Albumin Endocytosis Research Articles

The angiotensin II/type 1 angiotensin II receptor pathway is implicated in the dysfunction of albumin endocytosis in renal proximal tubule epithelial cells induced by high glucose levels

It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10−7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.

Renal handling of albumin in rats with early stage diabetes: A theoretical analysis.

In early diabetic nephropathy (DN), recent studies have shown that albuminuria stems mostly from alterations in tubular function rather than from glomerular damage. Several factors in DN, including hyperfiltration, hypertrophy and reduced abundance of the albumin receptors megalin and cubilin, affect albumin endocytosis in the proximal tubule (PT). To assess their respective contribution, we developed a model of albumin handling in the rat PT that couples the transport of albumin to that of water and solutes. Our simulations suggest that, under basal conditions, ∼75% of albumin is retrieved in the S1segment. The model predicts negligible uptake in S3, as observed experimentally. It also accurately predicts the impact of acute hyperglycaemia on urinary albumin excretion. Simulations reproduce observed increases in albumin excretion in early DN by considering the combined effects of increased glomerular filtration rate (GFR), osmotic diuresis, hypertrophy, and megalin and cubilin downregulation, without stipulating changes in glomerular permselectivity. The results indicate that in isolation, glucose-elicited osmotic diuresis and glucose transporter upregulation raise albumin excretion only slightly. Enlargement of PT diameter not only augments uptake via surface area expansion, but also reduces fluid velocity and thus shear stress-induced stimulation of endocytosis. Overall, our model predicts that downregulation of megalin and cubilin and hyperfiltration both contribute significantly to increasing albumin excretion in rats with early-stage diabetes. The results also suggest that acute sodium-glucose cotransporter 2 inhibition lowers albumin excretion only if GFR decreases sufficiently, and that angiotensin II receptor blockers mitigate urinary albumin loss in early DN in large part by upregulating albumin receptor abundance. KEY POINTS: The urinary excretion of albumin is increased in early diabetic nephropathy (DN). It is difficult to experimentally disentangle the multiple factors that affect the renal handling of albumin in DN. We developed a mathematical model of albumin transport in the rat proximal tubule (PT) to examine the impact of elevated plasma glucose, hyperfiltration, PT hypertrophy and reduced abundance of albumin receptors on albumin uptake and excretion in DN. Our model predicts that glucose-elicited osmotic diuresis per se raises albumin excretion only slightly. Conversely, increases in PT diameter and length favour reduced albumin excretion. Our results suggest that downregulation of the receptors megalin and cubilin in PT cells and hyperfiltration both contribute significantly to increasing albumin excretion in DN. The model helps to better understand the mechanisms underlying urinary loss of albumin in early-stage diabetes, and the impact of specific treatments thereupon.

A multi-trait GWAS identifies novel genes influencing albuminuria.

Albuminuria is common and associated with increased risks of end-stage kidney disease and cardiovascular diseases, yet its underlying mechanism remains obscure. Previous genome-wide association studies (GWAS) for albuminuria did not consider gene pleiotropy and primarily focused on European ancestry populations. This study adopted a multi-trait analysis of GWAS (MTAG) approach to jointly analyze two vital kidney traits, estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR) to identify and prioritize the genes associated with UACR. Data from the Taiwan Biobank from 2012 to 2023 were analyzed. GWAS of UACR and eGFR were performed separately and the summary statistics from these GWAS were jointly analyzed using MTAG. The polygenic risk scores (PRS) of UACR were constructed for validation. The UACR-associated loci were further fine-mapped and prioritized based on their deleteriousness, eQTL associations, and relatedness to Mendelian kidney diseases. MTAG analysis of the UACR revealed 15 genetic loci, including 12 novel loci. The PRS for UACR was significantly associated with urinary albumin level (P<0.001) and microalbuminuria (P=0.001 ∼ 0.045). A list of priority genes was generated. Twelve genes with high priority included the albumin endocytic receptor gene LRP2 and ciliary genes IFT172. The findings of this multi-trait GWAS suggest that primary cilia play a role in sensing mechanical stimuli, leading to albumin endocytosis. The priority list of genes warrants further translational investigation to reduce albuminuria.

Altered platelet-megakaryocyte endocytosis and trafficking of albumin and fibrinogen in RUNX1 haplodeficiency

AbstractPlatelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1-haplodeficiency.

Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins.

Proximal tubular epithelial cells (PTEC) are constantly exposed to potentially toxic metabolites and xenobiotics. The regenerative potential of the kidney enables the replacement of damaged cells either via the differentiation of stem cells or the re-acquisition of proliferative properties of the PTEC. Nevertheless, it is known that renal function declines, suggesting that the deteriorated cells are not replaced by fully functional cells. To understand the possible causes of this loss of kidney cell function, it is crucial to understand the role of toxins during the regeneration process. Therefore, we investigated the sensitivity and function of human induced pluripotent stem cells (hiPSC), hiPSC differentiating, and hiPSC differentiated into proximal tubular epithelial-like cells (PTELC) to known nephrotoxins. hiPSC were differentiated into PTELC, which exhibited similar morphology to PTEC, expressed prototypical PTEC markers, and were able to undergo albumin endocytosis. When treated with two nephrotoxins, hiPSC and differentiating hiPSC were more sensitive to cisplatin than differentiated PTELC, whereas all stages were equally sensitive to cyclosporin A. Both toxins also had an inhibitory effect on albumin uptake. Our results suggest a high sensitivity of differentiating cells towards toxins, which could have an unfavorable effect on regenerative processes. To study this, our model of hiPSC differentiating into PTELC appears suitable.

RUNX1 Haplodeficiency Reduces Platelet Endocytosis of Albumin and Fibrinogen and Impairs Megakaryocyte Intracellular Trafficking

Introduction:RUNX1 is a major hematopoietic transcription factor that regulates megakaryopoiesis and platelet formation and function. Germline RUNX1 haplodeficiency (RHD) is characterized by thrombocytopenia, impaired platelet function and granule deficiencies. Our previous studies in a patient with RUNX1 mutation showed that platelet albumin and fibrinogen were decreased (Sun et al, Blood 103: 948-54, 2004) .Human platelet α-granules contain numerous proteins, some synthesized (e.g., PF4 and VWF) and others (e.g., fibrinogen, albumin and IgG) are incorporated by endocytosis, a process poorly understood in platelets/megakaryocytes (MK). Methods: To understand the mechanisms leading to the decreased platelet albumin and fibrinogen, we performed studies on endocytosis in platelets from RHD patients, megakaryocytic HEL cells and MK differentiated from CD34 + hematopoietic stem and progenitor cells (HSPCs). We studied endocytosis of fluorescent-labeled albumin, fibrinogen and IgG in platelet suspensions (0-90 min) and in PMA-treated megakaryocytic HEL cells (up to 24 hrs) using flow cytometry and immunofluorescence microscopy, and assessed proteins linked to endocytosis. Results:Platelet uptake of albumin, fibrinogen and IgG was time- and concentration dependent. Uptake of all 3 was decreased in two patients (father and daughter) with RHD (c.352-1 G&amp;gt;T) (mean fluorescent intensity &amp;lt;50% of normal). In HEL cells, uptake of albumin and fibrinogen was time- and concentration-dependent. On siRNA RUNX1 knockdown (KD), uptake/retention of albumin and fibrinogen over 24 hrs was increased compared to control siRNA cells. Caveolin-1 and flotillin-1, two caveolae-associated membrane proteins linked to endocytosis, and LAMP2 (linked to lysosomal trafficking) and RAB11 (linked to recycling endosomes) were increased on RUNX1 KD on immunoblotting. Caveolin -1 downregulation (siRNA) in RUNX1-deficient HEL cells abrogated the increase in uptake of albumin, but not fibrinogen. At 30 min, in control cells albumin was strongly colocalized with caveolin-1 on immunofluorescence studies. This colocalization was abolished on CAV-1 KD. With RUNX1 KD there was a decrease in colocalization with caveolin-1, and an increase with flotillin-1 both at 30 (Figure 1a) and 120 min. Thus, RUNX1 KD alters endocytosis and trafficking of albumin from caveolin-1 to flotillin-1 bearing vesicles. In experiments where HEL cells were incubated in medium without albumin for 24 hours, cell albumin decreased over time in both control cells and RUNX1-deficient cells with lower levels in the latter. LAMP2 levels were increased on RUNX1 KD, which may provide a mechanism (lysosomal degradation) for this. With respect to fibrinogen, at 30 or 120 min, there was no colocalization of fibrinogen with caveolin-1 or flotillin-1, indicating fibrinogen uptake is not caveolin-dependent. In control cells, there was low/moderate fibrinogen colocalization with RAB11 (marker for recycling endosomes); this was markedly increased with RUNX1 KD (Figure 1b), along with increased RAB11 level on immunoblotting, suggesting that RUNX1 KD is associated with altered fibrinogen trafficking towards recycling endosomes. Recently, IFITM3 has been implicated in platelet fibrinogen uptake in nonviral sepsis. We found HEL cell IFITM3 was increased on RUNX1 KD; however, IFITM3 siRNA KD did not decrease fibrinogen uptake. We studied endocytosis in in vitro differentiated MKs for 12 days from human CD34 + HSPCs; upon lentiviral shRNA KD of RUNX1 the uptake of albumin and fibrinogen was increased compared to control cells . Conclusions: We provide the first evidence that 1) platelet endocytosis of albumin and fibrinogen is impaired in RHD and 2) RUNX1-deficient megakaryocytic cells have enhanced endocytosis but defective trafficking of albumin and fibrinogen, mediated by distinct mechanisms, and associated with increases in caveolin 1, flotillin-1, RAB11, LAMP2 and IFITM3. These studies provide new insights into mechanisms governing endocytosis by platelets and MKs relevant to the α-granule deficiencies in RHD.

Receptor-associated protein impairs ligand binding to megalin and megalin-dependent endocytic flux in proximal tubule cells.

Proximal tubule (PT) cells retrieve albumin and a broad array of other ligands from the glomerular ultrafiltrate. Efficient uptake of albumin requires PT expression of both megalin and cubilin receptors. Although most proteins engage cubilin selectively, megalin is required to maintain robust flux through the apical endocytic pathway. Receptor-associated protein (RAP) is a chaperone that directs megalin to the cell surface, and recombinant RAP dramatically inhibits the uptake of numerous megalin and cubilin ligands. The mechanism by which this occurs has been suggested to involve competitive inhibition of ligand binding and/or conformational changes in megalin that prevent interaction with ligands and/or with cubilin. To discriminate between these possibilities, we determined the effect of RAP on endocytosis of albumin, which binds to cubilin and megalin receptors with high and low affinity, respectively. Uptake was quantified in opossum kidney (OK) cells and in megalin or cubilin (Cubn) knockout (KO) clones. Surprisingly, RAP inhibited fluid-phase uptake in addition to receptor-mediated uptake in OK cells and Cubn KO cells but had no effect on endocytosis when megalin was absent. The apparent Ki for RAP inhibition of albumin uptake was 10-fold higher in Cubn KO cells compared with parental OK cells. We conclude that in addition to its predicted high-affinity competition for ligand binding to megalin, the primary effect of RAP on PT cell endocytosis is to globally dampen megalin-dependent endocytic flux. Our data explain the complex effects of RAP on binding and uptake of filtered proteins and reveal a novel role in modulating endocytosis in PT cells.NEW & NOTEWORTHY Receptor-associated protein inhibits binding and uptake of all known endogenous ligands by megalin and cubilin receptors via unknown mechanism(s). Here, we took advantage of recently generated knockout cell lines to dissect the effect of this protein on megalin- and cubilin-mediated endocytosis. Our study reveals a novel role for receptor-associated protein in blocking megalin-stimulated endocytic uptake of fluid-phase markers and receptor-bound ligands in proximal tubule cells in addition to its direct effect on ligand binding to megalin receptors.

Rapamycin treatment induces tubular proteinuria: role of megalin-mediated protein reabsorption.

Introduction: Rapamycin is an immunosuppressor that acts by inhibiting the serine/threonine kinase mechanistic target of rapamycin complex 1. Therapeutic use of rapamycin is limited by its adverse effects. Proteinuria is an important marker of kidney damage and a risk factor for kidney diseases progression and has been reported in patients and animal models treated with rapamycin. However, the mechanism underlying proteinuria induced by rapamycin is still an open matter. In this work, we investigated the effects of rapamycin on parameters of renal function and structure and on protein handling by proximal tubule epithelial cells (PTECs). Methods: Healthy BALB/c mice were treated with 1.5mg/kg rapamycin by oral gavage for 1, 3, or 7days. At the end of each treatment, the animals were kept in metabolic cages and renal function and structural parameters were analyzed. LLC-PK1 cell line was used as a model of PTECs to test specific effect of rapamycin. Results: Rapamycin treatment did not change parameters of glomerular structure and function. Conversely, there was a transient increase in 24-h proteinuria, urinary protein to creatinine ratio (UPCr), and albuminuria in the groups treated with rapamycin. In accordance with these findings, rapamycin treatment decreased albumin-fluorescein isothiocyanate uptake in the renal cortex. This effect was associated with reduced brush border expression and impaired subcellular distribution of megalin in PTECs. The effect of rapamycin seems to be specific for albumin endocytosis machinery because it did not modify renal sodium handling or (Na++K+)ATPase activity in BALB/c mice and in the LLC-PK1 cell line. A positive Pearson correlation was found between megalin expression and albumin uptake while an inverse correlation was shown between albumin uptake and UPCr or 24-h proteinuria. Despite its effect on albumin handling in PTECs, rapamycin treatment did not induce tubular injury measured by interstitial space and collagen deposition. Conclusion: These findings suggest that proteinuria induced by rapamycin could have a tubular rather than a glomerular origin. This effect involves a specific change in protein endocytosis machinery. Our results open new perspectives on understanding the undesired effect of proteinuria generated by rapamycin.

Fetal Reprogramming of Nutrient Surplus Signaling, O-GlcNAcylation, and the Evolution of CKD.

Fetal kidney development is characterized by increased uptake of glucose, ATP production by glycolysis, and upregulation of mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1 alpha (HIF-1 α ), which (acting in concert) promote nephrogenesis in a hypoxic low-tubular-workload environment. By contrast, the healthy adult kidney is characterized by upregulation of sirtuin-1 and adenosine monophosphate-activated protein kinase, which enhances ATP production through fatty acid oxidation to fulfill the needs of a normoxic high-tubular-workload environment. During stress or injury, the kidney reverts to a fetal signaling program, which is adaptive in the short term, but is deleterious if sustained for prolonged periods when both oxygen tension and tubular workload are heightened. Prolonged increases in glucose uptake in glomerular and proximal tubular cells lead to enhanced flux through the hexosamine biosynthesis pathway; its end product-uridine diphosphate N -acetylglucosamine-drives the rapid and reversible O-GlcNAcylation of thousands of intracellular proteins, typically those that are not membrane-bound or secreted. Both O-GlcNAcylation and phosphorylation act at serine/threonine residues, but whereas phosphorylation is regulated by hundreds of specific kinases and phosphatases, O-GlcNAcylation is regulated only by O-GlcNAc transferase and O-GlcNAcase, which adds or removes N-acetylglucosamine, respectively, from target proteins. Diabetic and nondiabetic CKD is characterized by fetal reprogramming (with upregulation of mTOR and HIF-1 α ) and increased O-GlcNAcylation, both experimentally and clinically. Augmentation of O-GlcNAcylation in the adult kidney enhances oxidative stress, cell cycle entry, apoptosis, and activation of proinflammatory and profibrotic pathways, and it inhibits megalin-mediated albumin endocytosis in glomerular mesangial and proximal tubular cells-effects that can be aggravated and attenuated by augmentation and muting of O-GlcNAcylation, respectively. In addition, drugs with known nephroprotective effects-angiotensin receptor blockers, mineralocorticoid receptor antagonists, and sodium-glucose cotransporter 2 inhibitors-are accompanied by diminished O-GlcNAcylation in the kidney, although the role of such suppression in mediating their benefits has not been explored. The available evidence supports further work on the role of uridine diphosphate N -acetylglucosamine as a critical nutrient surplus sensor (acting in concert with upregulated mTOR and HIF-1 α signaling) in the development of diabetic and nondiabetic CKD.

405-P: ROCK2 Activation and Its Role in Folic Acid–Induced Murine Model of Nephropathy

Over 2 million people currently receive treatment with dialysis or a kidney transplant throughout the world. Thus, developing a new strategy to treat chronic kidney diseases is significant from the perspective of a health economic and the prevention of cardiovascular death. The small GTPase Rho and its effector Rho-kinase (ROCK) are involved in the pathogenesis of diabetic nephropathy. In chronic kidney diseases including diabetic nephropathy, tubulointerstitial fibrosis is known to be highly associated with renal outcome. We have previously reported that ROCK inhibition prevented the progression of diabetic nephropathy in murine models. With regards to ROCK, there are two isoforms, which are ROCK1 and ROCK2. ROCK1 has been shown to be involved in the endocytosis of albumin in tubular epithelial cells via megalin/cubilin-dependent mechanism. However, the role of renal tubular ROCK2 remains to be elucidated. The aim of this study is to reveal the role of renal tubular ROCK2 in the pathogenesis of chronic kidney disease. To this end, we used a tubulointerstitial injury model known as folic acid-induced nephropathy (FAN). Importantly, ROCK2 was elevated at protein levels in FAN group compared with vehicle-treated group. We next generated tubular specific ROCK2 knockout mouse (TR2KO) by crossing Ksp-Cre mice with mice containing two loxP sites flanking in Exon 3 of ROCK2. In the Ksp-Cre transgenic mice, Cre recombinase has been demonstrated to be expressed in tubules, collecting ducts, and thick ascending limbs. Compared with vehicle-treated group, collagen deposition in the kidneys of FAN group was highly elevated as revealed by Masson’s trichrome staining. The increased collagen deposition was significantly improved in TR2KO. Our findings indicate that ROCK2 regulates the progression of tubulointerstitial fibrosis. ROCK2 may be a potential therapeutic target for the prevention of chronic kidney disease. Disclosure K.Sekiguchi: None. K.Matoba: None. S.Ohashi: None. E.Mitsuyoshi: None. Y.Nagai: None. Y.Takeda: None. R.Nishimura: Speaker's Bureau; Sanofi K.K., Medicure Inc., Boehringer Ingelheim Inc., Takeda Pharmaceutical Company Limited, Kissei Pharmaceutical Co., Ltd., Novartis Pharma K.K., Eli Lilly Japan K.K., Novo Nordisk, Merck &amp; Co., Inc., Abbott Japan Co., Ltd., Astellas Pharma Inc., Teijin Pharma Limited, Taisho Pharmaceutical Holdings Co., Ltd., Ono Pharmaceutical Co., Ltd., AstraZeneca.

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.

Tracing causes of tubule atrophy in vivo

Acute kidney injury (AKI) is a known risk factor for chronic kidney disease (CKD). The mechanisms for AKI-CKD transition may involve failed tubule repair and progressive interstitial fibrosis but are incompletely understood. Which events lead to failed tubule repair? Can injury spread over time? And are these processes steered by myofibroblasts and fibrotic tissue remodeling?Here, we combined serial intravital 2-photon microscopy and a novel model of partial ischemia-reperfusion injury (partial IRI), which allowed us to investigate injured tissue alongside uninjured tissue in the same kidney and over time. Thus, we occluded only one branch of the left renal artery for 21 min, rendering half the kidney ischemic. We then tracked tubule necrosis and remodeling alongside myofibroblast dynamics for up to 3 weeks. Necrotic cell death was detected by in vivo propidium iodide staining. PDGFRβ-CreERT2-Salsa6F mice (N=5) identified main myofibroblast precursor cells, by tdTomato-expression. Myofibroblast-differentiation was confirmed by ex vivo αSMA staining. Proximal tubule (PT) function was assessed by in vivo uptake of fluorescent albumin.Necrotic cell death was solely detectable upon reperfusion. Border areas in between post-ischemic and non-ischemic tissue presented severely necrotic PTs alongside uninjured PTs. From 1 day after partial IRI, we observed formation of granular casts in the lumen of necrotic tubules, which 1-2 days later also appeared in initially non-necrotic PTs downstream. Tubule necrosis and granular cast accumulation preceded thinned epithelium that lacked albumin endocytosis (day 3-7) and recruitment of myofibroblasts (peaking D4-10). Of note, non-necrotic tubules, without granular cast accumulation, didn’t remodel nor recruit myofibroblasts.Among all remodeling tubule segments, 24% turned atrophic. Importantly, preceding necrotic cell death was higher in atrophic than in recovering tubules (55±13% vs. 39±18% necrotic cells/total segment cell number, (mean±SD), p&lt;.0001). Preceding granular casts accounted for 38±15% vs. 12±11% (mean ±SD) of the luminal area in atrophic and recovering tubules, respectively (p&lt;.0001). Ex vivo staining for pro-fibrotic VCAM-1 demonstrated selective positivity in atrophic tubules, which appeared scattered at D8 while at D21, entire atrophic tubules stained positive (n=3, each). Consistently, we detected persistent myofibroblast enclosure of atrophic tubule, while recruitment around recovered PTs resolved by day 14 (50±21% vs. 23±18% of coverage, p&lt;.001).Tracking the same tubules in vivo and over time, we demonstrated early proximal tubule necrosis, which induced luminal granular cast formation and injury propagation. These events preceded myofibroblast accumulation and predicted tubule atrophy. We further documented de novo expression of pro-inflammatory VCAM-1 and persistent myofibroblast recruitment selectively in atrophic tubules, suggesting an active role of this tubule state in AKI-CKD transition. Novo Nordisk Foundation, Augustinus Foundation, Aarhus University Research Foundation (AUFF) 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.

Comparison of Three Transcytotic Pathways for Distribution to Brain Metastases of Breast Cancer.

Advances in drug treatments for brain metastases of breast cancer have improved progression free survival but new, more efficacious strategies are needed. Most chemotherapeutic drugs infiltrate brain metastases by moving between brain capillary endothelial cells, paracellular distribution, resulting in heterogeneous distribution, lower than that of systemic metastases. Herein, we tested three well-known transcytotic pathways through brain capillary endothelial cells as potential avenues for drug access: Transferrin receptor (TfR) peptide, Low density lipoprotein receptor 1 (LRP1) peptide, Albumin. Each was far-red labeled, injected into two hematogenous models of brain metastases, circulated for two different times, and their uptake quantified in metastases and uninvolved (nonmetastatic) brain. Surprisingly, all three pathways demonstrated distinct distribution patterns in vivo. Two were suboptimal: TfR distributed to uninvolved brain but poorly in metastases, while LRP1 was poorly distributed. Albumin distributed to virtually all metastases in both model systems, significantly greater than in uninvolved brain (P <0.0001). Further experiments revealed that albumin entered both macrometastases and micrometastases, the targets of treatment and prevention translational strategies. Albumin uptake into brain metastases was not correlated with the uptake of a paracellular probe (biocytin). We identified a novel mechanism of albumin endocytosis through the endothelia of brain metastases consistent with clathrin-independent endocytosis (CIE), involving the neonatal Fc receptor (FcRn), galectin-3 (Gal-3) and glycosphingolipids. Components of the CIE process were found on metastatic endothelial cells in human craniotomies. The data suggest a reconsideration of albumin as a translational mechanism for improved drug delivery to brain metastases and possibly other CNS cancers.

Gold nanoparticles reduce tubule-interstitial injury and proteinuria in a murine model of subclinical acute kidney injury

Subclinical acute kidney injury (subAKI) is characterized by tubule-interstitial injury without significant changes in glomerular function. SubAKI is associated with the pathogenesis and progression of acute and chronic kidney diseases. Currently, therapeutic strategies to treat subAKI are limited. The use of gold nanoparticles (AuNPs) has shown promising benefits in different models of diseases. However, their possible effects on subAKI are still unknown. Here, we investigated the effects of AuNPs on a mouse model of subAKI. Animals with subAKI showed increased functional and histopathologic markers of tubular injury. There were no changes in glomerular function and structure. The animals with subAKI also presented an inflammatory profile demonstrated by activation of Th1 and Th17 cells in the renal cortex. This phenotype was associated with decreased megalin-mediated albumin endocytosis and expression of proximal tubular megalin. AuNP treatment prevented tubule-interstitial injury induced by subAKI. This effect was associated with a shift to an anti-inflammatory Th2 response. Furthermore, AuNP treatment preserved megalin-mediated albumin endocytosis in vivo and in vitro. AuNPs were not nephrotoxic in healthy mice. These results suggest that AuNPs have a protective effect in the tubule-interstitial injury observed in subAKI, highlighting a promising strategy as a future antiproteinuric treatment.

Megalin Orchestrates FcRn Endocytosis and Trafficking.

The neonatal Fc receptor (FcRn) is highly expressed in the renal proximal tubule and is important for the reclamation of albumin by cellular transcytosis to prevent its loss in the urine. The initial event of this transcellular transport mechanism is the endocytosis of albumin by the apical scavenger receptors megalin and cubilin. An interaction of megalin and FcRn was postulated, however, evidence is still missing. Similarly, the intracellular trafficking of FcRn remains unknown and shall be identified in our study. Using a Venus-based bimolecular fluorescence complementation system, we detected an interaction between megalin and FcRn in the endosomal compartment, which significantly increased with the induction of endocytosis using albumin or lactoglobulin as a ligand. The interaction between megalin and FcRn occurred at a neutral and acidic pH between the extracellular domains of both proteins. Amnionless, another transmembrane acceptor of cubilin, revealed no interaction with FcRn. With the induction of endocytosis by albumin or lactoglobulin, super resolution microscopy demonstrated a redistribution of megalin and FcRn into clathrin vesicles and early endosomes. This trafficking into clathrin vesicles was impaired in megalin-deficient cells upon albumin-induced endocytosis, supporting the role of megalin in FcRn redistribution. Our results indicate that megalin and FcRn specifically bind and interact within their extracellular domains. The availability of megalin is necessary for the redistribution of FcRn. Megalin, therefore, orchestrates FcRn endocytosis and intracellular trafficking as an early event intranscytosis.

Beta-Hydroxybutyric Acid Inhibits Renal Tubular Reabsorption via the AKT/DAB2/Megalin Signalling Pathway.

Patients with diabetic ketosis often exhibit albuminuria. We previously found that acute hyperglycaemia can cause nephrotoxic injury. Here, we explored whether an excessive ketone body level causes kidney injury and the potential underlying mechanism. Fifty-six type 2 diabetes without ketosis (NDK group), 81 type 2 diabetes with ketosis (DK group), and 38 healthy controls (NC group) were enrolled. Clinical data were collected before and after controlling diabetic ketosis. Beta-hydroxybutyric acid (BOHB), an AKT activator, an AKT inhibitor, or plasmids encoding DAB2 were transformed into human renal proximal tubule epithelial cells (HK-2 cells). The urinary albumin-to-creatinine ratio (ACR), transferrin (TF), immunoglobulin G (IgG), Beta2-microglobulin (β2-MG), retinol-binding protein (RBP), N-acetyl-beta-glucosaminidase (NAG), and Beta-galactosidase (GAL) were higher in the DK than NC and NDK groups. The proportion of patients with an increased urinary level of TF, IgG, β2-MG, RBP, NAG, or GAL was higher in the DK group too. After controlling ketosis, urinary microalbumin, TF, IgG, β2-MG, and RBP decreased significantly. In HK-2 cells, albumin endocytosis and megalin expression decreased with increasing BOHB concentration. Compared with BOHB treatment, BOHB with AKT activator significantly increased the DAB2, megalin levels and albumin endocytosis; the AKT inhibitor treatment exhibited the opposite effects. Compared with BOHB treatment, megalin expression and albumin endocytosis were significantly increased after BOHB with DAB2 overexpression treatment. Patients with diabetic ketosis may suffer from glomerular and tubular injuries that recover after ketosis control. High concentrations of BOHB downregulate megalin expression by inhibiting the AKT/DAB2/megalin signalling pathway and albumin endocytosis in proximal renal tubules.

Hyperinsulinemia impairs the metabolic switch to ketone body utilization in proximal renal tubular epithelial cells under energy crisis via the inhibition of the SIRT3/SMCT1 pathway.

ObjectiveTo investigate the effects and mechanism of hyperinsulinemia on the metabolic switch to β‐hydroxybutyrate (BHB) absorption and utilization under a starvation or hypoxic environment in proximal tubular epithelial cells.MethodsA high-fat diet-induced hyperinsulinemia model in ZDF rats was used to test the expression of key enzymes/proteins of ketone body metabolism in the kidney. Notably, 12-week-old renal tubule SMCT1 specific knockout mice (SMCT1 flox/floxCre+) and control mice (SMCT1 flox/floxCre-) were used to confirm the roles of SMCT1 in kidney protection under starvation. The changes of key enzymes/proteins of energy metabolism, mitochondrial function, and albumin endocytosis in HK2 cells under low glucose/hypoxic environments with or without 50 ng/mL insulin were studied. Silent information regulation 2 homolog 3 (SIRT3) was overexpressed to evaluate the effect of hyperinsulinemia on the metabolic switch to BHB absorption and utilization through the SIRT3/SMCT1 pathway in HK2 cells.ResultsIn ZDF rats, the expression of HMGCS2 increased, the SMCT1 expression decreased, while SCOT remained unchanged. In renal tubule SMCT1 gene-specific knockout mice, starvation for 48 h induced an increase in the levels of urine retinol-binding protein, N-acetyl-β-glucosaminidase, and transferrin, which reflected tubular damages. In HK2 cells under an environment of starvation and hypoxia, the levels of key enzymes related to fatty acid oxidation and ketone body metabolism were increased, whereas glucose glycolysis did not change. The addition of 2 mmol/l BHB improved ATP production, mitochondrial biosynthesis, and endocytic albumin function, while cell apoptosis was reduced in HK2 cells. The addition of 50 ng/ml insulin resulted in the decreased expression of SMCT1 along with an impaired mitochondrial function, decreased ATP production, and increased apoptosis. The overexpression of SIRT3 or SMCT1 reversed these alterations induced by a high level of insulin both in low-glucose and hypoxic environments.ConclusionsThe increased absorption and utilization of BHB is part of the metabolic flexibility of renal tubular epithelial cells under starvation and hypoxic environments, which exhibits a protective effect on renal tubular epithelial cells by improving the mitochondrial function and cell survival. Moreover, hyperinsulinemia inhibits the absorption of BHB through the inhibition of the SIRT3/SMCT1 pathway.

TMBIM6 promotes diabetic tubular epithelial cell survival and albumin endocytosis by inhibiting the endoplasmic reticulum stress sensor, IRE1α

Reduced albumin reabsorption in proximal tubular epithelial cells (PTECs), resulting from decreased megalin plasma membrane (PM) localization due to prolonged endoplasmic reticulum (ER) stress, potentially contributes to albuminuria in early diabetic kidney disease (DKD). To examine this possibility, we investigated the cytoprotective effect of TMBIM6 in promoting diabetic PTEC survival and albumin endocytosis by attenuating ER stress with an IRE1α inhibitor, KIRA6. Renal TMBIM6 distribution and expression were determined by immunohistochemistry, western blotting, and qPCR, whereas tubular injury was evaluated in db/db mice. High-glucose (HG)-treated HK-2 cells were either treated with KIRA6 or transduced with a lentiviral vector for TMBIM6 overexpression. ER stress was measured by western blotting and ER-Tracker Red staining, whereas apoptosis was determined by performing TUNEL assays. Megalin expression was measured by immunofluorescence, and albumin endocytosis was evaluated after incubating cells with FITC-labeled albumin. Tubular injury and TMBIM6 downregulation occurred in db/db mouse renal cortical tissues. Both KIRA6 treatment and TMBIM6 overexpression inhibited ER stress by decreasing the levels of phosphorylated IRE1α, XBP1s, GRP78, and CHOP, and stabilizing ER expansion in HG-treated HK-2 cells. TUNEL assays performed with KIRA6-treated or TMBIM6-overexpressing cells showed a significant decrease in apoptosis, consistent with the significant downregulation of BAX and upregulation of BCL-2, as measured by immunoblotting. Both KIRA6 and TMBIM6 overexpression promoted megalin PM localization and restored albumin endocytosis in HG-treated HK-2 cells. TMBIM6 promoted diabetic PTEC survival and albumin endocytosis by negatively regulating the IRE1α branch of ER stress.

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.

388-P: Renal ROCK2 Activity in Folic Acid-Induced Nephropathy

The small GTPase Rho and its effector Rho-kinase (ROCK) are involved in the pathogenesis of diabetic nephropathy. In chronic kidney diseases including diabetic nephropathy, tubulointerstitial fibrosis is closely related to renal outcome. We have previously reported that ROCK inhibition suppressed the progression of diabetic nephropathy in murine models. ROCK has two isoforms, ROCK1 and ROCK2. ROCK1 is known to be involved in the endocytosis of albumin in tubular epithelial cells via megalin/cubilin-dependent mechanism. On the other hand, the role of renal tubular ROCK2 has not been elucidated.In the present study, we aimed to establish the role of renal tubular ROCK2 in the pathogenesis of chronic kidney disease using a tubulointerstitial injury model known as folic acid-induced nephropathy (FAN) . Masson’s trichrome staining showed an elevated collagen deposition in the kidneys of FAN group compared with vehicle-treated group. We detected a relative upregulation of ROCK2 at protein levels in FAN group, compared with vehicle-treated group. Immunoprecipitation assay demonstrated that the kinase activity of ROCK2 relatively increased in FAN group. Based on these findings, we conclude that ROCK2 has an important contribution to tubulointerstitial fibrosis. ROCK2 may become a therapeutic target for the treatment of chronic kidney disease. Disclosure K.Sekiguchi: None. K.Matoba: None. R.Ukichi: None. Y.Nagai: None. Y.Takeda: None. Y.Kanazawa: None. K.Utsunomiya: None. R.Nishimura: Speaker's Bureau; Abbott Diabetes, Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Medtronic, Merck &amp; Co., Inc., Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Holdings Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Terumo Corporation.