Tubular cell and keratinocyte single-cell transcriptomics applied to lupus nephritis reveal type I IFN and fibrosis relevant pathways.

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.

Activation of the YAP/KLF5 transcriptional cascade in renal tubular cells aggravates kidney injury.

The Hippo/YAP pathway plays a critical role in tissue homeostasis. Our previous work demonstrated that renal tubular YAP activation induced by double knockout (dKO) of the upstream Hippo kinases Mst1 and Mst2 promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Here, we show that tubular YAP was already activated 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell overproliferation, tubular injury, and renal inflammation induced by UUO or cisplatin. YAP promoted the transcription of the transcription factor KLF5. Consistent with this, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell overproliferation, tubular injury, and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, where tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell overproliferation, tubular injury, and renal inflammation.

DNA-binding protein-A: a multitool in tubular epithelial cells.

Acute kidney injury is still associated with high morbidity and mortality. Reichardt et al. investigated DNA-binding protein-A (Ybx3) in acute kidney injury induced by ischemia-reperfusion injury and found that mice lacking Ybx3 have altered mitochondrial function and increased antioxidant activity, making them more resistant to ischemia-reperfusion injury-acute kidney injury. The study highlights a new role of the multifaceted protein DNA-binding protein-A, which could be potentially therapeutically exploited.

Commentary: the perspectives of harnessing the power of scattered tubular-like cells for renal repair.

The commentary discusses the regenerative capacity of the kidneys; recent studies reveal that renal cells can regenerate when exposed to certain conditions. A major focus is on scattered tubular-like cells (STCs), which can dedifferentiate and acquire progenitor-like properties in response to injury. These cells exhibit a glycolytic metabolism, making them resilient to hypoxic conditions and capable of repairing damaged renal tissues. Despite their potential, STCs are difficult to isolate and exist in small numbers. Here we highlight the need for more research into STC function, metabolic profiles, mechanisms limiting STC injury repair capacity, and methods of their pharmacological activation. Understanding these mechanisms could lead to novel therapies for kidney diseases.

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.

Usp9x contributes to the development of sepsis-induced acute kidney injury by promoting inflammation and apoptosis in renal tubular epithelial cells via activation of the TLR4/nf-κb pathway.

As a pattern recognition receptor, Toll-like receptor 4 (TLR4) is crucial for the development and progression of acute kidney injury (AKI). This study aims to explore whether the deubiquitinase Usp9x influences the TLR4/NF-B pathway to cause sepsis-induced acute kidney injury (S-AKI). The model of AKI was established in Sprague-Dawley rats using the cecal ligation and puncture (CLP) method, while renal tubular epithelial cell NRK-52E was stimulated with lipopolysaccharide (LPS) in vitro. All plasmids were transfected into NRK-52E cells according to the indicated group. The deubiquitinase of TLR4 was predicted by the online prediction software Ubibrowser. Subsequently, Western blot and Pearson correlation analysis identified Usp9x protein as a potential candidate. Co-IP analysis verified the interaction between TLR4 and Usp9x. Further research revealed that overexpression of Usp9x inhibited degradation of TLR4 protein by downregulating its ubiquitination modification levels. Both in vivo and in vitro experiments observed that interference with Usp9x effectively alleviated the inflammatory response and apoptosis of renal tubular epithelial cells (RTECs) induced by CLP or LPS, whereas overexpression of TLR4 reversed this situation. Transfection with sh-Usp9x in NRK-52E cells suppressed the expression of proteins associated with the TLR4/NF-κB pathway induced by LPS. Moreover, the overexpression of TLR4 reversed the effect of sh-Usp9x transfection. Therefore, the deubiquitinase Usp9x interacts with TLR4, leading to the upregulation of its expression through deubiquitination modification, and the activation of the TLR4/NF-κB signaling pathway, thereby promoting inflammation and apoptosis in renal tubular epithelial cells and contributing to sepsis-induced acute kidney injury.

Wogonin Inhibits Apoptosis and Necroptosis Induced by Nephropathogenic Infectious Bronchitis Virus in Chicken Renal Tubular Epithelial Cells.

NIBV is an acute and highly contagious virus that has a major impact on the poultry industry. Wogonin, as a flavonoid drug, has antiviral effects, but there have been no reports indicating its role in renal injury caused by NIBV infection. The aim of this study is to investigate the antiviral effect of wogonin against NIBV. Renal tubular epithelial cells were isolated and cultured, and divided into four groups: Con, Con+Wog, NIBV and NIBV+Wog. We found that wogonin significantly inhibited the copy number of NIBV and significantly alleviated NIBV-induced cell apoptosis and necrosis. Moreover, wogonin inhibited the reduction in mitochondrial membrane potential and the aberrant opening of mPTP caused by NIBV. In conclusion, wogonin can protect renal tubular epithelial cells from damage by inhibiting the replication of NIBV and preventing mitochondrial apoptosis and necroptosis induced by NIBV.

Hedgehog interacting protein activates sodium-glucose cotransporter 2 expression and promotes renal tubular epithelial cell senescence in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipRT-KO), hypothesising that local deficiency of HHIP in the renal tubules would attenuate tubular cell senescence, thereby preventing DKD tubulopathy. METHODS: Low-dose streptozotocin was employed to induce diabetes in both HhipRT-KO and control (Hhipfl/fl) mice. Transgenic mice overexpressing Hhip in renal proximal tubular cells (RPTC) (HhipRPTC-Tg) were used for validation, and primary RPTCs and human RPTCs (HK2) were used for in vitro studies. Kidney morphology/function, tubular senescence and the relevant molecular measurements were assessed. RESULTS: Compared with Hhipfl/fl mice with diabetes, HhipRT-KO mice with diabetes displayed lower blood glucose levels, normalised GFR, ameliorated urinary albumin/creatinine ratio and less severe DKD, including tubulopathy. Sodium-glucose cotransporter 2 (SGLT2) expression was attenuated in RPTCs of HhipRT-KO mice with diabetes compared with Hhipfl/fl mice with diabetes. In parallel, an increased tubular senescence-associated secretory phenotype involving release of inflammatory cytokines (IL-1ß, IL-6 and monocyte chemoattractant protein-1) and activation of senescence markers (p16, p21, p53) in Hhipfl/fl mice with diabetes was attenuated in HhipRT-KO mice with diabetes. In contrast, HhipRPTC-Tg mice had increased tubular senescence, which was inhibited by canagliflozin in primary RPTCs. In HK2 cells, HHIP overexpression or recombinant HHIP increased SGLT2 protein expression and promoted cellular senescence by targeting both ataxia-telangiectasia mutated and ataxia-telangiectasia and Rad3-related-mediated cell arrest. CONCLUSIONS/INTERPRETATION: Tubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.

Soluble Urokinase Receptor and Acute Kidney Injury.

BACKGROUND: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. METHODS: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. RESULTS: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. CONCLUSIONS: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).

Exosomal miR-7002-5p derived from highglucose-induced macrophages suppresses autophagy in tubular epithelial cells by targeting Atg9b.

Macrophage infiltration plays an important role in the progression of diabetic nephropathy (DN). Previously, we demonstrated that highglucose-stimulated macrophage-derived exosomes (HG-exo) induces proliferation and extracellular matrix accumulation in glomerular mesangial cells, but its effect on tubular cells is unclear. This study aimed to explore the role of HG-exo on renal tubular injury in DN. The results show that HG-exo could induce dysfunction, autophagy inhibition, and inflammation in mouse tubular epithelial cell (mTEC) and C57 mouse kidney. Moreover, miR-7002-5p was differentially expressed in HG-exo based on miRNAs sequencing and bioinformatics analysis. A dual-luciferase reporter assay confirmed that Atg9b was the direct target gene of miR-7002-5p. Further experimentation showed that miR-7002-5p inhibition in vivo and vitro reserves HG-exo effects. These results demonstrated that HG-exo carries excessive miR-7002-5p and inhibits autophagy through targeting Atg9b; this process then induces renal tubular dysfunction and inflammation. In conclusion, our study clarifies the important role of macrophage-derived exosomes in DN and is expected to provide new insight on DN prevention and treatment.

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule.

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.

Molecular detection of maturation stages in the developing kidney.

Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.

Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney.

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.

Extracellular vesicles regulate purinergic signaling and epithelial sodium channel expression in renal collecting duct cells.

Purinergic signaling regulates several renal physiological and pathophysiological processes. Extracellular vesicles (EVs) are nanoparticles released by most cell types, which, in non-renal tissues, modulate purinergic signaling. The aim of this study was to investigate the effect of EVs from renal proximal tubule (HK2) and collecting duct cells (HCD) on intra- and intersegment modulation of extracellular ATP levels, the underlying molecular mechanisms, and the impact on the expression of the alpha subunit of the epithelial sodium channel (αENaC). HK2 cells were exposed to HK2 EVs, while HCD cells were exposed to HK2 and HCD EVs. Extracellular ATP levels and αENaC expression were measured by chemiluminescence and qRT-PCR, respectively. ATPases in EV populations were identified by mass spectrometry. The effect of aldosterone was assessed using EVs from aldosterone-treated cells and urinary EVs (uEVs) from primary aldosteronism (PA) patients. HK2 EVs downregulated ectonucleoside-triphosphate-diphosphohydrolase-1 (ENTPD1) expression, increased extracellular ATP and downregulated αENaC expression in HCD cells. ENTPD1 downregulation could be attributed to increased miR-205-3p and miR-505 levels. Conversely, HCD EVs decreased extracellular ATP levels and upregulated αENaC expression in HCD cells, probably due to enrichment of 14-3-3 isoforms with ATPase activity. Pretreatment of donor cells with aldosterone or exposure to uEVs from PA patients enhanced the effects on extracellular ATP and αENaC expression. We demonstrated inter- and intrasegment modulation of renal purinergic signaling by EVs. Our findings postulate EVs as carriers of information along the renal tubules, whereby processes affecting EV release and/or cargo may impact on purinergically regulated processes.

Icariin inhibits epithelial mesenchymal transition of renal tubular epithelial cells via regulating the miR-122-5p/FOXP2 axis in diabetic nephropathy rats.

Epithelial mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs) dominates the pathology of diabetic nephropathy (DN). microRNAs (miRNAs) can influence the fate of DN via regulation of EMT. This study aimed to analyze the role of Icariin (ICA) in EMT of RTECs, hoping to provide theoretical basis for DN management. The DN rat model was established using streptozocin, followed by ICA treatment, histopathological observation, and detection of creatinine and blood urea nitrogen. In vitro cell models were established using high glucose (HG), followed by assessment of cell proliferation, apoptosis, and migration, and E-cadherin, α-SMA, miR-122-5p, and FOXP2 expressions. Cells were transfected with miR-122-5p mimics or si-FOXP2 for joint experiments with ICA. The targeting relationship between miR-122-5p and FOXP2 was verified. ICA repaired renal dysfunctions and glomerular structure abnormities of DN rats in a dose-dependent manner. In vitro, ICA improved proliferation while suppressed migration, apoptosis, and EMT of RTECs. miR-122-5p was up-regulated in DN rats and suppressed by ICA, and miR-122-5p targeted FOXP2. miR-122-5p up-regulation or FOXP2 down-regulation reversed the protective effects of ICA on HG-induced RTECs. Overall, our finding ascertained that ICA inhibited miR-122-5p to promote FOXP2 transcription, thereby attenuating EMT of RTECs and renal injury in DN rats.

Differential lipidomics of HK-2 cells and exosomes under high glucose stimulation.

Abnormal cellular lipid metabolism has a very important role in the occurrence and progression of diabetic kidney disease (DKD). However, the lipid composition and differential expression by high glucose stimulation of renal tubular cells and their exosomes, which is a vital part of the development of DKD, are largely unknown. In this study, based on targeted lipid analysis by isotope labeling and tandem mass spectrometry, a total of 421 and 218 lipid species were quantified in HK-2 cells and exosomes, respectively. More importantly, results showed that GM3 d18:1/22:0, GM3 d18:1/16:0, GM3 d18:0/16:0, GM3 d18:1/22:1 were significantly increased, while LPE18:1, LPE, CL66:4 (16:1), BMP36:3, CL70:7 (16:1), CL74:8 (16:1) were significantly decreased in high glucose-stimulated HK-2 cells. Also, PG36:1, FFA22:5, PC38:3, SM d18:1/16:1, CE-16:1, CE-18:3, CE-20:5, and CE-22:6 were significantly increased, while GM3 d18:1/24:1, GM3 were significantly decreased in exosomes secreted by high glucose-stimulated HK-2 cells. Furthermore, TAG, PC, CL were decreased significantly in the exosomes comparing with the HK-2 cells, and LPA18:2, LPI22:5, PG32:2, FFA16:1, GM3 d18:1/18:1, GM3 d18:1/20:1, GM3 d18:0/20:0, PC40:6p, TAG52:1(18:1), TAG52:0(18:0), CE-20:5, CE-20:4, CE-22:6 were only found in exosomes. In addition, the expression of PI4P in HK-2 cells decreased under a high glucose state. These data may be useful to provide new targets for exploring the mechanisms of DKD.

Abnormally decreased renal Klotho is linked to endoplasmic reticulum-associated degradation in mice.

Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded proteins retained in the ER, is implicated in various diseases including chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial fibrosis (RIF). Methods: Following establishment of a mouse RIF model by unilateral ureteral obstruction (UUO), a specific ERAD inhibitor, Eeyarestatin I (EerI), was administered to experimental animals by intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by enzyme-linked immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1, glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and protein disulfide isomerase (PDI). HK-2 cells were exposed to transforming growth factor (TGF)-ß1 with or without EerI, and expressions of related proteins including Klotho, Derlin-1, GRP78, ATF4 and PDI were determined by western blotting analyses. Results: UUO induced severe kidney injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-ß1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded proteins, subsequently contributing to the additional production of mature Klotho protein. Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.

The Therapeutic Potential of Zinc-Alpha2-Glycoprotein (AZGP1) in Fibrotic Kidney Disease.

Chronic kidney disease (CKD) is characterized by a long-term loss of kidney function and, in most cases, by progressive fibrosis. Zinc-alpha2-glycoprotein (AZGP1) is a secreted protein, which is expressed in many different tissues and has been associated with a variety of functions. In a previous study, we have shown in cell culture and in AZGP1 deficient mice that AZGP1 has protective anti-fibrotic effects. In the present study, we tested the therapeutic potential of an experimental increase in AZGP1 using two different strategies. (1) C57Bl/6J mice were treated systemically with recombinant AZGP1, and (2) a transgenic mouse strain was generated to overexpress AZGP1 conditionally in proximal tubular cells. Mice underwent unilateral uretic obstruction as a pro-fibrotic kidney stress model, and kidneys were examined after 14 days. Recombinant AZGP1 treatment was accompanied by better preservation of tubular integrity, reduced collagen deposition, and lower expression of injury and fibrosis markers. Weaker but similar tendencies were observed in transgenic AZGP1 overexpressing mice. Higher AZGP1 levels led to a significant reduction in stress-induced accumulation of tubular lipid droplets, which was paralleled by improved expression of key players in lipid metabolism and fatty acid oxidation. Together these data show beneficial effects of elevated AZGP1 levels in fibrotic kidney disease and highlight a novel link to tubular cell lipid metabolism, which might open up new opportunities for CKD treatment.

Protective Effect of Quercetin 3-O-Glucuronide against Cisplatin Cytotoxicity in Renal Tubular Cells.

Quercetin, a flavonoid with promising therapeutic potential, has been shown to protect from cisplatin nephrotoxicity in rats following intraperitoneal injection, but its low bioavailability curtails its prospective clinical utility in oral therapy. We recently developed a micellar formulation (P-quercetin) with enhanced solubility and bioavailability, and identical nephroprotective properties. As a first aim, we herein evaluated the oral treatment with P-quercetin in rats, which displayed no nephroprotection. In order to unravel this discrepancy, quercetin and its main metabolites were measured by HPLC in the blood and urine after intraperitoneal and oral administrations. Whilst quercetin was absorbed similarly, the profile of its metabolites was different, which led us to hypothesize that nephroprotection might be exerted in vivo by a metabolic derivate. Consequently, we then aimed to evaluate the cytoprotective capacity of quercetin and its main metabolites (quercetin 3-O-glucoside, rutin, tamarixetin, isorhamnetin and quercetin 3-O-glucuronide) against cisplatin toxicity, in HK-2 and NRK-52E tubular cell lines. Cells were incubated for 6 h with quercetin, its metabolites or vehicle (pretreatment), and subsequently 18 h in cotreatment with 10-300 µM cisplatin. Immediately after treatment, cell cultures were subject to the MTT technique as an index of cytotoxicity and photographed under light microscopy for phenotypic assessment. Quercetin afforded no direct cytoprotection and quercetin-3-O-glucuronide was the only metabolite partially preventing the effect of cisplatin in cultured tubule cells. Our results identify a metabolic derivative of quercetin contributing to its nephroprotection and prompt to further explore exogenous quercetin-3-O-glucuronide in the prophylaxis of tubular nephrotoxicity.

A new view of macula densa cell microanatomy.

Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation, and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells, were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy of the rat, rabbit, and human kidney. An elaborate network of major and minor cell processes, here named maculapodia, were found at the cell base, projecting toward other MD cells and the glomerular vascular pole. The extent of maculapodia showed upregulation by low dietary salt intake and the female sex. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. Electron microscopy of rat, rabbit, and human kidneys and three-dimensional volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD cells and between MD and other target cells.NEW & NOTEWORTHY This study illuminated a physiologically regulated dense network of basal cell major and minor processes (maculapodia) in macula densa (MD) cells. The newly identified dynamic and secretory features of these microanatomical structures suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD and other target cells. Detailed characterization of the function and molecular details of MD cell intercellular communications and their role in physiology and disease warrant further studies.