SARS-CoV-2 infection causes a decline in renal megalin expression and affects vitamin D metabolism in the kidney of K18-hACE2 mice.

Patients with coronavirus disease 2019 (COVID-19) often experience acute kidney injury, linked to disease severity or mortality, along with renal tubular dysfunction and megalin loss in proximal tubules. Megalin plays a crucial role in kidney vitamin D metabolism. However, the impact of megalin loss on vitamin D metabolism during COVID-19 is unclear. This study investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection reduces megalin expression in proximal tubules and its subsequent effect on vitamin D metabolism in mice expressing human angiotensin converting enzyme 2 (K18-hACE2 mice). Histological and immunohistochemical staining analyses revealed glomerular and capillary congestion, and elevated renal neutrophil gelatinase-associated lipocalin levels, indicative of acute kidney injury in K18-hACE2 mice. In SARS-CoV-2-infected mice, immunohistochemical staining revealed suppressed megalin protein levels. Decreased vitamin D receptor (VDR) localization in the nucleus and increased mRNA expression of VDR, CYP27B1, and CYP24A1 were observed by quantitative PCR in SARS-CoV-2-infected mice. Serum vitamin D levels remained similar in infected and vehicle-treated mice, but an increase in tumor necrosis factor-alpha and a decrease in IL-4 mRNA expression were observed in the kidneys of the SARS-CoV-2 group. These findings suggest that megalin loss in SARS-CoV-2 infection may impact the local role of vitamin D in kidney immunomodulation, even when blood vitamin D levels remain unchanged.

Dichloroacetate reduces cisplatin-induced apoptosis by inhibiting the JNK/14-3-3/Bax/caspase-9 pathway and suppressing caspase-8 activation via cFLIP in murine tubular cells.

Cisplatin-induced injury to renal proximal tubular cells stems from mitochondrial damage-induced apoptosis and inflammation. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, a potential generator of ROS and ATP, protects against cisplatin-induced nephrotoxicity by promoting the TCA cycle. However, its effects on apoptotic pathways and ROS production in renal tubular cells remain unclear. Here, we investigated the detailed molecular mechanisms of the DCA's effects by immunoblot, RT-PCR, RNA-sequencing, and RNA-silencing in a murine renal proximal tubular (mProx) cell line and mouse kidneys. In mProx cells, DCA suppressed cisplatin-induced apoptosis by attenuating the JNK/14-3-3/Bax/caspase-9 and death receptor/ligand/caspase-8 pathways without impeding inflammatory signaling. RNA-sequencing demonstrated that DCA increased the cisplatin-reduced expression of cFLIP, a caspase-8 inactivator, and decreased the expression of almost all oxidative phosphorylation (OXPHOS) genes. DCA also increased NF-kB activation and ROS production, probably enhancing the cFLIP induction and OXPHOS gene reduction, respectively. Furthermore, cFLIP silencing weakened the DCA's anti-apoptotic effects. Finally, in mouse kidneys, DCA attenuated cisplatin-caused injuries such as functional and histological damages, caspase activation, JNK/14-3-3 activation, and cFLIP reduction. Conclusively, DCA mitigates cisplatin-induced nephrotoxicity by attenuating the JNK/14-3-3/Bax/caspase-9 pathway and inhibiting the caspase-8 pathways via cFLIP induction, probably outweighing the cisplatin plus DCA-derived cytotoxic effects including ROS.

CHIP drives proteasomal degradation of NUR77 to alleviate oxidative stress and intrinsic apoptosis in cisplatin-induced nephropathy.

Carboxy-terminus of Hsc70-interacting protein (CHIP), an E3 ligase, modulates the stability of its targeted proteins to alleviate various pathological perturbations in various organ systems. Cisplatin is a widely used chemotherapeutic agent, but it is also known for its alarming renal toxicity. The role of CHIP in the pathogenesis of cisplatin-induced acute kidney injury (AKI) has not been adequately investigated. Herein, we demonstrated that CHIP was abundantly expressed in the renal proximal tubular epithelia, and its expression was downregulated in cisplatin-induced AKI. Further investigation revealed that CHIP overexpression or activation alleviated, while its gene disruption promoted, oxidative stress and apoptosis in renal proximal tubular epithelia induced by cisplatin. In terms of mechanism, CHIP interacted with and ubiquitinated NUR77 to promote its degradation, which consequently shielded BCL2 to maintain mitochondrial permeability of renal proximal tubular cells in the presence of cisplatin. Also, we demonstrated that CHIP interacted with NUR77 via its central coiled-coil (CC) domain, a non-canonical interactive pattern. In conclusion, these findings indicated that CHIP ubiquitinated and degraded its substrate NUR77 to attenuate intrinsic apoptosis in cisplatin-treated renal proximal tubular epithelia, thus providing a novel insight for the pathogenesis of cisplatin-induced AKI.

Sustained alterations in proximal tubule gene expression in primary culture associate with HNF4A loss.

Primary cultures of proximal tubule cells are widely used to model the behavior of kidney epithelial cells in vitro. However, de-differentiation of primary cells upon culture has been observed and appreciated for decades, yet the mechanisms driving this phenomenon remain poorly understood. This confounds the interpretation of experiments using primary kidney epithelial cells and prevents their use to engineer functional kidney tissue ex vivo. In this report, we measure the dynamics of cell-state transformations in early primary culture of mouse proximal tubules to identify key pathways and processes that correlate with and may drive de-differentiation. Our data show that the loss of proximal-tubule-specific genes is rapid, uniform, and sustained even after confluent, polarized epithelial monolayers develop. This de-differentiation occurs uniformly across many common culture condition variations. Changes in early culture were strongly associated with the loss of HNF4A. Exogenous re-expression of HNF4A can promote expression of a subset of proximal tubule genes in a de-differentiated proximal tubule cell line. Using genetically labeled proximal tubule cells, we show that selective pressures very early in culture influence which cells grow to confluence. Together, these data indicate that the loss of in vivo function in proximal tubule cultures occurs very early and suggest that the sustained loss of HNF4A is a key regulatory event mediating this change.

Protective effect and mechanism of CoQ10 in mitochondrial dysfunction in diquat-induced renal proximal tubular injury.

Coenzyme Q10 (CoQ10) plays an important role in improving mitochondrial function and has many beneficial effects on the kidney. However, whether CoQ10 protects against diquat (DQ)-induced acute kidney injury (AKI) remains unclear. In this study, we investigated the protective effects and mechanism of action of CoQ10 against DQ-induced AKI. Institute of Cancer Research (ICR) mice were intraperitoneally injected with DQ to induce AKI. The expression levels of serum creatinine (Cr), urea, and kidney injury molecule-1 (KIM-1) increased, those of aquaporin 1 (AQP-1) decreased, and those of mitochondrial reactive oxygen species (ROS) increased with increased depolarization of mitochondrial membranes and mitochondrial rupture. In contrast, treatment with CoQ10 significantly improved DQ-induced AKI. CoQ10 treatment reduced serum Cr, urea, and KIM-1 contents, increased the AQP-1 expression, and reduced ROS contents in mice with DQ poisoning. Our results suggest that AKI caused by DQ poisoning may be related to the disruption of mitochondrial homeostasis and that CoQ10 treatment protects against AKI caused by DQ poisoning by improving mitochondrial kinetic homeostasis. Thus, CoQ10 represents a new therapeutic option for the prevention and treatment of AKI caused by DQ poisoning.

Elevated serum GDF15 level as an early indicator of proximal tubular cell injury in acute kidney injury.

Acute kidney injury (AKI) is a high-burden medical condition, and current diagnostic criteria can only assess AKI after full manifestation. Stress marker growth differentiation factor 15 (GDF15) was reported to have a role in kidney injury of critical patients. Herein, we evaluated dynamic changes in GDF15 across diverse AKI scenarios and explored the underlying mechanisms of its induction. Serum parameters and renal lesions were analyzed in mouse models of unilateral ischemia-reperfusion injury (uni-IRI) and unilateral ureteral obstruction (UUO). The human proximal tubular (HK-2) cell line was stimulated with various conditions, and induction of GDF15 expression was determined. Serum GDF15 levels were rapidly induced within hours after injury in both animal models and declined thereafter. Renal GDF15 expression exhibited a temporary and early increased induction and was mainly located in aquaporin 1-positive proximal tubules in both unilateral AKI model tissues. In cell experiments, rapid GDF15 production was highly induced by t-BHP and CoCl2. Treatment with either an antioxidant or mitogen-activated protein kinase inhibitors abolished t-BHP- and CoCl2-mediated GDF15 expression. In addition, silencing nuclear factor erythroid 2-related factor 2 expression also reduced the basal and t-BHP- or CoCl2-mediated GDF15 expression level in HK-2 cells. Our data showed that elevated serum GDF15 levels could be detected early in unilateral AKI models without notable alterations in kidney function parameters. GDF15 expression was associated with oxidative stress- and hypoxia-mediated proximal tubular cell injury. These data document that elevated serum GDF15 can possibly serve as an early biomarker for proximal tubular cell injury in AKI.

Sex-specific proximal tubular cell differentiation pathways identified by single-nucleus RNA sequencing.

Postnatal kidney growth is substantial and involves expansion in kidney tubules without growth of new nephrons, which are the functional units of the kidney. Proliferation and differentiation pathways underpinning nephron elongation are not well defined. To address this, we performed sequential characterization of mouse kidney transcriptomics at the single cell level. Single nuclear RNA sequencing (snRNA-seq) was performed on kidney tissue from male and female mice at 1, 2, 4 and 12 weeks of age using the 10x Chromium platform. Unbiased clustering was performed on 68,775 nuclei from 16 animals. 31 discrete cellular clusters were seen, which were identified through comparison of their gene expression profiles to canonical markers of kidney cell populations. High levels of proliferation were evident at early time points in some cell types, especially tubular cells, but not in other cell types, for example podocytes. Proliferation was especially evident in Proximal Tubular Cells (PTCs) which are the most abundant cell type in the adult kidney. Uniquely when compared to other kidney cell types, PTCs demonstrated sex-specific expression profiles at late, but not early, time points. Mapping of PTC differentiation pathways using techniques including trajectory and RNA Velocity analyses delineated increasing PTC specialization and sex-specific phenotype specification. Our single-cell transcriptomics data characterise cellular states observed during kidney growth. We have identified PTC differentiation pathways that lead to sex-specific tubular cell phenotypes. Tubular proliferative responses are of central importance in postnatal kidney growth and have also been linked to kidney recovery versus fibrosis following injury. Our unbiased and comprehensive dataset of tubular cell development can be used to identify candidate pathways for therapeutic targeting.

NRF2 Activation by AR-20007 Preserves Renal Tubular Epithelial Cells from Antimycin A-Induced Cell Death via Glutathione Metabolism Regulation.

Oxidative stress plays a crucial role in the development and progression of various kidney diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is the primary transcription factor that protects cells from oxidative stress by regulating cytoprotective genes including those involved in the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and cell death. Antimycin A, an inhibitor of complex III of the electron transport chain, causes oxidative stress and reduces GSH levels. In this study, we induced mitochondrial damage in rat renal proximal tubular cells using antimycin A and investigated cellular viability and levels of NRF2 and GSH. Treatment with antimycin A altered the expression of antioxidant genes, including reduction in the transcription of glutathione-cysteine ligase subunits (Gclc and Gclm) and glutathione reductase (Gsr1), followed by a reduction in total GSH content with a concomitant decrease in NRF2 protein expression. AR-20007, previously described as an NRF2 activator, stabilizes and increases NRF2 protein expression in cells. By stimulating NRF2, AR-20007 increased the expression of antioxidant and detoxifying enzymes, thereby enhancing protection against oxidative stress induced by antimycin A. These data suggest that NRF2 activation effectively inhibits antimycin A-induced oxidative stress and that NRF2 may be a promising therapeutic target for preventing cell death during acute kidney injury.

Cilastatin, a new therapeutic promise for acute kidney injury.

Acute kidney injury is a devasting clinical syndrome resulting from multiple causes, characterized by an abrupt deterioration of kidney function for which there is no pharmacologic treatment. Cilastatin has demonstrated direct nephroprotective effects in acute kidney injury and now is shown to be effective to specifically target therapeutically loaded nanoparticles to the proximal tubule to treat acute kidney injury.

Podocyte-specific KLF6 primes proximal tubule CaMK1D signaling to attenuate diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease worldwide. While injury to the podocytes, visceral epithelial cells that comprise the glomerular filtration barrier, drives albuminuria, proximal tubule (PT) dysfunction is the critical mediator of DKD progression. Here, we report that the podocyte-specific induction of human KLF6, a zinc-finger binding transcription factor, attenuates podocyte loss, PT dysfunction, and eventual interstitial fibrosis in a male murine model of DKD. Utilizing combination of snRNA-seq, snATAC-seq, and tandem mass spectrometry, we demonstrate that podocyte-specific KLF6 triggers the release of secretory ApoJ to activate calcium/calmodulin dependent protein kinase 1D (CaMK1D) signaling in neighboring PT cells. CaMK1D is enriched in the first segment of the PT, proximal to the podocytes, and is critical to attenuating mitochondrial fission and restoring mitochondrial function under diabetic conditions. Targeting podocyte-PT signaling by enhancing ApoJ-CaMK1D might be a key therapeutic strategy in attenuating the progression of DKD.

Cylindromatosis lysine 63 deubiquitinase (CYLD) suppress TLR3-mediated CCL5 expression in human renal proximal tubular epithelial cells.

BACKGROUND: One of the causes of tubulointerstitial nephritis is viral infection, with innate immune responses affecting its pathogenesis. Toll-like receptor 3 (TLR3) recognizes viral infections and acts antivirally by activating signaling to produce inflammatory cytokines/chemokines, including C-C motif chemokine ligand 5 (CCL5) and interferon-ß (IFN-ß). Although cylindromatosis lysine 63 deubiquitinase (CYLD) is known to be associated with tubulointerstitial nephritis and renal function, its role in the antiviral innate immune response in tubular epithelial cells remains unknown. In this study, we investigated the association between CYLD and TLR3-mediated CCL5 production in cultured human renal proximal tubular epithelial cells (hRPTECs). METHODS AND RESULTS: Polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand, was used to stimulate hRPTECs. mRNA expression was measured using reverse transcription-quantitative polymerase chain reaction. Protein expression was assayed using western blotting or an enzyme-linked immunosorbent assay. Knockdown of IFN-ß, nuclear factor-kappa B (NF-κB) p65, and CYLD was performed by transfecting cells with specific small interfering RNAs. The intracellular localization of CYLD in hRPTECs was analyzed using immunofluorescence. Poly IC induced CCL5 expression in a time- and concentration-dependent manner, and knockdown of either IFN-ß or p65 reduced poly IC-induced CCL5 expression. CYLD knockdown increased the poly IC-induced CCL5, phosphorylated IκB kinase α/ß (IKK complex), and phosphorylated p65 expression. The CYLD protein was localized in the cytoplasm, and poly IC did not alter its expression. CONCLUSION: CYLD may prevent excessive inflammation due to an antiviral innate immune response by suppressing IKK complex and NF-κB activation downstream of TLR3 in hRPTECs.

Spatial transcriptomics defines injury specific microenvironments and cellular interactions in kidney regeneration and disease.

Kidney injury disrupts the intricate renal architecture and triggers limited regeneration, together with injury-invoked inflammation and fibrosis. Deciphering the molecular pathways and cellular interactions driving these processes is challenging due to the complex tissue structure. Here, we apply single cell spatial transcriptomics to examine ischemia-reperfusion injury in the mouse kidney. Spatial transcriptomics reveals injury-specific and spatially-dependent gene expression patterns in distinct cellular microenvironments within the kidney and predicts Clcf1-Crfl1 in a molecular interplay between persistently injured proximal tubule cells and their neighboring fibroblasts. Immune cell types play a critical role in organ repair. Spatial analysis identifies cellular microenvironments resembling early tertiary lymphoid structures and associated molecular pathways. Collectively, this study supports a focus on molecular interactions in cellular microenvironments to enhance understanding of injury, repair and disease.

Lambda light chain - restricted non - crystalline proximal tubulopathy with cast nephropathy in multiple myeloma: a case report and literature review.

BACKGROUND: Multiple myeloma (MM) often causes renal tubular damage, such as the light chain cast nephropathy (LCCN) and the light chain proximal tubulopathy (LCPT). The excessive light chains deposited in the proximal and distal tubules usually manifest with different characteristics, leading to a rare coexistence of the two pathological conditions. Here we report a unique case of a patient with multiple myeloma (MM) who presented with acute kidney injury (AKI) due to dual conditions of λ light chain-restricted non-crystalline LCPT and LCCN. This report reviews the clinical presentation and histological findings, comparing them with previously published cases. CASE PRESENTATION: A 49-year-old male patient was admitted with a chief complaint of "fatigue, loss of appetite for 40 days and elevated blood creatinine for 10 days." In serum and urine, the λ light chain level and the ratio of κ to λ free light chain were 1235 mg/dl and 93.25 mg/dl, 0.0022 and 0.0316, respectively. Additionally, serum protein electrophoresis showed an M-spike with monoclonal IgD-λ. Bone marrow puncture revealed 30.5% primitive naive plasma cells, indicative of IgD-λ MM. Light microscopy of kidney biopsy specimen showed periodic acid-Schiff (PAS)-negative cytoplasm in some proximal tubules and PAS-negative casts with a rigid appearance in some distal tubule lumens. On immunofluorescence, these proximal tubular epithelial cells cytoplasm and casts stained exclusively with λ-light chains. Electron microscopy did not reveal any crystalline inclusions. Given the clinical and bone marrow puncture findings, the overall pathological presentation was LCPT with LCCN secondary to IgD-λ MM. After chemotherapy and dialysis, the patient's condition was improved and he was tracked in follow-ups. CONCLUSION: In some tubular renal injuries caused by MM, the morphological changes are subtle and often overlooked. In this paper, we present a rare case of LCPT with LCCN showing λ restriction in patient with MM. Through the clinicopathological analysis of patients, the understanding of the disease can be deepened and the diagnosis rate improved.

Evaluating nephrotoxicity reduction in a novel polymyxin B formulation: insights from a 3D kidney-on-a-chip model.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels (P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase (P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly (P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced (P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics.

PARVB deficiency alleviates cisplatin-induced tubular injury by inhibiting TAK1 signaling.

Cisplatin-induced renal tubular injury largely restricts the wide-spread usage of cisplatin in the treatment of malignancies. Identifying the key signaling pathways that regulate cisplatin-induced renal tubular injury is thus clinically important. PARVB, a focal adhesion protein, plays a crucial role in tumorigenesis. However, the function of PARVB in kidney disease is largely unknown. To investigate whether and how PARVB contributes to cisplatin-induced renal tubular injury, a mouse model (PARVB cKO) was generated in which PARVB gene was specifically deleted from proximal tubular epithelial cells using the Cre-LoxP system. In this study, we found depletion of PARVB in proximal tubular epithelial cells significantly attenuates cisplatin-induced renal tubular injury, including tubular cell death and inflammation. Mechanistically, PARVB associates with transforming growth factor-ß-activated kinase 1 (TAK1), a central regulator of cell survival and inflammation that is critically involved in mediating cisplatin-induced renal tubular injury. Depletion of PARVB promotes cisplatin-induced TAK1 degradation, inhibits TAK1 downstream signaling, and ultimately alleviates cisplatin-induced tubular cell damage. Restoration of PARVB or TAK1 in PARVB-deficient cells aggravates cisplatin-induced tubular cell injury. Finally, we demonstrated that PARVB regulates TAK1 protein expression through an E3 ligase ITCH-dependent pathway. PARVB prevents ITCH association with TAK1 to block its ubiquitination. Our study reveals that PARVB deficiency protects against cisplatin-induced tubular injury through regulation of TAK1 signaling and indicates targeting this pathway may provide a novel therapeutic strategy to alleviate cisplatin-induced kidney damage.

COMMD5 counteracts cisplatin-induced nephrotoxicity by maintaining tubular epithelial integrity and autophagy flux.

Oxidative stress mediated by reactive oxygen species (ROS) contributes to apoptosis of tubular epithelial cells (TECs) and renal inflammation during acute kidney injury (AKI). Copper metabolism MURR1 domain-containing 5 [COMMD5/hypertension-related, calcium-regulated gene (HCaRG)] shows strong cytoprotective properties. COMMD5 is highly expressed in proximal tubules (PTs), where it controls cell differentiation. We assessed its role in cisplatin-induced AKI using transgenic mice in which COMMD5 is overexpressed in the PTs. Cisplatin caused the accumulation of damaged mitochondria and cellular waste in PTs, thus increasing the apoptosis of TECs. COMMD5 overexpression effectively protected TECs from cisplatin nephrotoxicity by decreasing intracellular ROS levels, mitochondrial dysfunction, and apoptosis through the preservation of tubular epithelial integrity, thus alleviating morphological and functional kidney damage. Excessive ROS production by hydrogen peroxide led to long-term autophagy activation through an increased burden on the autophagy/lysosome degradation system in TECs, and autophagic elimination of damaged mitochondria and cellular waste was compromised. COMMD5 attenuated oxidative injury by increasing autophagy flux, possibly due to a reduction of intracellular ROS levels through maintained tubular epithelial integrity, which decreased JNK/caspase-3-dependent apoptosis. Meanwhile, COMMD5 inhibition by siRNA reduced the resistance of TECs to cisplatin cytotoxicity, as shown by disrupted tubular epithelial integrity and cell viability. These data indicated that COMMD5 protects TECs from drug-induced oxidative stress and toxicity by maintaining tubular epithelial integrity and autophagy flux and ultimately decreases mitochondrial dysfunction and apoptosis. Increasing COMMD5 content in PTs is proposed as a new protective and therapeutic strategy against AKI.NEW & NOTEWORTHY Oxidative stress overload by drug treatment causes the accumulation of damaged mitochondria that could contribute to tubulopathy. However, effective preventive treatment for drug-induced acute kidney injury remains incompletely understood. Our study showed that copper metabolism MURR1 domain-containing 5 (COMMD5) reduced mitochondrial dysfunction and increased autophagy flux by alleviating reactive oxygen species production through maintaining tubular epithelial integrity when tubular epithelial cells were under oxidative stress, thus ameliorating renal function in cisplatin-treated mice. These results uncover a novel renoprotective mechanism underlying tubular epithelial integrity and autophagy flux.

A Focus on the Proximal Tubule Dysfunction in Dent Disease Type 1.

Dent disease type 1 is a rare X-linked recessive inherited renal disorder affecting mainly young males, generally leading to end-stage renal failure and for which there is no cure. It is caused by inactivating mutations in the gene encoding ClC-5, a 2Cl-/H+ exchanger found on endosomes in the renal proximal tubule. This transporter participates in reabsorbing all filtered plasma proteins, which justifies why proteinuria is commonly observed when ClC-5 is defective. In the context of Dent disease type 1, a proximal tubule dedifferentiation was shown to be accompanied by a dysfunctional cell metabolism. However, the exact mechanisms linking such alterations to chronic kidney disease are still unclear. In this review, we gather knowledge from several Dent disease type 1 models to summarize the current hypotheses generated to understand the progression of this disorder. We also highlight some urinary biomarkers for Dent disease type 1 suggested in different studies.

Malate dehydrogenase-2 inhibition shields renal tubular epithelial cells from anoxia-reoxygenation injury by reducing reactive oxygen species.

Ischemia-reperfusion (I-R) injury is the most common cause of acute kidney injury. In experiments involving primary human renal proximal tubular epithelial cells (RPTECs) exposed to anoxia-reoxygenation, we explored the hypothesis that mitochondrial malate dehydrogenase-2 (MDH-2) inhibition redirects malate metabolism from the mitochondria to the cytoplasm, towards the malate-pyruvate cycle and reversed malate-aspartate shuttle. Colorimetry, fluorometry, and western blotting showed that MDH2 inhibition accelerates the malate-pyruvate cycle enhancing cytoplasmic NADPH, thereby regenerating the potent antioxidant reduced glutathione. It also reversed the malate-aspartate shuttle and potentially diminished mitochondrial reactive oxygen species (ROS) production by transferring electrons, in the form of NADH, from the mitochondria to the cytoplasm. The excessive ROS production induced by anoxia-reoxygenation led to DNA damage and protein modification, triggering DNA damage and unfolded protein response, ultimately resulting in apoptosis and senescence. Additionally, ROS induced lipid peroxidation, which may contribute to the process of ferroptosis. Inhibiting MDH-2 proved effective in mitigating ROS overproduction during anoxia-reoxygenation, thereby rescuing RPTECs from death or senescence. Thus, targeting MDH-2 holds promise as a pharmaceutical strategy against I-R injury.

Spns1 is an iron transporter essential for megalin-dependent endocytosis.

Proximal tubule endocytosis is essential to produce protein-free urine as well as to regulate system-wide metabolic pathways, such as the activation of Vitamin D. We have determined that the proximal tubule expresses an endolysosomal membrane protein, protein spinster homolog1 (Spns1), which engenders a novel iron conductance that is indispensable during embryonic development. Conditional knockout of Spns1 with a novel Cre-LoxP construct specific to megalin-expressing cells led to the arrest of megalin receptor-mediated endocytosis as well as dextran pinocytosis in proximal tubules. The endocytic defect was accompanied by changes in megalin phosphorylation as well as enlargement of lysosomes, confirming previous findings in Drosophila and Zebrafish. The endocytic defect was also accompanied by iron overload in proximal tubules. Remarkably, iron levels regulated the Spns1 phenotypes because feeding an iron-deficient diet or mating Spns1 knockout with divalent metal transporter1 knockout rescued the phenotypes. Conversely, iron-loading wild-type mice reproduced the endocytic defect. These data demonstrate a reversible, negative feedback for apical endocytosis and raise the possibility that regulation of endocytosis, pinocytosis, megalin activation, and organellar size and function is nutrient-responsive.NEW & NOTEWORTHY Spns1 mediates a novel iron conductance essential during embryogenesis. Spns1 knockout leads to endocytic and lysosomal defects, accompanied by iron overload in the kidney. Reversal of iron overload by restricting dietary iron or by concurrent knockout of the iron transporter, DMT1 rescued the endocytic and organellar defects and reverted markers of iron overload. These data suggest feedback between iron and proximal tubule endocytosis.

Pik3c3 expression profiling in the mouse kidney and its role in proximal tubule cell physiology.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, coimmunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology. NEW & NOTEWORTHY This is the first report defining the class 3 phosphatidylinositol 3-kinase (Pik3c3) expression profile in the kidney. Pik3c3 is nearly absent in renal interstitial cells, glomerular mesangial cells, and endothelial cells. Remarkably, glomerular podocytes express the highest Pik3c3 level in the kidney. However, the proximal tubule exhibits the highest expression level among all renal tubules. This study also unveils the pivotal role of Pik3c3 in regulating EGFR degradation and signaling termination in RPTCs, furthering our understanding of Pik3c3 in renal cell physiology.