Proximal Tubule Cells Research Articles

Integrin β6 mediates epithelial–mesenchymal transition in diabetic kidney disease

The progression of diabetic kidney disease (DKD) is associated with increased fibronectin (FN) levels in proximal tubular epithelial cells. Bioinformatics analysis showed that integrin β6 and cell adhesion function were significantly changed in the cortices of db/db mice. Remodelling of cell adhesion is one of the core changes during epithelial–mesenchymal transition (EMT) in DKD. Integrin is a family of transmembrane proteins that regulates cell adhesion and migration, and extracellular FN is the major ligand of integrin β6. We found that the expression of integrin β6 was elevated in the proximal tubules of db/db mice and FN-induced renal proximal tubule cells. The levels of EMT were also significantly increased in vivo and in vitro. In addition, FN treatment activated the Fak/Src pathway, increased the expression of p-YAP, and then upregulated the Notch1 pathway in diabetic proximal tubules. Knockdown of integrin β6 or Notch1 reduced the EMT aggravation induced by FN. Furthermore, urinary integrin β6 was significantly increased in DKD patients. Our findings reveal a critical role of integrin β6 in regulating EMT in proximal tubular epithelial cells and identify a novel direction for the detection and treatment of DKD.

Acrolein produced during acute kidney injury promotes tubular cell death

Acute kidney injury is an important global health concern as it is associated with high morbidity and mortality. Polyamines, essential for cell growth and proliferation, are known to inhibit cardiovascular disease. However, under conditions of cellular damage, toxic acrolein is produced from polyamines by the enzyme spermine oxidase (SMOX). We used a mouse renal ischemia-reperfusion model and human proximal tubule cells (HK-2) to investigate whether acrolein exacerbates acute kidney injury by renal tubular cell death. Acrolein visualized by acroleinRED was increased in ischemia-reperfusion kidneys, particularly in tubular cells. When HK-2 cells were cultured under 1% oxygen for 24 h, then switched to 21% oxygen for 24 h (hypoxia-reoxygenation), acrolein accumulated and SMOX mRNA and protein levels were increased. Acrolein induced cell death and fibrosis-related TGFB1 mRNA in HK-2 cells. Administration of the acrolein scavenger cysteamine suppressed the acrolein-induced upregulation of TGFB1 mRNA. Cysteamine also inhibited a decrease in the mitochondrial membrane potential observed by MitoTrackerCMXRos, and cell death induced by hypoxia-reoxygenation. The siRNA-mediated knockdown of SMOX also suppressed hypoxia-reoxygenation-induced acrolein accumulation and cell death. Our study suggests that acrolein exacerbates acute kidney injury by promoting tubular cell death during ischemia-reperfusion injury. Treatment to control the accumulation of acrolein might be an effective therapeutic option for renal ischemia-reperfusion injury.

A single-cell multiomic analysis of kidney organoid differentiation

Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.

Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

Of late, numerous microphysiological systems have been employed to model the renal proximal tubule. Yet there is lack of research on refining the functions of the proximal tubule epithelial layer—selective filtration and reabsorption. In this report, pseudo proximal tubule cells extracted from human-induced pluripotent stem cell-derived kidney organoids are combined and cultured with immortalized proximal tubule cells. It is shown that the cocultured tissue is an impervious epithelium that offers improved levels of certain transporters, extracellular matrix proteins collagen and laminin, and superior glucose transport and P-glycoprotein activity. mRNA expression levels higher than those obtained from each cell type were detected, suggesting an anomalous synergistic crosstalk between the two. Alongside, the improvements in morphological characteristics and performance of the immortalized proximal tubule tissue layer exposed, upon maturation, to human umbilical vein endothelial cells are thoroughly quantified and compared. Glucose and albumin reabsorption, as well as xenobiotic efflux rates through P-glycoprotein were all improved. The data presented abreast highlight the advantages of the cocultured epithelial layer and the non-iPSC-based bilayer. The in vitro models presented herein can be helpful in personalized nephrotoxicity studies.

HNF1A binds and regulates the expression of SLC51B to facilitate the uptake of estrone sulfate in human renal proximal tubule epithelial cells

Renal defects in maturity onset diabetes of the young 3 (MODY3) patients and Hnf1a-/- mice suggest an involvement of HNF1A in kidney development and/or its function. Although numerous studies have leveraged on Hnf1α-/- mice to infer some transcriptional targets and function of HNF1A in mouse kidneys, species-specific differences obviate a straightforward extrapolation of findings to the human kidney. Additionally, genome-wide targets of HNF1A in human kidney cells have yet to be identified. Here, we leveraged on human in vitro kidney cell models to characterize the expression profile of HNF1A during renal differentiation and in adult kidney cells. We found HNF1A to be increasingly expressed during renal differentiation, with peak expression on day 28 in the proximal tubule cells. HNF1A ChIP-Sequencing (ChIP-Seq) performed on human pluripotent stem cell (hPSC)-derived kidney organoids identified its genome-wide putative targets. Together with a qPCR screen, we found HNF1A to activate the expression of SLC51B, CD24, and RNF186 genes. Importantly, HNF1A-depleted human renal proximal tubule epithelial cells (RPTECs) and MODY3 human induced pluripotent stem cell (hiPSC)-derived kidney organoids expressed lower levels of SLC51B. SLC51B-mediated estrone sulfate (E1S) uptake in proximal tubule cells was abrogated in these HNF1A-deficient cells. MODY3 patients also exhibit significantly higher excretion of urinary E1S. Overall, we report that SLC51B is a target of HNF1A responsible for E1S uptake in human proximal tubule cells. As E1S serves as the main storage form of nephroprotective estradiol in the human body, lowered E1S uptake and increased E1S excretion may reduce the availability of nephroprotective estradiol in the kidneys, contributing to the development of renal disease in MODY3 patients.

Glucose and cell confluency regulation of HDGF and beta-catenin nuclear localization

Introduction: Diabetes mellitus (DM) is the seventh leading cause of death in the US and diabetic kidney disease (DKD)is the cause of nearly 50% of individuals receiving dialysis. Kidney proximal tubule (PT) cells are sensitive to injury under diabetic conditions of high glucose. Maladaptive repair can cause fibrosis and loss of PT function contributing to end-stage renal disease. PT cell proliferation re-establishes the monolayer responsible for PT functionality. Preliminary lab findings showed increased abundance and nuclear localization of Hepatoma-derived growth factor (HDGF), known to regulate proliferation, in cells grown at low (<40%) confluency in high glucose supplemented growth media. This change associated with altered nuclear co-localization with β-catenin (ß-Cat), involved in renal fibrosis, suggesting crosstalk between both factors may be involved in PT cell responses in DKD. Hypothesis: Cell confluency and glucose concentrations regulate the spatial and absolute abundance/degradation of HDGF altering cell signaling pathways in PT cells. Methods: Cultured human kidney PT cells (HK-11 cell line) were grown to low and high confluency under normal (5mM) and high (25mM) glucose conditions. RNA was collected via column purification for analysis of cell signaling. RNASeq and bioinformatic analysis. revealed transcript abundances and respective statistical significances between normal and high glucose conditions grown to low confluency. MetaCore and STRING were utilized to examine fold change differences of the transcriptome and affected pathways. To determine regulation of protein abundance, HK-11 cells were cultured in normal glucose media to 80-90% confluency and treated with no agent (control), DMSO, MG132 (proteasome inhibitor), and chloroquine (lysosome inhibitor), and abundance of HDGF and β-Cat activation analyzed by immunoblot. Results: HDGF transcripts from conditions of normal and high glucose grown to low confluency were found in relatively equal abundance (q ≤ 1). Pathway analyses revealed genes associated with multiple pathways including nuclear signaling, ER stress, proliferation and ubiquitination exhibited differential transcript abundance at statistically significant values. Immunoblot analysis suggested that HDGF is degraded via the lysosome. Conclusions: HDGF regulation by diabetes-like high glucose does not occur at the level of transcription and but through lysosomal degradation. ß-Cat nuclear localization is increased under conditions of high glucose and low cellular confluency. To confirm HDGF degradation and ß-Cat activation, more replicates are needed for statistical analysis. Future studies will determine the role of β-cat activation on HDGF abundance and nuclear localization. NIH T35-DK072923, P20-GM103436, NIH P20 GM113226, P50 AA024337, P30 ES030283 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Signaling crosstalk between Dipeptidyl Peptidase 4 and Angiotensin II-AT1R plays a role in proximal tubule NHE3 regulation and salt sensitive hypertension

Background: Salt sensitivity to hypertension occurs in 50-60% of all hypertensive patients and is more common in overweight/obese, females and aging population. In salt sensitive hypertension, the renin-angiotensin system (RAS) is known to play a major role and in particular, Angiotensin II (Ang II) regulation of the sodium hydrogen exchanger (NHE3) in the proximal tubule may mediate the pathophysiology. Dipeptidyl peptidase 4 (DPP4), an exopeptidase expressed abundantly on the brush border, binds to NHE3 and DPP4 inhibition reduces NHE3-mediated sodium reabsorption in proximal tubule cells. Interestingly, we observed both in vitro and in vivo that Ang II stimulates DPP4 activity. However, the mechanisms mediating the interplay between Ang II and DPP4 and its role in NHE3 regulation remain unknown. Therefore, we hypothesized that Ang II stimulation of DPP4 activity via crosstalk between AT1R and DPP4 may account for NHE3 modulation and, consequently, salt-sensitive hypertension. Methods: To address this hypothesis, we stimulated OK-AT1R (stable expressing rat AT1R) proximal tubule cells with Ang II (10 -8 to 10 -10 M), OKP cells with Ang II 10 -8 M and microperfused rat nephrons (Ang II 10 -10 M). In addition, sodium balance studies were conducted on DPP4-/- mice with high salt (1% sodium choride in drinking water) and low salt (0.02% in diet) Results: In OK-AT1R cells, we observed a dose-dependent increase in DPP4 activity, which was inhibited with AT1R blockade, and a battery of kinase inhibitors, including those for Src, PI3K, mTOR, S6K, ERK, and EGFR. Blockade with the DPP4 inhibitor MK-0626 not only suppressed DPP4 activity but partially suppressed Ang II-mediated signaling via EGFR, src, PI3K, mTOR and S6K. Further dissection of the pathways revealed that the blockade of src, which binds to both AT1R and DPP4, mimicked many of the effects of AT1R and DPP4 blockade. Mechanistically, blockade of motor proteins myosin II and myosin VI, which have been shown to redistribute with DPP4 and NHE3 along the microvilli microdomains, did not inhibit Ang II-induced DPP4 activity suggesting other mechanisms (phosphorylation) may underlie Ang II-DPP4 crosstalk in proximal tubule cells. Importantly, we found that DPP4 inhibition blocked Ang II-mediated stimulation of NHE3 activity (pHi) in vitro (OKP cells) and JHCO 3 - in vivo (rat nephrons). In addition, we observed that DPP4 gene-deleted mice lose more salt under both high and low salt ingestion and that DPP4 deficiency lowers BP under conditions of high salt, suggesting that DPP4 may regulate salt-sensitive hypertension. In conclusion, Ang II-AT1R and DPP4 likely crosstalk via binding to src and/or PI3-K, and inhibition of DPP4 suppresses Ang II-AT1R signaling and downstream effectors, including NHE3. Moreover, inhibition of signaling downstream of AT1R can suppress DPP4 activity, suggesting that AT1R and DPP4 act in concert but not via subcellular redistribution via motor proteins. The Sao Paulo Research Foundation - FAPESP (2021/14534-3), Sao Paulo, SP, Brazil to Flavia Martins and Dr. Adriana Girardi; NIH/NIDDK - K08 DK115886 to Dr. Ravi Nistala This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute and reversible modulation of kidney proximal tubule endocytic capacity by fluid shear stress

The proximal tubule (PT) of the kidney is comprised of cells that are uniquely specialized for efficient apical uptake of albumin and other proteins that escape the glomerular filtration barrier. These proteins bind to the multiligand receptors megalin and cubilin and are internalized via clathrin-dependent endocytosis into apical early endosomes that rapidly mature into larger apical vacuoles (AVs). Dissociated ligands are delivered from AVs to lysosomes for degradation, while receptors are collected into dense apical tubules for recycling to the cell surface. When cells are grown under continuous fluid shear stress (FSS), the endocytic uptake is approximately 9-fold greater than static cells. Additionally, cells cultured in this manner rapidly and reversibly adjust their endocytic capacity in response to changes in FSS. We hypothesize flow-dependent modulation of endocytic capacity enables PT cells in vivo to preserve uptake efficiency in response to changes in glomerular filtration. We are combining biochemical, imaging, and mathematical modeling approaches to determine the mechanism(s) by which cells chronically and acutely modulate endocytic uptake. Increased levels of megalin transcripts (~2x) and protein (~4x) is insufficient to account for the large increase in endocytic uptake observed in cells cultured under FSS vs static conditions. We used cell surface biotinylation experiments to compare the fraction of megalin at the surface, the fraction endocytosed, and the half-life of surface-tagged megalin under FSS and static conditions. These data were used to adapt a kinetic model that describes kinetics of megalin biosynthesis, trafficking, and degradation in cells. Overall, the cells grown under FSS have a greater endocytic rate and recycling rate compared with static grown cells. By contrast, megalin degradation is faster than recycling in static-grown cells. Colocalization of megalin with known markers of apical compartments will enable us to extend our model to determine whether changes in endosomal maturation rates contribute to these differences. Additionally, cells cultured under continuous FSS rapidly and reversibly adjust their endocytic capacity in response to changes in shear stress. We hypothesize flow-dependent modulation of endocytic capacity enables PT cells in vivo to preserve uptake efficiency in response to changes in glomerular filtration. We are currently adapting our model to determine how acute changes in shear stress trigger alterations in endocytic uptake. National Institute of Health: 5T32GM133353-03, R01 DK118726, R01 DK125049 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Western diet exacerbates aristolochic acid-induced nephropathy in mice

Aristolochic acid (AA) ingestion causes Balkan nephropathy, characterized by tubular injury and progression to chronic kidney disease (CKD). AA is taken up by proximal tubule cells via organic anion transport (OAT3) and induces p53-mediated DNA damage, but little is known about modulating factors. The Western diet (WD) is rich in saturated fats, sugars, and salt and contributes to the development of metabolic disorders such as obesity and diabetes, but also to the progression of CKD. The aim of this study was to elucidate the impact of WD on AA-induced kidney injury. 5-week-old male C57BL6 mice were fed with WD or normal chow (NC) for 8 weeks, followed by administration of AA every 3 days at a dose of 3 mg/kg for 3 weeks. Samples were collected after a 3-week recovery period (n=4/group for vehicle; n=7-8/group for AA). The AA-induced increase in plasma creatinine and the reduction of hematocrit were significantly greater in WD vs NC. This was associated with increased kidney gene expression in WD vs NC of markers of DNA damage ( p53), injury ( Kim1 and Ngal), and inflammation ( Tnfa). The WD group had a greater body weight and, therefore, received a higher total dose of AA (0.73 and 0.6 mg), which may have worsened kidney injury. Therefore, in a second series, the same total dose of AA (0.9 mg) was administered to WD and NC groups. WD similarly increased the AA-induced fall in hematocrit and rise in plasma creatinine and renal p53, Kim1, Ngal, and Tnfa mRNA expression compared to the NC group. Moreover, WD increased renal mRNA expression of Oat3. These findings suggest that WD increases the susceptibility to AA nephrotoxicity, possibly through upregulation of OAT3. This model may be useful for studying the mechanistic impact of Western diet on the development and progression of CKD caused by nephrotoxic agents. Supported by National Institutes of Health (NIH) Grants RF1AG061296 (PM, VV), R01DK132690 (VV), and P30 DK079337 (VV), the Department of Veterans Affairs, and a fellowship of the Manpei Suzuki Diabetes Foundation (YO). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A myogenic program is enacted by cardiac protein CSRP3 in human and mouse kidney

Introduction: Cardiac injury with renal injury (acute cardiorenal syndrome) can induce renal fibrosis, but mechanisms by which heart injury signals to kidney are poorly understood. The myocardiocyte differentiation factor cardiac LIM protein (CSRP3) is released into plasma after cardiac injury, and taken up by megalin (Lrp2) in renal proximal tubular epithelial cells in vivo. We evaluated the relationship between cardiac injury and renal fibrosis in a mouse model and in human cells by characterizing novel primary culture of human tubular epithelial cells and then testing the hypothesis that CSRP3 alters PTEC transcription of myogenesis, transport, and extracellular matrix genes in vitro and in vivo. Methods: In mice, cardiac arrest (CA) was induced with potassium chloride and resuscitated with epinephrine and chest compressions (CPR) as previously described. SnRNAseq was conducted on mouse kidney cells harvested 24h after CA/CPR and analyzed using Seurat. Research-designated human kidneys were obtained under IRB exemption for non-human subjects research and underwent dissection, digestion and FACS sorting for PTEC selection. hPTEC were passaged up to 4x before transporters were characterized by qPCR, immunoblot, and immunofluorescence. Recombinant human CSRP3 (Origene) was administered to hPTEC followed by RNASeq and qPCR. CSRP3 (5μg) was retroorbitally injected in mice and renal fibrosis was assessed 7 weeks later. Results: In mice, snRNAseq demonstrated that CA/CPR significantly upregulated muscle cell differentiation ontologies in proximal tubule cells, driven by regulation of 110/162 myogenesis genes. Following FACS sorting for CD10 and CD13, hPTEC were 95% pure PTEC and expressed RNA and protein for PT transporters Lrp2, Aqp1, Slc34a1, and Slc5a2. In hPTEC, CSRP3 induced myogenesis differentiation factor 1 (MyoD1)>30 fold, and 20 myogenesis pathway and extracellular matrix genes. 8/20 genes were coordinately regulated between mouse kidney after CA/CPR and hPTEC after CSRP3. CSRP3 injection caused tubulointerstitial fibrosis in mice (V αSMA /V kidney (%): 1.05 (CSRP3) vs 0.68 (control), n=8/group, p<0.01). Conclusion: hPTEC cultured from research-designated donor kidneys express a PT phenotype. The myocardiocyte differentiation factor CSRP3 causes transcriptional change in hPTEC, resulting in myogenic differentiation which is highly similar to that observed in mouse kidney after CA/CPR with coordinate regulation of myogenesis genes. Intravenous administration of CSRP3 to mice causes tubular interstitial fibrosis. Together these data strongly suggest CSRP3 may drive renal fibrosis via the myogenesis pathway. Merit grant I01BX004288-01 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Developing a multiscale mathematical model of protein uptake along the mouse proximal tubule

The proximal tubule (PT) efficiently reclaims albumin and other proteins that escape the glomerular filtration barrier to maintain a protein free urine. The recovery of filtered proteins occurs via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. PT health is impacted by excess albumin uptake that occurs when the glomerular barrier is breached and by nephrotoxic drugs that enter cells in a megalin/cubilin dependent manner. Understanding where and how endocytic uptake occurs along the PT axis is key to predicting disease outcomes and to devising strategies to protect PT cells from nephrotoxic insults. Transcriptomic and proteomic studies in male rodents have demonstrated a difference in the expression of megalin and cubilin across the S1, S2, and S3 sub-segments that comprise the PT. Based on these differences in expression, we previously developed an axial model of protein uptake in mice which predicts that most uptake under normal conditions occurs in the S1 segment, with later PT segments providing excess uptake capacity under nephrotic conditions. Separately, we created a kinetic model that predicts rates of megalin trafficking rates in S1 segment cells. This model demonstrated that both the actual level and the fraction of total receptor present at the apical surface are critical determinants of endocytic capacity. To integrate our cell-based kinetic model of megalin traffic into the axial model, we used imaging-based approaches to quantify the expression and distribution of megalin and cubilin in SGLT2-positive S1 and SGLT2-negative S2 segments in fixed cortical kidney sections from wild-type C57BL/6 age-matched male and female mice. The fraction of megalin and cubilin at the apical surface was determined by colocalization with the brush border marker, villin, in regions where AP2, a marker for endocytic vesicles, was excluded. These data will be incorporated into our current axial model to develop a multiscale model of protein uptake along the length of the PT. The expanded model will improve our understanding of how endocytic capacity along the PT in vivo is regulated under normal and disease conditions and allow us to determine whether there are sex-based differences in the recovery of filtered proteins. National Institutes of Health T32 DK007052, F31 DK121394, R01 DK125049, R01 DK118726 and ASN Foundation for Kidney Research Pre-Doctoral Fellowship Award This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteinuria observed post-uninephrectomy stems from partial suppression of protein endocytosis along the proximal tubule

Living kidney donors are at higher risk of developing proteinuria, yet few studies have addressed the etiology. To investigate whether post-uninephrectomy (UNX) proteinuria was due to increased filtration and/or reduced endocytosis/reabsorption along the proximal tubule (PT), we compared renal function, morphology, and PT endocytic machinery in male Sprague Dawley rats before and after UNX.At 12 wk post-UNX, the weight of the remnant kidney rose by 50%, PT diameters by 30%, and single nephron GFR by 50% (based on 26% fall in creatinine clearance and 50% decrease in the number of nephrons). Urinary albumin excretion increased 10-fold post-UNX. Since the filtered load of proteins decreases in parallel with GFR post-UNX, increased albumin excretion likely reflects decreased endocytosis along the PT, a notion supported by a 50% reduction in abundance of albumin and plasminogen in renal cortex homogenates from remnant versus explant kidneys. We tested the hypothesis that the protein endocytic machinery was reduced following UNX. Immunoblot assays of renal cortex revealed decreased abundance of megalin and Dab2, two key mediators of protein endocytosis along the PT, and no change in cubilin abundance. Immunohistochemistry confirmed a 50% fall in megalin along the PT apical membrane.Mathematical modeling predicted that albumin uptake per nephron increases post-UNX, owing to higher single nephron GFR and PT hypertrophy, but is attenuated by the decrease in megalin abundance; moreover, modeling predicts that if the endocytic machinery were not partly suppressed, overall albumin excretion would remain at pre-UNX levels.In addition to albumin, urinary plasminogen and angiotensinogen were similarly increased post-UNX, while uromodulin (synthesized and secreted distally) was decreased post-UNX. Urinary biomarkers of renal injury, RBP4 and KIM-1, were increased post-UNX likely reflecting the impact of the increased endocytosis of albumin which can carry bound toxic lipids into the PT.Taken together, our findings suggest that the blunting of albumin endocytosis post-UNX, apparent as increased albuminuria, may serve to protect PT cells from albumin-induced cytotoxicity. DK083785; Keck School of Medicine Dean’s Pilot Funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Selenium deficiency causes hypertension by increasing renal AT1 receptor expression via GPx1/H2O2/NF-κB pathway.

Epidemiological studies show an association between low body selenium and the risk of hypertension. However, whether selenium deficiency causes hypertension remains unknown. Here, we report that Sprague-Dawley rats fed a selenium-deficient diet for 16 weeks developed hypertension, accompanied with decreased sodium excretion. The hypertension of selenium-deficient rats was associated with increased renal angiotensin II type 1 receptor (AT1R) expression and function that was reflected by the increase in sodium excretion after the intrarenal infusion of the AT1R antagonist candesartan. Selenium-deficient rats had increased systemic and renal oxidative stress; treatment with the antioxidant tempol for 4 weeks decreased the elevated blood pressure, increased sodium excretion, and normalized renal AT1R expression. Among the altered selenoproteins in selenium-deficient rats, the decrease in renal glutathione peroxidase 1 (GPx1) expression was most prominent. GPx1, via regulation of NF-κB p65 expression and activity, was involved in the regulation of renal AT1R expression because treatment with dithiocarbamate (PDTC), an NF-κB inhibitor, reversed the up-regulation of AT1R expression in selenium-deficient renal proximal tubule (RPT) cells. The up-regulation of AT1R expression with GPx1 silencing was restored by PDTC. Moreover, treatment with ebselen, a GPX1 mimic, reduced the increased renal AT1R expression, Na+-K+-ATPase activity, hydrogen peroxide (H2O2) generation, and the nuclear translocation of NF-κB p65 protein in selenium-deficient RPT cells. Our results demonstrated that long-term selenium deficiency causes hypertension, which is due, at least in part, to decreased urine sodium excretion. Selenium deficiency increases H2O2 production by reducing GPx1 expression, which enhances NF-κB activity, increases renal AT1R expression, causes sodium retention and consequently increases blood pressure.

Nonmuscle myosin II proteins, Myh9 and Myh10, interacts with unconventional myosin Myo18A in immortalized mouse thick ascending limb epithelial cells

Conventional nonmuscle myosin II (NM2) and unconventional myosin 18A (Myo18A) belong to an evolutionarily conserved family of molecular motor proteins that slide or walk on the actin cytoskeleton to facilitate a myriad of cellular functions including cargo transport pathways. NM2 and Myo18A are known to copolymerize into filaments in vitro in cell-free systems and in cells. Our previous work demonstrated the crucial role for Myh9&10 proteins in regulating transport of uromodulin (UMOD) and the sodium, potassium, chloride cotransporter (NKCC2) in the thick ascending limb (TAL) of mouse kidney using a Myh9&10 conditional knockout mouse model (Otterpohl, et al; 2020). Myo18A is expressed in the proximal tubule and TAL cells in the murine kidney and localizes to the apical membrane. Our results show reduced expression of Myo18A in the TAL cells of the Myh9&10 cKO mouse kidney sections. Furthermore, we identified Myo18A as a major interaction partner to the Myh10 heavy chain in a BioID screen performed in renal epithelial cells. Given that Myo18A alpha is the only myosin with a PDZ domain, we hypothesize that Myo18A coordinates with Myh9&10 in the TAL cells to regulate membrane remodeling during vesicular transport, including the targeting and stabilization of TAL proteins like UMOD and NKCC2. To test this hypothesis, we stably expressed GFP-tagged Myo18A alpha construct in immortalized, mouse thick ascending limb (imTAL) cells generated in the lab. We performed coimmunoprecipitation (Co-IP) experiments that showed interaction between Myo18A alpha, Myh9 and Myh10 proteins. Immunostaining experiments show colocalization of GFP-Myo18A alpha with Myh9 and Myh10 in imTAL cells. These results strongly support the coordinated functions of Myo18A and Myh9&10 proteins in TAL epithelia and warrants further experiments to determine their role in regulating UMOD and NKCC2 transport in the TAL cells. 1R01DK131020-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low dose Taxol ameliorated renal fibrosis in mice with diabetic kidney disease by downregulation of HIPK2.

Our previous studies reported that low-dose paclitaxel (Taxol) ameliorated renal fibrosis in the unilateral ureteral obstruction and remnant kidney models. However, the regulatory role of Taxol in diabetic kidney disease (DKD) is still unclear. Herein, we observed that low-dose Taxol attenuated high glucose-increased expression of fibronectin, collagen I and collagen IV in Boston University mouse proximal tubule cells. Mechanistically, Taxol suppressed the expression of homeodomain-interacting protein kinase 2 (HIPK2) via disrupting the binding of Smad3 to HIPK2 promoter region, and consequently inhibited the activation of p53. Besides, Taxol ameliorated RF in Streptozotocin mice and db/db-induced DKD via suppression of Smad3/HIPK2 axis as well as inactivation of p53. Altogether, these results suggest that Taxol can block Smad3-HIPK2/p53 axis, thereby attenuating the progression of DKD. Hence, Taxol is a promising therapeutic drug for DKD.

Different effects of fenoldopam and angiotensin II on autophagy in vascular smooth muscle cells

Both dopamine and angiotensin II are essential neurotransmitters/hormones with pleiotropic functions in vascular smooth muscle cells (VSMCs). Autophagy, an intracellular self-degradative process that delivers cytoplasmic constituents to lysosomes, plays an important role in physiological homeostasis in VSMCs. Fenoldopam (Fen), a dopamine D 1 /D 5 receptor agonist, increases autophagic flux in renal proximal tubule cells, however, in VSMCs the roles of these two neurotransmitters on autophagy are unknown. In rat aortic VSMCs, Fen increased autophagy, determined by the protein expression of microtubule-associated protein 1 light chain (LC)3-II and autophagy related 5 (ATG5), in a concentration- and time-dependent manner. By contrast, angiotensin II (Ang II), the endogenous AT 1 R agonist, decreased the protein expression of LC3-II and ATG5 in a concentration-and time-dependent manner. Further studies showed that Fen (1 μM, 15 min) increased while Ang II (100 nM, 15 min) decreased cyclic AMP (cAMP) production in VSMCs (Vehicle: 100±35.4%, Fen: 513.7±95.8%, Ang II: 24.6±8.0%, n=6). Pre-treatment with Rp-cAMPS (50 μM, 6 hr), a PKA inhibitor, attenuated the Fen-mediated increase in LC3-II protein expression (Fen: 1 μM, 6 hr) (Vehicle: 100±15.0%, Fen: 251.8±37.2%, Fen+Rp-cAMP: 96.9±16.7%, n=6) and reversed the Ang II-mediated decrease in autophagy (Ang II: 100 nM, 6 hr) (Vehicle: 100±8.9%, Ang II: 44.9±6.3%, Ang II+Rp-cAMP: 95.4±11.0%, n=4). These results demonstrate distinct roles of Fen and Ang II on autophagy through regulation of cAMP/PKA signaling by their corresponding receptors in VSMCs. This study was supported by National Institutes of Health of US and National Natural Science Foundation of China. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High glucose-stimulated secretion of Semaphorin-3G by proximal tubule cells inhibits angiogenesis in peritubular endothelial cells

Hyperglycemia in the diabetic kidney causes tubular and microvascular abnormalities that eventually lead to kidney damage. Given the close association between the proximal tubule and peritubular capillaries, a thorough understanding of their interaction could help identify mechanisms of diabetic kidney disease. Microvascular abnormalities are known to occur in diabetic patients and animal models. Despite known growth factors produced by the proximal tubule that could potentially target neighboring endothelial cells, it is not well understood whether the proximal tubule regulates angiogenesis. Here we propose that the proximal tubule secretes Semaphorin-3G (Sema3G), an anti-angiogenic protein. We hypothesize, that high glucose stimulates Sema3G secretion by proximal tubule cells and inhibits angiogenesis in peritubular endothelial cells.To study the role of secreted Sema3G, we collected the conditioned media from cultured polarized proximal tubule cells (RPTEC cell line) where we silenced Sema3G via lentivirus-delivered shRNAs. To study angiogenesis, we developed primary cultures of mouse kidney peritubular endothelial cells and analyzed angiogenesis via tubulogenesis assays in matrigel.We observed that the basolateral conditioned medium from RPTEC cells stimulated angiogenesis of peritubular endothelial cells in matrigel over 8 hours (p<0.01). However, RPTEC cells pre-grown on high glucose (25 mM) lost this ability. The apical media did not have any effect. To identify a potential proximal tubule anti-angiogenic factor, we measured release of Sema3G from polarized RPTEC cells. We found that RPTEC cells secreted Sema3G basolaterally and high glucose stimulated this by 5.5-fold. (p<0.01). Next, we tested the anti-angiogenic potential of Sema3G in peritubular endothelial cells. While adding recombinant Sema3G (200ng/ml) produced a modest inhibition (28%, p<0.05) of baseline peritubular endothelial cell angiogenesis, instead it completely blocked the stimulatory effect of a dose-response treatment (50–100ng/ml) with the potent angiogenic factor VEGF (p<0.01). Finally, to test the role of endogenous proximal tubule Sema3G, we silenced Sema3G via lentivirus-delivered shRNA in RPTEC cells. We then collected the conditioned medium from cells on normal or high glucose and performed angiogenesis assays. We found that silencing Sema3G in RPTEC cells on high glucose restored the ability of the conditioned medium to stimulate peritubular endothelial angiogenesis.We conclude that RPTEC proximal tubule cells secrete Sema3G, which is stimulated by high glucose, and this anti-angiogenic factor inhibits angiogenesis in mouse peritubular endothelial cells. Henry Ford Hospital, American Heart Association This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal proximal tubule cells: power and finesse

The proximal tubule is the high-capacity reabsorptive powerhouse of the kidney. Two papers in recent issues of the JCI highlight mechanisms of more delicate effects of the proximal tubule. Yoon et al. demonstrated the intracellular mechanism by which parathyroid hormone (PTH) increases production of 1,25-vitamin D. Activation of PTH receptor 1/cAMP/PKA signaling inhibited salt-inducible kinase 2 (SIK2) and SIK3, which increased CYB27B1 transcription and 1,25-vitamin D production. Replication of these effects with small-molecule SIK inhibitors suggests possible therapeutic applications for patients with disorders characterized by 1,25-vitamin D deficiency. Zhou et al. discovered that proximal tubule glycolysis acts as a phosphate sensor that regulates fibroblast growth factor 23 production in bone. They described several kidney-specific metabolic modifications that enabled glycolysis to be deployed as a phosphate sensor. The provocative results raise intriguing questions with implications for patients with disorders of phosphate homeostasis, including chronic kidney disease.

Megalin, cubilin, and Dab2 drive endocytic flux in kidney proximal tubule cells.

The kidney proximal tubule (PT) elaborates a uniquely high-capacity apical endocytic pathway to retrieve albumin and other proteins that escape the glomerular filtration barrier. Megalin and cubilin/amnionless (CUBAM) receptors engage Dab2 in these cells to mediate clathrin-dependent uptake of filtered ligands. Knockout of megalin or Dab2 profoundly inhibits apical endocytosis and is believed to atrophy the endocytic pathway. We generated CRISPR/Cas9 knockout (KO) clones lacking cubilin, megalin, or Dab2 expression in highly differentiated PT cells and determined the impact on albumin internalization and endocytic pathway function. KO of each component had different effects on the concentration dependence of albumin uptake as well its distribution within PT cells. Reduced uptake of a fluid phase marker was also observed, with megalin KO cells having the most dramatic decline. Surprisingly, protein levels and distribution of key endocytic proteins were preserved in KO PT cell lines and in megalin KO mice, despite the reduced endocytic activity. Our data highlight specific functions of megalin, cubilin, and Dab2 in apical endocytosis and demonstrate that these proteins drive endocytic flux without compromising the physical integrity of the apical endocytic pathway. Our studies suggest a novel model to explain how these components coordinate endocytic uptake in PT cells.

Single-cell RNA sequencing reveals the transcriptomic landscape of kidneys in patients with ischemic acute kidney injury.

Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys. In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining. 15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI. Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.