Proximal Tubule Injury Research Articles

Proximal Tubule Secretory Clearance, Injury, and Kidney Viability in Cirrhosis.

Cirrhosis affects all structures of the kidney, in particular the tubules which are responsible for secretion of protein-bound metabolites and electrolyte/water homeostasis. Yet, prevailing assessments of kidney function focus solely on glomerular filtration rate (GFR) which may incompletely reflect these processes. We sought to characterize markers of tubular function, injury, and viability in patients with and without cirrhosis. We recruited outpatients undergoing liver transplantation evaluation for a collection of plasma and 24-hour urine, matching by GFR to control participants without cirrhosis. We measured urinary kidney injury molecule-1 (KIM-1), a marker of proximal tubular injury, as well as epidermal growth factor (EGF), a marker of viability necessary for tubular epithelial cell proliferation after injury. We also estimated secretory clearance by measuring several highly secreted endogenous metabolites in urine and plasma. We recruited 39 patients with cirrhosis (mean MELD-Na 17±4, Child-Pugh 8±2, eGFR 66±20 ml/min/1.73m2) and 58 GFR-matched controls without cirrhosis (eGFR 66±21 ml/min/1.73m2). Urinary KIM-1 was 4.4-fold higher than controls (95% CI:2.9-6.5), and EGF averaged 7.41-fold higher than controls (95% CI:2.15-25.53). We found that of 8 solutes, 5 had significantly greater kidney clearance in cirrhosis (1.3-2.1-fold higher): indoxyl sulfate, p-cresol sulfate, pyridoxic acid, tiglylglycine, and xanthosine. Cirrhosis was characterized by molecular signs of tubular injury in stable outpatients without acute kidney injury, accompanied by largely preserved tubular secretory clearance and greater signs of tubular viability. Within the limitations of the study, this suggests a phenotype of chronic ischemic injury but with initial preservation of tubular function in cirrhosis.

Distal convoluted tubule-specific disruption of the COP9 signalosome but not its regulatory target cullin 3 causes tubular injury.

The disease familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome) is caused by aberrant accumulation of with-no-lysine kinase (WNK4) activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We used Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in the DCT and examined the mice after short- and long-term deletion. Short-term DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, pSPAKS373, and pNCCT53 abundance. However, neither model demonstrated changes in plasma K+, Cl-, or total CO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance and a higher abundance of kidney injury molecule-1, a marker of proximal tubule injury. No injury or reduction in NCC or parvalbumin was observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.NEW & NOTEWORTHY CUL3 degrades WNK4, which prevents activation of NCC in the DCT. CSN regulation of CUL3 is impaired in the disease FHHt, causing accumulation of WNK4. Short-term DCT-specific disruption of CUL3 or the CSN in mice resulted in activation of the WNK4-SPAK-NCC pathway but not hyperkalemic metabolic acidosis found in FHHt. Tubule injury was observed only after long-term CSN disruption. The data suggest that disruption of other cullins may be the cause for the injury.

#1333 SGLT2 inhibitors as a treatment for Fanconi-Bickel syndrome: a case report

Abstract Background and Aims Fanconi-Bickel syndrome is an ultra-rare genetic disease characterized by SLC2A2 mutation encoding for the basolateral glucose transporter (GLUT2) in the proximal tubule of the nephron. This defective transport ultimately leads to accumulation of glycogen and dysfunction of proximal tubule cells. This manifests clinically as the renal Fanconi syndrome and eventually kidney failure. Systemic complications of proximal tubule injury include metabolic acidosis, bone demineralization and, for some not completely elucidated reasons, dysglycemia. Method We present the case of a 43-year-old man who was diagnosed with Fanconi-Bickel syndrome (homozygous splice site mutation in SLC2A2). He had a history of infantile-onset renal Fanconi syndrome, hypercalciuria and bilateral nephrolithiasis, osteoporosis, bilateral sensorineural deafness and non-insulin dependent type 2 diabetes. At referral the patient presented with CKD G2, non-anion gap metabolic acidosis, slightly elevated serum calcium, decreased serum phosphate, suppressed PTH and hypercalciuria (Table 1), despite oral supplementation of phosphate, bicarbonate and vitamin D. We hypothesized that SGLT2i in this patient may halt the progressive proximal tubule injury by preventive glucose uptake and help the management of diabetes. Results Fractional excretion of phosphate (FEPO4) and urinary retinol-binding protein (RBP)-creatinine ratio, two markers of proximal tubular injury, were stable after 2 months of SGLT2i therapy (Fig. 1). Conclusion To date, Fanconi-Bickel syndrome does not have a specific treatment. Standard of care includes supplements of fluids and electrolytes to counterbalance proximal tubular injury and management of complications such as diabetes. SGLT2i use has been described in a mouse model of another glycogen storage disease (GSD1b), whose underlying defect is different from that of Fanconi-Bickel but presents with a similar phenotype [2]. Data showed that gliflozins prevents glycogen accumulation and restores proximal tubule cells’ function. Using the same rationale, the administration of SGLT2i in adult patients with Fanconi-Bickel syndrome may prevent further proximal damage and slow the progression towards end-stage kidney disease. In conclusion, we hypothesize a potential benefit of SGLT2i in adult patients affected by Fanconi-Bickel syndrome.

#1284 Proteinuria causes excessive proliferation and senescence in proximal tubular cells via receptor-mediated endocytosis

Abstract Background and Aims Proteinuria induces proximal tubule (PT) injury, oxidative stress, lysosomal dysfunction, and inflammation, which contribute to the progression of CKD. Cellular senescence and the associated secretory phenotype, where cells secrete proinflammatory and profibrotic mediators, have been linked to renal interstitial inflammation and fibrosis. Premature senescence is induced by excessive proliferation, oxidative stress, or DNA injury. In kidneys, hyperglycemia or hypertension have been reported to elicit this response. However, the direct association between proteinuria-induced PT protein overload and cellular senescence is unknown. We aimed to clarify the effect of protein overload on cellular proliferation and senescence in proteinuric mice with or without knockout (KO) of the receptors responsible for protein endocytosis, megalin and cubilin. Method Podocin-KO (proteinuric mouse model) and megalin-cubilin-podocin triple-KO mice were used in study. Kidneys from these mice were analyzed using immunohistochemical and immunofluorescent staining and the mice were injected with EdU to investigate renal proliferation. mRNA expression of different markers in renal cortex were evaluated with quantitative rt-PCR. To investigate albumin induced cellular proliferation and senescence, immortalized proximal tubular cells (RPTEC/TERT1, ATCC) were treated with human serum albumin (HSA; Sigma), fatty acid-free HSA (Sigma), and transferrin (Sigma). Western blotting (WB) and quantitative rt-PCR, of different markers were conducted and staining for senescence-associated beta-galactosidase (SA-β-gal) was performed. Results PCNA, a proliferation marker, was detected at a higher level in PTs of podocin-KO mice compared to wild-type (WT) mice, and triple-KO mice. Furthermore, we evaluated EdU DNA incorporation, in mosaic megalin and cubilin-expressing triple-KO mice. Coimmunostaining of megalin (Fig. 1-a) or cubilin (Fig. 1-b) with incorporated EdU revealed that most of the EdU-positive nuclei was observed in megalin or cubilin-positive tubules (white arrowheads). A few EdU positive nuclei was detected in megalin or cubilin-negative tubules or the interstitium (yellow arrowheads). The mRNA expression of PDGF- β, a growth factor, was more upregulated in the cortex of podocin-KO mice than in triple-KO mice. These data indicate that protein uptake via endocytic receptors induces secretion of PDGF-β from PT cells, acting in a paracrine manner potentially inducing proliferation. Additionally, immunofluorescent staining revealed that senescence markers, p21 and γ-H2AX, were detected in PTs of podocin-KO mice at a higher level than in triple-KO mice. The mRNA expression of TGF-β (profibrotic) and MCP-1 (proinflammatory) were higher in podocin-KO, than in triple-KO mice in accordance with more senescent cells in podocin KO mice. Upregulation of p21, p16, and γ-H2AX as well as positive staining for SA-β-gal in RPTEC/TERT1 cells, were only detected in HSA (containing free fatty acids)-treated cells in a dose dependent manner and not in cells treated with HSA depleted for free fatty acids or transferrin. Our results show that excessive protein reabsorption generates senescent cells in proximal tubules potentially through increased proliferation elicited by paracrine signaling. The increase in senescent cells was associated to profibrotic and proinflammatory signaling in line with a senescent secretory phenotype. Our studies in PRTEC/TERT1 confirmed our animal experiments and showed that senescence was induced only by fatty acid bound albumin. Conclusion The present study showed that proteinuria directly induced cellular senescence in PTECs via megalin/cubilin endocytosis of filtered protein. Fatty acid bound-albumin is suggested to cause the cellular senescence.

Inhibition of SGL2 Prevents Albumin Induced Proximal Tubule Inflammation

Albuminuria is an independent risk factor for cardiovascular disease and renal damage in hypertension. It is associated with a higher risk of cardiovascular morbidity and mortality, and with declining kidney function. The Na+-glucose cotransporter-2 (SGLT2) plays a crucial role in glucose and sodium reabsorption in the proximal tubule. Clinical trials have shown that SGLT2 inhibitors can reduce cardiovascular events, lower blood pressure, and decrease the risk of end-stage kidney disease in individuals with type 2 diabetes mellitus. These trials have also demonstrated the potential of SGLT2 inhibitors to provide additional benefits beyond their glucose control, including decreased albuminuria. In this study, we hypothesized that exposure to elevated albumin causes proximal tubule injury and the release of cytokines from the basolateral membrane. We propose that by reducing transport in the proximal tubule, SGLT2 inhibitors may alleviate kidney damage by reducing cytokine production and subsequently renal inflammation. To test our hypothesis, we conducted experiments using Dahl salt-sensitive (SS) rats and immortalized human proximal tubule cells (RPTEC-TERT1). To determine how early albuminuria and proximal injury occur in the development of salt-sensitive hypertension, 24-hour urine was collected from SS rats fed either a control salt (CS, 0.4% NaCl, n=10) or a high salt (HS, 4.0% NaCl, n=11) diet for 7 days. Significant increases in albuminuria were observed after 1 day of HS (CS: 25±7 vs. HS: 59±13 mg/day p=0.016). This was followed by significant increases in urinary kidney injury molecule-1 (Kim-1) after 2 days of HS (CS: 13±2 vs. HS: 23±3 pg/day p=0.021). Preliminary analysis found substantial increases in urinary CCL2 after 4 days of HS (CS: 58±7 vs. HS: 135±25 pg/mg, n=5). To investigate whether the renoprotective effects of SGLT2 inhibitors are in part due to their ability to reduce inflammation in the proximal tubule, we exposed the apical membrane of RPTEC-TERT1 cells to human serum albumin (HSA, 10mg/ml, n=6) with and without Dapagliflozin (Dapa, 15ng/ml, n=5) for 24-hours and measured the levels of CCL2 in the basolateral media. HSA caused a significant increase in CCL2 levels relative to control cells (Control: 42.5±5.6 vs. HSA: 84.1±8.7 pg/mg, p=0.00). This increase was prevented by co-treatment with Dapa and CCL2 levels were equivalent to those in the control cells (HSA+Dapa: 44.0±4.5 pg/mg, p=0.988). In conclusion, our findings support the hypothesis that albuminuria precedes proximal tubule injury and may contribute to the development of renal inflammation in salt-sensitive hypertension. The ability of Dapa to reduce prevent albumin induced CCL2 production in RPTEC-TERT1 cells suggests that SGLT2 inhibitors may reduce renal inflammation in salt-sensitive hypertension and is an area of ongoing studies. Studies funded by R01HL152166 to LE. This is the full abstract presented at the American Physiology Summit 2024 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.

Allogeneic bone marrow mesenchymal stem cell-derived exosomes alleviate human hypoxic AKI-on-a-Chip within a tight treatment window

BackgroundAcute hypoxic proximal tubule (PT) injury and subsequent maladaptive repair present high mortality and increased risk of acute kidney injury (AKI) - chronic kidney disease (CKD) transition. Human bone marrow mesenchymal stem cell-derived exosomes (hBMMSC-Exos) as potential cell therapeutics can be translated into clinics if drawbacks on safety and efficacy are clarified. Here, we determined the real-time effective dose and treatment window of allogeneic hBMMSC-Exos, evaluated their performance on the structural and functional integrity of 3D microfluidic acute hypoxic PT injury platform.MethodshBMMSC-Exos were isolated and characterized. Real-time impedance-based cell proliferation analysis (RTCA) determined the effective dose and treatment window for acute hypoxic PT injury. A 2-lane 3D gravity-driven microfluidic platform was set to mimic PT in vitro. ZO-1, acetylated α-tubulin immunolabelling, and permeability index assessed structural; cell proliferation by WST-1 measured functional integrity of PT.ResultshBMMSC-Exos induced PT proliferation with ED50 of 172,582 µg/ml at the 26th hour. Hypoxia significantly decreased ZO-1, increased permeability index, and decreased cell proliferation rate on 24–48 h in the microfluidic platform. hBMMSC-Exos reinforced polarity by a 1.72-fold increase in ZO-1, restored permeability by 20/45-fold against 20/155 kDa dextran and increased epithelial proliferation 3-fold compared to control.ConclusionsThe real-time potency assay and 3D gravity-driven microfluidic acute hypoxic PT injury platform precisely demonstrated the therapeutic performance window of allogeneic hBMMSC-Exos on ischemic AKI based on structural and functional cellular data. The novel standardized, non-invasive two-step system validates the cell-based personalized theragnostic tool in a real-time physiological microenvironment prior to safe and efficient clinical usage in nephrology.

Zoledronic acid and ibandronate-induced nephrotoxicity in 2D and 3D proximal tubule cells derived from human and rat.

Drug-induced proximal tubule (PT) injury remains a serious safety concern throughout drug development. Traditional in vitro 2-dimensional (2D) and preclinical in vivo models often fail to predict drug-related injuries presented in clinical trials. Various 3-dimensional (3D) microphysiological systems (MPSs) have been developed to mimic physiologically relevant properties, enabling them to be more predictive toward nephrotoxicity. To explore the capabilities of an MPS across species, we compared cytotoxicity in hRPTEC/TERT1s and rat primary proximal tubular epithelial cells (rPPTECs) following exposure to zoledronic acid and ibandronate (62.5-500 µM), and antibiotic polymyxin B (PMB) (50 and 250 µM, respectively). For comparison, we investigated cytotoxicity using 2D cultured hRPTEC/TERT1s and rPPTECs following exposure to the same drugs, including overlapping concentrations, as their 3D counterparts. Regardless of the in vitro model, bisphosphonate-exposed rPPTECs exhibited cytotoxicity quicker than hRPTEC/TERT1s. PMB was less sensitive toward nephrotoxicity in rPPTECs than hRPTEC/TERT1s, demonstrating differences in species sensitivity within both 3D and 2D models. Generally, 2D cultured cells experienced faster drug-induced cytotoxicity compared to the MPSs, suggesting that MPSs can be advantageous for longer-term drug-exposure studies, if warranted. Furthermore, ibandronate-exposed hRPTEC/TERT1s and rPPTECs produced higher levels of inflammatory and kidney injury biomarkers compared to zoledronic acid, indicating that ibandronate induces acute kidney injury, but also a potential protective response since ibandronate is less toxic than zoledronic acid. Our study suggests that the MPS model can be used for preclinical screening of compounds prior to animal studies and human clinical trials.

Canagliflozin ameliorates high glucose-induced apoptosis in NRK-52E cells via inhibiting oxidative stress and activating AMPK/mTOR-mediated autophagy.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors, as a new type of hypoglycemic drug, can prevent proximal renal tubule injury related to glucose toxicity and play a renoprotective role. Canagliflozin, a recognized SGLT-2 inhibitor, has been proved to have potential protection in diabetic nephropathy (DN), but its mechanism has not been fully elucidated. In this study, the protective effect of canagliflozin against high glucose (HG)-induced renal tubular epithelial cell (NRK-52E) injury in vitro was assessed. The viability and apoptosis of NRK-52E cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, oxidative stress-related proteins (NOX4 and Nrf2), autophagy marker light chain 3 (LC3) I/II, and adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway-related proteins were evaluated by Western blot. Reactive oxygen species (ROS) level was evaluated by dihydroethidium (DHE) reactive oxygen species assay, the activities of SOD, CAT, GSH-Px and MDA were analyzed using kits. The changes of morphology and red fluorescent protein (RFP)-LC3 fluorescence were observed under microscopy. Canagliflozin significantly ameliorated HG-induced NRK-52E cell apoptosis and caspase-3 cleavage. Furthermore, canagliflozin markedly ameliorated HG-induced NRK-52E cell oxidative stress. Moreover, canagliflozin significantly increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Finally, canagliflozin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C abolished canagliflozin-induced autophagy activation, as well as the anti-apoptotic effect of canagliflozin. Canagliflozin effectively ameliorate HG-induced apoptosis of NRK-52E cells in vitro that involved its antioxidant effect and induction of autophagy through the AMPK/mTOR pathway.

3D Cell Printing of Advanced Vascularized Proximal Tubule-on-a-Chip for Drug Induced Nephrotoxicity Advancement.

Renal dysfunction due to drug-induced nephrotoxicity (DIN) affects >20% of the adult population worldwide. The vascularized proximal tubule is a complex structure that is often the primary site of drug-induced kidney injury. Herein, a vascularized proximal tubule-on-a-chip (Vas-POAC) was fabricated, demonstrating improved physiological emulation over earlier single-cell proximal tubule models. A perfusable model of vascularized proximal tubules permits the growth and proliferation of renal proximal tubule cells and adjacent endothelial cells under various conditions. An in vitro Vas-POAC showed mature expressions of the tubule and endothelial cell markers in the mature epithelium and endothelium lumens after 7 days of culture. Expression in the mature proximal tubule epithelium resembled the polarized expression of sodium-glucose cotransporter-2 and the de novo synthesis of ECM proteins. These perfusable Vas-POACs display significantly improved functional properties relative to the proximal tubules-on-a-chip (POAC), which lacks vascular components. Furthermore, the developed Vas-POAC model evaluated the cisplatin-induced nephrotoxicity and revealed enhanced drug receptivity compared to POAC. We further evaluated the capability of the developed proximal tubule model to act as a functional platform that targets screening drug doses that can cause renal proximal tubule injury in adults. Thus, our cell-printed models may prove valuable for screening, thoughtful mechanistic investigations of DIN, and discovery of drugs that interfere with tubule formation.

#6485 UNI-494 LOWERS URINARY B2-MICROGLOBULIN LEVELS IN RATS

Abstract Background and Aims Mitochondrial dysfunction in renal cells play a critical role in the pathophysiology of acute kidney injury (AKI) and chronic kidney disease (CKD). The proximal tubule is the primary sensor and effector in CKD progression and AKI. The measurement of urine Β2-microglobulin (Β2M) is a sensitive assay for proximal tubule injury. Nicorandil, a selective mitochondrial ATP-sensitive potassium channel activator, may be a promising AKI treatment, but its clinical use is limited by serious gastrointestinal side effects and rapid absorption and elimination. UNI-494, a novel nicorandil prodrug designed to improve its pharmacologic properties, may increase the short half-life and improve the safety profile of nicorandil. This study presents the efficacy data on the effects of UNI-494 in a rat ischemia-reperfusion (I/R) model, evaluating biomarkers of renal injury. Method 49 male Sprague-Dawley rats were randomly assigned to 4 groups to evaluate the in vivo efficacy of UNI-494 in a bilateral renal (I/R) model. Group 1, the Sham group, consisted of 10 subjects while groups 2–4 each had 13. Group 1 received no treatment, Group 2 a vehicle, Group 3 10mg/kg of UNI-494, and Group 4 20mg/kg of UNI-494. Treatments were administered as a single dose on Day 0, 1 hour prior to modeling. Body weights were measured on Days -1, 0, and 1. Urine Β2M levels and urinary albumin-to-creatinine ratio (UACR) samples were collected within 24 hours after the surgery using the metabolic cages. Serum blood urea nitrogen (BUN) and creatinine samples were collected on Day -1 (pre-modeling) and 24 hours after modeling. T-tests were used to evaluate statistical differences between groups. Results Β2M levels were significantly lower for the 20 mg/kg UNI-494 dose group compared to the vehicle group, and Β2M content in both UNI-494 dose groups (10 and 20 mg/kg) was significantly lower compared to the vehicle group (Fig. 1). Β2M levels and content were lower for the 20 mg/kg UNI-494 dose group than for the 10 mg/kg dose group (Fig. 1). There was no difference in body weight changes between the UNI-494 dose groups and the vehicle group. There were no statistically significant differences among all groups for serum creatinine and BUN pre-modeling and in UACR. Conclusion Lower levels of urine Β2M levels in the higher UNI-494 dose group compared to the other groups indicate that UNI-494 may have a renoprotective effect. Additionally, the lower levels of Β2M in the higher dose group compared to the lower dose group indicate a dose-response trend. Our study results are consistent with previously published data with nicorandil in a similar rat model of I/R. This potential renoprotective effect should be further investigated.

NDUFV1 attenuates renal ischemia-reperfusion injury by improving mitochondrial homeostasis.

Impaired mitochondrial function and dysregulated energy metabolism have been shown to be involved in the pathological progression of kidney diseases such as acute kidney injury (AKI) and diabetic nephropathy. Hence, improving mitochondrial function is a promising strategy for treating renal dysfunction. NADH: ubiquinone oxidoreductase core subunit V1 (NDUFV1) is an important subunit of mitochondrial complex I. In the present study, we found that NDUFV1 was reduced in kidneys of renal ischemia/reperfusion (I/R) mice. Meanwhile, renal I/R induced kidney dysfunction as evidenced by increases in BUN and serum creatinine, severe injury of proximal renal tubules, oxidative stress, and cell apoptosis. All these detrimental outcomes were attenuated by increased expression of NDUFV1 in kidneys. Moreover, knockdown of Ndufv1 aggravated cell insults induced by H2 O2 in TCMK-1 cells, which further confirmed the renoprotective roles of NDUFV1. Mechanistically, NDUFV1 improved the integrity and function of mitochondria, leading to reduced oxidative stress and cell apoptosis. Overall, our data indicate that NDUFV1 has an ability to maintain mitochondrial homeostasis in AKI, suggesting therapies by targeting mitochondria are useful approaches for dealing with mitochondrial dysfunction associated renal diseases such as AKI.

The β2-adrenergic receptor agonist formoterol restores mitochondrial homeostasis in glucose-induced renal proximal tubule injury through separate integrated pathways

Mitochondrial dysfunction drives the development and progression of diabetic kidney disease (DKD). Previously, we discovered that the β2-adrenergic receptor (AR) agonist formoterol regulates mitochondrial dynamics in the hyperglycemic renal proximal tubule. The goal of this study was to identify signaling mechanisms through which formoterol restores the mitochondrial fission/fusion proteins Drp1 and Mfn1. Using primary renal proximal tubule cells (RPTC), the effect of chronic high glucose on RhoA/ROCK1/Drp1 and Raf/MEK1/2/ERK1/2/Mfn1 signaling was determined. In glucose-treated RPTC, RhoA became hyperactive, leading to ROCK1-induced activation of Drp1. Treatment with formoterol and/or pharmacological inhibitors targeting RhoA, ROCK1 and Drp1 blocked RhoA and Drp1 hyperactivity. Inhibiting this pathway also restored maximal mitochondrial respiration. By preventing Gβγ signaling with gallein, we determined that formoterol signals through the Gβγ subunit of the β2-AR to restore RhoA and Drp1. Furthermore, formoterol restored this pathway by blocking binding of RhoA with the guanine nucleotide exchange factor p114RhoGEF. Formoterol also restored the mitochondrial fusion protein Mfn1 through a second Gβγ-dependent mechanism composed of Raf/MEK1/2/ERK1/2/Mfn1. Glucose-treated RPTC exhibited decreased Mfn1 activity, which was restored with formoterol. Pharmacological inhibition of Gβγ, Raf and MEK1/2 also restored Mfn1 activity. We demonstrate that glucose promotes the interaction between RhoA and p114RhoGEF, leading to increased RhoA and ROCK1-mediated activation of Drp1, and decreases Mfn1 activity through Raf/MEK1/2/ERK1/2. Formoterol restores these pathways and mitochondrial function in response to elevated glucose by activating separate yet integrative pathways that promote mitochondrial biogenesis, decreased fission and increased fusion in RPTC, further supporting its potential as a therapeutic for DKD.

491 - Perilous drug-induced proximal tubule injury leading to intensive care unit admission

491 - Perilous drug-induced proximal tubule injury leading to intensive care unit admission

Uromodulin Deficiency Shapes AKI Severity by Enhanced Oxidative Injury and Stress Response in Proximal Tubules With Early Activation of the Inflammasome

Uromodulin Deficiency Shapes AKI Severity by Enhanced Oxidative Injury and Stress Response in Proximal Tubules With Early Activation of the Inflammasome

Multimodal single cell sequencing implicates chromatin accessibility and genetic background in diabetic kidney disease progression

The proximal tubule is a key regulator of kidney function and glucose metabolism. Diabetic kidney disease leads to proximal tubule injury and changes in chromatin accessibility that modify the activity of transcription factors involved in glucose metabolism and inflammation. Here we use single nucleus RNA and ATAC sequencing to show that diabetic kidney disease leads to reduced accessibility of glucocorticoid receptor binding sites and an injury-associated expression signature in the proximal tubule. We hypothesize that chromatin accessibility is regulated by genetic background and closely-intertwined with metabolic memory, which pre-programs the proximal tubule to respond differently to external stimuli. Glucocorticoid excess has long been known to increase risk for type 2 diabetes, which raises the possibility that glucocorticoid receptor inhibition may mitigate the adverse metabolic effects of diabetic kidney disease.

Abstract P082: Exposure To Excess Albumin Causes Inflammatory Activation Of The Proximal Tubule.

In previous studies, we used chronic servo control experiments to show that renal inflammation is a secondary event in salt-sensitive hypertension, with pressure amplifying renal immune cell infiltration in Dahl salt-sensitive (SS) rats. In contrast, albuminuria occurs early in the development of hypertension and facilitates its progression. In this study, we tested the hypothesis that albuminuria causes release of pro-inflammatory cytokines in the proximal tubule, which in turn may initiate renal inflammation.We exposed the apical membrane of human proximal tubule cell monolayers (RPTEC-TERT1) to human serum albumin (HSA, 10 mg/ml, n=8) for 24 and 48hrs. Control cells were kept in standard media without HSA (n=8). The concentrations of 47 cytokines were measured in the basolateral (BM) and apical (AM) media using Luminex assays. After 24hrs we found that 18 cytokines were significantly higher in the BM and 14 in the AM relative to controls. At 48hrs, 17 cytokines were significantly higher in the BM and 15 in the AM. Notably, we found significant increases in basolateral release of several chemokines, including CCL2 (24hrs: 6.1±0.9 vs. 40.6±8.9, 48hrs: 20.1±4.5 vs. 85.8±15.6 pg/mg), CXCL1 (24hrs: 1.3±0.3 vs. 39.5±12.2, 48hrs: 3.4±1.1 vs. 94.2±41.4 pg/mg), and IL-8 (24hrs: 1.3±0.4 vs. 11.6±3.7, 48hrs: 2.1±0.5 vs. 10.1±3 pg/mg). We propose that HSA-induced chemokine secretion facilitates immune cell recruitment and the initiation of renal inflammation. To track the progression of albuminuria and proximal tubule injury in SS rats we performed daily urine collections in rats fed either a high-salt (HS, 4.0% NaCl, n=6) or a control-salt (CS, 0.4% NaCl, n=5) diet for 7-days. In SS rats fed HS, albuminuria preceded proximal tubule injury. Urinary albumin was elevated after 2-days of HS (22.6±6.5 vs. 61.1±18.7 mg/day) whereas Kidney Injury Molecule-1 (KIM-1) was increased after 5-days of HS (15.5±1.5 vs. 31.3±5.6 ng/day).To conclude, in vitro exposure to excess albumin causes significant increases in the levels of multiple cytokines involved in the initiation of inflammation. In SS rats fed HS, albuminuria precedes proximal tubule injury. Together these data suggest that albuminuria may facilitate hypertension through the initiation of renal inflammation.

Abstract P319: Genetic Interactions Between Novel Genes Arhgef11 And Cgnl1 Are Involved In Hypertensive Chronic Kidney Disease

Patients with hypertension are at higher risk for developing chronic kidney disease (CKD) and ultimately kidney failure. Previous genetic studies involving the Dahl salt-sensitive (SS) rat, a well-established model of hypertensive CKD, identified multiple regions linked to kidney injury. Extensive work on Chr.2 identified and characterized the role of Arhgef11 in the onset and progression of CKD using SS- Arhgef11 -/- rats. The identification of Arhgef11 and mechanism (regulation of actin cytoskeleton components) has provided an opportunity to expedite gene identification on other chromosomes based on known genetic interactions between loci, such as with Chr. 8. The effect of the Chr. 8 locus on kidney injury was previously validated using a congenic strain [S.SHR(8)]. Bioinformatics analysis of genes residing within the confidence interval, and those mechanistically linked with Arhgef11 , identified cingulin-like 1 ( Cgnl1 ) as a candidate. Cgnl1 plays a role in the regulation of cell-cell contacts via adherens and tight junctions through several interacting proteins [e.g. ZO-1 and PLEKHA7 (linked with hypertension by GWAS and an SS- Plekha7 mutant model)]. Recent studies using a novel model (SS- Cgnl1 -/- ) demonstrates that knockout of Cgnl1 leads to loss of salt-sensitive blood pressure and reduced renal injury compared to wildtype SS, supporting the congenic studies. Using multi-omics approaches incorporating bulk RNAseq, single nuclei RNAseq, spatial transcriptomics, small RNAseq, and mass spectrometry discovery proteomics, a putative mechanism connecting proximal tubule injury to alterations in sodium solute transporters and hypertension was established. The direct physiological interaction between Arhgef11 and Cgnl1 on hypertension and CKD is currently underway using a double knockout model (SS- Arhgef11 -/- Cgnl1 -/- ). In addition, to better elucidate the impact of loss of Arhgef11 , Cgnl1 , or both genes together, gene-edited cell lines using CRISPR technology has been generated and are also being studied. It is anticipated that completion of this work will lead to a better understanding of the role of genetics in hypertensive CKD, the influence of factors that complicate kidney disease, and potential new therapeutic targets.

Glycosylation of a key cubilin Asn residue results in reduced binding to albumin

Kidney disease often manifests with an increase in proteinuria, which can result from both glomerular and/or proximal tubule injury. The proximal tubules are the major site of protein and peptide endocytosis of the glomerular filtrate, and cubilin is the proximal tubule brush border membrane glycoprotein receptor that binds filtered albumin and initiates its processing in proximal tubules. Albumin also undergoes multiple modifications depending upon the physiologic state. We previously documented that carbamylated albumin had reduced cubilin binding, but the effects of cubilin modifications on binding albumin remain unclear. Here, we investigate the cubilin-albumin binding interaction to define the impact of cubilin glycosylation and map the key glycosylation sites while also targeting specific changes in a rat model of proteinuria. We identified a key Asn residue, N1285, that when glycosylated reduced albumin binding. In addition, we found a pH-induced conformation change may contribute to ligand release. To further define the albumin-cubilin binding site, we determined the solution structure of cubilin’s albumin-binding domain, CUB7,8, using small-angle X-ray scattering and molecular modeling. We combined this information with mass spectrometry crosslinking experiments of CUB7,8 and albumin that provides a model of the key amino acids required for cubilin-albumin binding. Together, our data supports an important role for glycosylation in regulating the cubilin interaction with albumin, which is altered in proteinuria and provides new insight into the binding interface necessary for the cubilin–albumin interaction.

Aristolochic acid I induces proximal tubule injury through ROS/HMGB1/mt DNA mediated activation of TLRs.

Aristolochic acids (AAs) are extracted from certain plants as folk remedies for centuries until their nephrotoxicity and carcinogenicity were recognized. Aristolochic acid I (AAI) is one of the main pathogenic compounds, and it has nephrotoxic, carcinogenic and mutagenic effects. Previous studies have shown that AAI acts mainly on proximal renal tubular epithelial cells; however, the mechanisms of AAI‐induced proximal tubule cell damage are still not fully characterized. We exposed human kidney proximal tubule cells (PTCs; HK2 cell line) to AAI in vitro at different time/dose conditions and assessed cell proliferation, reactive oxygen species (ROS) generation, nitric oxide (NO) production, m‐RNA/ protein expressions and mitochondrial dysfunction. AAI exposure decreased proliferation and increased apoptosis, ROS generation / NO production in PTCs significantly at 24 h. Gene/ protein expression studies demonstrated activation of innate immunity (TLRs 2, 3, 4 and 9, HMGB1), inflammatory (IL6, TNFA, IL1B, IL18, TGFB and NLRP3) and kidney injury (LCN2) markers. AAI also induced epithelial‐mesenchymal transition (EMT) and mitochondrial dysfunction in HK2 cells. TLR9 knock‐down and ROS inhibition were able to ameliorate the toxic effect of AAI. In conclusion, AAI treatment caused injury to PTCs through ROS‐HMGB1/mitochondrial DNA (mt DNA)‐mediated activation of TLRs and inflammatory response.

Acute Kidney Injury Associates with Long-Term Increases in Plasma TNFR1, TNFR2, and KIM-1: Findings from the CRIC Study.

Some markers of inflammation-TNF receptors 1 and 2 (TNFR1 and TNFR2)-are independently associated with progressive CKD, as is a marker of proximal tubule injury, kidney injury molecule 1 (KIM-1). However, whether an episode of hospitalized AKI may cause long-term changes in these biomarkers is unknown. Among adult participants in the Chronic Renal Insufficiency Cohort (CRIC) study, we identified 198 episodes of hospitalized AKI (defined as peak/nadir inpatient serum creatinine values ≥1.5). For each AKI hospitalization, we found the best matched non-AKI hospitalization (unique patients), using prehospitalization characteristics, including eGFR and urine protein/creatinine ratio. We measured TNFR1, TNFR2, and KIM-1 in banked plasma samples collected at annual CRIC study visits before and after the hospitalization (a median of 7 months before and 5 months after hospitalization). In the AKI and non-AKI groups, we found similar prehospitalization median levels of TNFR1 (1373 pg/ml versus 1371 pg/ml, for AKI and non-AKI, respectively), TNFR2 (47,141 pg/ml versus 46,135 pg/ml, respectively), and KIM-1 (857 pg/ml versus 719 pg/ml, respectively). Compared with matched study participants who did not experience AKI, study participants who did experience AKI had greater increases in TNFR1 (23% versus 10%, P<0.01), TNFR2 (10% versus 3%, P<0.01), and KIM-1 (13% versus -2%, P<0.01). Among patients with CKD, AKI during hospitalization was associated with increases in plasma TNFR1, TNFR2, and KIM-1 several months after their hospitalization. These results highlight a potential mechanism by which AKI may contribute to more rapid loss of kidney function months to years after the acute insult.