24A1 expression was increased with 1,25-VitD3 under normal as well as uraemic serum, although to a lesser extent. ABCB1/P-gp mRNA expression increased under normal and uraemic serum, with exposure to 1,25-VitD3. SLCO4C1/OATP4C1 exhibited increased mRNA but decreased protein expression, under uraemic serum + 1,25-VitD3. Functional assessments of transport showed no changes regardless of exposure to serum or 1,25-VitD3. Key findings indicate that uraemic serum and VitD treatment led to differential effects on the functional expression of CYPs and transporters in hPTECs.

NFAT5 Is Involved in GRP-Enhanced Secretion of GLP-1 by Sodium.

Gastrin, secreted by G-cells, and glucagon-like peptide-1 (GLP-1), secreted by L-cells, may participate in the regulation of sodium balance. We studied the effect of sodium in mice in vivo and mouse ileum and human L-cells, on GLP-1 secretion, and the role of NFAT5 and gastrin-releasing peptide receptor (GRPR) in this process. A high-sodium diet increases serum GLP-1 levels in mice. Increasing sodium concentration stimulates GLP-1 secretion from mouse ileum and L-cells. GRP enhances the high sodium-induced increase in GLP-1 secretion. High sodium increases cellular GLP-1 expression, while low and high sodium concentrations increase NFAT5 and GRPR expression. Silencing NFAT5 in L-cells abrogates the stimulatory effect of GRP on the high sodium-induced GLP-1 secretion and protein expression, and the sodium-induced increase in GRPR expression. GLP-1 and gastrin decrease the expression of Na+-K+/ATPase and increase the phosphorylation of sodium/hydrogen exchanger type 3 (NHE3) in human renal proximal tubule cells (hRPTCs). This study gives a new perspective on the mechanisms of GLP-1 secretion, especially that engendered by ingested sodium, and the ability of GLP-1, with gastrin, to decrease Na+-K+/ATPase expression and NHE3 function in hRPTCs. These results may contribute to the better utilization of current and future GLP-1-based drugs in the treatment of hypertension.

Stratifin promotes renal dysfunction in ischemic and nephrotoxic AKI mouse models via enhancing RIPK3-mediated necroptosis.

Stratifin (SFN) is a member of the 14-3-3 family of highly conserved soluble acidic proteins, which regulates a variety of cellular activities such as cell cycle, cell growth and development, cell survival and death, and gene transcription. Acute kidney injury (AKI) is prevalent disorder characterized by inflammatory response, oxidative stress, and programmed cell death in renal tubular epithelial cells, but there is still a lack of effective therapeutic target for AKI. In this study, we investigated the role of SFN in AKI and the underlying mechanisms. We established ischemic and nephrotoxic AKI mouse models caused by ischemia-reperfusion (I/R) and cisplatin, respectively. We conducted proteomic and immunohistochemical analyses and found that SFN expression levels were significantly increased in AKI patients, cisplatin- or I/R-induced AKI mice. In cisplatin- or hypoxia/reoxygenation (H/R)-treated human proximal tubule epithelial cells (HK2), we showed that knockdown of SFN significantly reduced the expression of kidney injury marker Kim-1, attenuated programmed cell death and inflammatory response. Knockdown of SFN also significantly alleviated the decline of renal function and histological damage in cisplatin-caused AKI mice in vivo. We further revealed that SFN bound to RIPK3, a key signaling modulator in necroptosis, to induce necroptosis and the subsequent inflammation in cisplatin- or H/R-treated HK2 cells. Overexpression of SFN increased Kim-1 protein levels in cisplatin-treated MTEC cells, which was suppressed by RIPK3 knockout. Taken together, our results demonstrate that SFN that enhances cisplatin- or I/R-caused programmed cell death and inflammation via interacting with RIPK3 may serve as a promising therapeutic target for AKI treatment.

Dopamine D5 receptor-mediated decreases in mitochondrial reactive oxygen species production are cAMP and autophagy dependent.

Overproduction of reactive oxygen species (ROS) plays an important role in the pathogenesis of hypertension. The dopamine D5 receptor (D5R) is known to decrease ROS production, but the mechanism is not completely understood. In HEK293 cells overexpressing D5R, fenoldopam, an agonist of the two D1-like receptors, D1R and D5R, decreased the production of mitochondria-derived ROS (mito-ROS). The fenoldopam-mediated decrease in mito-ROS production was mimicked by Sp-cAMPS but blocked by Rp-cAMPS. In human renal proximal tubule cells with DRD1 gene silencing to eliminate the confounding effect of D1R, fenoldopam still decreased mito-ROS production. By contrast, Sch23390, a D1R and D5R antagonist, increased mito-ROS production in the absence of D1R, D5R is constitutively active. The fenoldopam-mediated inhibition of mito-ROS production may have been related to autophagy because fenoldopam increased the expression of the autophagy hallmark proteins, autophagy protein 5 (ATG5), and the microtubule-associated protein 1 light chain (LC)3-II. In the presence of chloroquine or spautin-1, inhibitors of autophagy, fenoldopam further increased ATG5 and LC3-II expression, indicating an important role of D5R in the positive regulation of autophagy. However, when autophagy was inhibited, fenoldopam was unable to inhibit ROS production. Indeed, the levels of these autophagy hallmark proteins were decreased in the kidney cortices of Drd5-/- mice. Moreover, ROS production was increased in mitochondria isolated from the kidney cortices of Drd5-/- mice, relative to Drd5+/+ littermates. In conclusion, D5R-mediated activation of autophagy plays a role in the D5R-mediated inhibition of mito-ROS production in the kidneys.

Reduced Thiamine Availability and Hyperglycemia Impair Thiamine Transport in Renal Glomerular Cells through Modulation of Thiamine Transporter 2.

Thiamine helps transketolase in removing toxic metabolites, counteracting high glucose-induced damage in microvascular cells, and progression of diabetic retinopathy/nephropathy in diabetic animals. Diabetic subjects show reduced thiamine levels. Hyperglycemia and reduced thiamine availability concur in impairing thiamine transport inside the blood-retinal barrier, with thiamine transporter-2 (THTR2) primarily involved. Here, we examined the behavior of thiamine transporter-1 (THTR1), THTR2, and their transcription factor Sp1 in response to high glucose and altered thiamine availability in renal cells involved in diabetic nephropathy. Human proximal tubule epithelial cells, podocytes, glomerular endothelial, and mesangial cells were exposed to high glucose and/or thiamine deficiency/oversupplementation. Localization and modulation of THTR1, THTR2, and Sp1; intracellular thiamine; transketolase activity; and permeability to thiamine were examined. Reduced thiamine availability and hyperglycemia impaired thiamine transport and THTR2/Sp1 expression. Intracellular thiamine, transketolase activity, and permeability were strongly dependent on thiamine concentrations and, partly, excess glucose. Glomerular endothelial cells were the most affected by the microenvironmental conditions. Our results confirmed the primary role of THTR2 in altered thiamine transport in cells involved in diabetic microvascular complications. Lack of thiamine concurs with hyperglycemia in impairing thiamine transport. Thiamine supplementation could represent a therapeutic option to prevent or slow the progression of these complications.

Downregulation of miR-17 suppresses TGF-β1-mediated renal fibrosis through targeting Smad7.

MiR-17 is found upregulated in diabetic mice; however, its effect(s) on renal fibrosis of diabetic nephropathy remain(s) unknown. This study aimed to explore the mechanism underlying the downregulation of miR-17 in renal fibrosis of diabetic nephropathy (DN). Patients with diabetes mellitus (DM) and DN and normal healthy individual controls, mice (db/db, db/m), and human mesangial cells (HMCs) and human proximal tubule epithelial cells (HK-2) were used as research subjects in the study. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of miR-17 in the serum samples, renal tissues and cells. Acid-Schiff (PAS) and Masson staining experiments were performed to detect glomerular mesangial matrix and collagen deposition. Levels of fibrosis-related proteins (E-Cadherin (E-cad), vimentin, fibronectin and collagen I) were measured by Western blot (WB). The target gene of miR-17 was predicted by TargetScan 7.2 and confirmed by dual-luciferase reporter analysis. The study found that miR-17 expression was elevated in the serums of DN patients as well as in the serums and kidney tissues of db/db mice. db/db mice showed a severe renal fibrosis condition. The levels of E-cad in db/db mice, HMC and HK-2 cells were increased by downregulating miR-17 expression, while expressions of vimentin, fibronectin and collagen I were reduced. Smad7 was predicted to be the target gene of miR-17, and its expression was promoted by downregulation of miR-17. Moreover, the reduced Smad7 expression could inhibit the expressions of fibrosis-related proteins, which, however, can be ameliorated by the downregulation of miR-17. In addition, downregulation of miR-17 could suppress renal fibrosis mediated by TGF-β1 through targeting Smad7, which might be a clinical therapeutic target for patients with DN.

Regulation of endoplasmic reticulum stress on the damage and apoptosis of renal tubular epithelial cells induced by calcium oxalate crystals.

This study aimed to observe whether calcium oxalate (CaOx) crystals can induce the activation of endoplasmic reticulum (ER) stress in human renal cortex proximal tubule epithelial (HK-2) cells and to explore the regulatory of ER stress on the damage and apoptosis of HK-2 cells induced by CaOx crystals. We detected the optimal CaOx crystal concentration and intervention time by Western blot. ER stress modifiers tunicamycin (TM) and 4-phenylbutyric acid (4-PBA) were used to regulate the ER stress of HK-2 cells. The activities of ER stress marker proteins GRP78 and CHOP were evaluated by Western blot and immunohistochemistry. Western blot and TUNEL staining were used to detect cell apoptosis. We observed cell-crystal adhesion with an optical microscope. Lactate dehydrogenase (LDH) test kit and IL-1β enzyme-linked immunosorbent assay kit were used to detect and evaluate HK-2 cell damage. We found that the expression of ER stress marker proteins GRP78 and CHOP gradually increased with the increase in CaOx crystal concentration and intervention time and reached the maximum at 2.0mmol/L and 24h. The use of ER stress modifiers TM and 4-PBA can effectively regulate the ER stress level induced by CaOx crystals, and the level of apoptosis is positively correlated with the level of ER stress. 4-PBA pretreatment remarkably reduced cell-crystal adhesion and the secretions of IL-1β and LDH, whereas the results of TM pretreatment were the opposite. In summary, the damage and apoptosis of HK-2 cells induced by CaOx crystals are closely related to the level of ER stress. Inhibiting the ER stress of HK-2 cells can substantially reduce the cell damage and apoptosis induced by CaOx crystals.

The critical role of FXR is associated with the regulation of autophagy and apoptosis in the progression of AKI to CKD

Autophagy is important for cells to break down and recycle cellular proteins, remove damaged organelles, and especially, for recovery from acute kidney injury (AKI). Despite research on the role and cellular mechanism of autophagy in AKI, the role of autophagy in the progression to chronic kidney disease (CKD) remains poorly understood. Here, using farnesoid X receptor (FXR) knockout (KO) mice, we determined whether FXR prevents the progression of AKI to CKD after renal ischemic-reperfusion (such as I/R) injury through the regulation of renal autophagy and apoptosis. FXR regulated genes that participate in renal autophagy under feeding and fasting conditions, such as hepatic autophagy, and the activation of FXR by agonists, such as GW4064 and INT-747, attenuated the increased autophagy and apoptosis of hypoxia-induced human renal proximal tubule epithelial (HK2) cells. The expression levels of autophagy-related and apoptosis-related proteins in FXR KO mice were increased compared with those in wild-type (WT) mice. We also showed that the increase in reactive oxidative species (ROS) in hypoxia-treated HK2 cells was attenuated by treatment with FXR agonist or by FXR overexpression, and that the level of ROS was elevated in FXR-deficient cells and mice. At 28 days after I/R injury, the autophagy levels were still elevated in FXR KO mice, and the expression levels of fibrosis-related proteins and ROS deposits were higher than those in WT mice. In conclusion, the regulation of renal autophagy and apoptosis by FXR may be a therapeutic target for the early stages of kidney damage, and the progression of AKI to CKD.