Development of osmotic vacuolization of proximal tubular epithelial cells following treatment with sodium-glucose transport protein 2 inhibitors in type II diabetes mellitus patients-3 case reports.

We encountered 3 cases of acute kidney injury that occurred after treatment with a SGLT2 inhibitor. In case 1, serum creatinine increased from 1.65 to 3.0 mg/dL, in case 2, serum creatinine increased from 1.03 to 1.21 mg/dL, and in case 3, serum creatinine increased from 0.8 to 1.1 mg/dL. Renal biopsy showed isometric vacuolization on tubules, that was completely negative for Periodic acid-Schiff (PAS) stain in case 1, and was partially negative for PAS stain in case 2 and 3, consistent with osmotic vacuolization. Immunohistochemical analysis showed positive staining for CD138 and CD10 indicating the proximal tubules in the vacuolar lesions. 3 patients were obese with body mass index of more than 30, and showed an increase in serum renin. In conclusion, in type II diabetes mellitus (T2DM), individuals that remain within their standard weight range, SGLT2 inhibitor treatment does not result in osmotic vacuolization of proximal tubular epithelial cells and AKI. However, treatment with a SGLT2 inhibitor may cause damage of the proximal tubules resulting in AKI in T2DM individuals who do not remain within their standard weight range, due to an overdose lavage of sugar in the urine and dehydration.

Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3.

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.

Involvement of necroptosis in contrast-induced nephropathy in a rat CKD model.

BACKGROUND: The risk of contrast-induced nephropathy (CIN) is high in patients with chronic kidney disease (CKD). However, the mechanism of CIN in CKD is not fully understood. Here, we prepared a clinically relevant model of CIN and examined the role of necroptosis, which potentially cross-talks with autophagy, in CIN. METHODS: In Sprague-Dawley rats, CKD was induced by subtotal nephrectomy (SNx, 5/6 nephrectomy) 4 weeks before induction of CIN. CIN was induced by administration of a contrast medium (CM), iohexol, following administration of indomethacin and N-omega-Nitro-L-arginine methyl ester. Renal function and tissue injuries were assessed 48 h after CM injection. RESULTS: Serum creatinine (s-Cre) and BUN were increased from 0.28 ± 0.01 to 0.52 ± 0.02 mg/dl and from 15.1 ± 0.7 to 29.2 ± 1.2 mg/dl, respectively, after SNx alone. CM further increased s-Cre and BUN to 0.69 ± 0.03 and 37.2 ± 2.1, respectively. In the renal tissue after CM injection, protein levels of receptor-interacting serine/threonine-protein kinase (RIP) 1, RIP3, cleaved caspase 3, and caspase 8 were increased by 64 ~ 212%, while there was reduction in LC3-II and accumulation of p62. Necrostatin-1, an RIP1 inhibitor, administered before and 24 h after CM injection significantly suppressed elevation of s-Cre, BUN and urinary albumin levels, kidney injury molecule-1 expression and infiltration of CD68-positive macrophages in renal tissues after CM injection. CONCLUSION: The results suggest that necroptosis of proximal tubular cells contributes to CIN in CKD and that suppression of protective autophagy by pro-necroptotic signaling may also be involved.

Characterization of ferroptosis in kidney tubular cell death under diabetic conditions.

Kidney tubular cell death induced by transforming growth factor-ß1 (TGF-ß1) is known to contribute to diabetic nephropathy, a major complication of diabetes. Caspase-3-dependent apoptosis and caspase-1-dependent pyroptosis are also involved in tubular cell death under diabetic conditions. Recently, ferroptosis, an atypical form of iron-dependent cell death, was reported to cause kidney disease, including acute kidney injury. Ferroptosis is primed by lipid peroxide accumulation through the cystine/glutamate antiporter system Xc- (xCT) and glutathione peroxidase 4 (GPX4)-dependent mechanisms. The aim of this study was to evaluate the role of ferroptosis in diabetes-induced tubular injury. TGF-ß1-stimulated proximal tubular epithelial cells and diabetic mice models were used for in vitro and in vivo experiments, respectively. xCT and GPX4 expression, cell viability, glutathione concentration, and lipid peroxidation were quantified to indicate ferroptosis. The effect of ferroptosis inhibition was also assessed. In kidney biopsy samples from diabetic patients, xCT and GPX4 mRNA expression was decreased compared to nondiabetic samples. In TGF-ß1-stimulated tubular cells, intracellular glutathione concentration was reduced and lipid peroxidation was enhanced, both of which are related to ferroptosis-related cell death. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, alleviated TGF-ß1-induced ferroptosis. In diabetic mice, kidney mRNA and protein expressions of xCT and GPX4 were reduced compared to control. Kidney glutathione concentration was decreased, while lipid peroxidation was increased in these mice, and these changes were alleviated by Fer-1 treatment. Ferroptosis is involved in kidney tubular cell death under diabetic conditions. Ferroptosis inhibition could be a therapeutic option for diabetic nephropathy.

Reduced lifespan of erythrocytes in Dahl/Salt sensitive rats is the cause of the renal proximal tubule damage.

We studied the mechanisms of anemia and the influence of anemia on renal pathology in Dahl/Salt Sensitive (Dahl/SS) rat, a model of cardio-renal-anemia syndrome. Erythrocyte lifespan was shortened and associated with decreased hemoglobin level in the Dahl/SS rats given high-salt diet. Serum haptoglobin decreased, reticulocytes increased, and erythropoiesis in the bone marrow and extramedullary hematopoiesis in the spleen was markedly stimulated by increased serum erythropoietin in them. As a mechanism of hemolysis, we investigated the incidence of eryptosis, suicidal death of erythrocytes. Eryptosis was increased, and red blood cell-derived microparticles, small particle which are generated in hemolytic disease, were also increased in Dahl/SS rats fed with high-salt diet. Deposition of hemosiderin and mitochondrial morphologic abnormality, a sign of ferroptosis, in proximal renal tubules was associated with intravascular hemolysis. Treatment with deferasirox, an oral iron chelator, reduced the renal proximal tubular injury and the glomerular sclerosis in Dahl/SS rats fed with high-salt diet. In conclusion, reduced half-life of erythrocytes induced by hemolysis is the major cause of anemia in Dahl/SS rat. Iron accumulation induced by hemolysis causes renal proximal tubule injury and accelerates renal damage in this model.

Calcifying nanoparticles induce cytotoxicity mediated by ROS-JNK signaling pathways.

Calcifying nanoparticles (CNPs) play an important role in kidney stone formation, but the mechanism(s) are unclear. CNPs were isolated and cultured from midstream urine of patients with kidney stones. CNP morphology and characteristics were examined by electron microscopy and electrophoresis analysis. Chemical composition was analyzed using energy-dispersive X-ray microanalysis and Western blotting. Human renal proximal convoluted tubule cell (HK-2) cultures were exposed to CNPs for 0, 12 and 72 h, and production of reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis levels were evaluated. CNPs isolated from patients showed classical morphology, the size range of CNPs were 15-500 nm and negative charge; they were found to contain fetuin-A. Exposure of HK-2 cells to CNPs induced ROS production, decreased mitochondrial membrane potential and decreased cell viability. Transmission electron microscopy showed that CNPs can enter the cell by phagocytosis, and micrographs revealed signs of apoptosis and autophagy. CNPs increased the proportion of apoptotic cells, down-regulated Bcl-2 expression and up-regulated Bax expression. CNPs also up-regulated expression of LC3-B, Beclin-1and p-JNK.CNPs are phagocytosed by HK-2 cells, leading to autophagy, apoptosis and ROS production, in part through activation of JNK signaling pathways. ROS and JNK pathways may contribute to CNP-induced cell injury and kidney stone formation.

Monoclonal light chain crystalline podocytopathy and tubulopathy associated with monoclonal gammopathy of renal significance: a case report and literature review.

BACKGROUND: Monoclonal gammopathy of renal significance (MGRS) is a recently defined group of renal diseases caused by monoclonal immunoglobulin secreted by nonmalignant proliferative B cell or plasma cell. Monoclonal immunoglobulin can form different types of structures deposited in renal tissue, including fibrils, granules, microtubules, crystals and casts, and has mostly been reported in multiple myeloma patients. Here we report a rare case with κ light chain crystals in both podocytes and tubular epithelial cells associated with MGRS, which adds more information to the spectrum of MGRS-related renal diseases. CASE PRESENTATION: A 53-year old woman presented with albumin-predominant moderate proteinuria and renal failure. She had monoclonal IgGκ in the serum and monoclonal IgGκ plus free κ in the urine. Multiple myeloma and lymphoproliferative disorders were excluded. Renal biopsy confirmed κ-restricted crystal-storing renal disease involving the podocytes and proximal tubular epithelial cells. The patient was treated with bortezomib followed by lenalidomide-based chemotherapy, and renal function was stable after 1 year of follow-up. CONCLUSIONS: This is a rare case of combined crystalline podocytopathy and tubulopathy associated with MGRS, in which diagnosis was dependent on electron and immuno-electron microscopy.

Ultrastructural identification of a proximal tubulopathy without crystals in a relapsed multiple myeloma patient.

A multiple myeloma patient, who had been treated with a hematopoietic stem cell transplant, underwent a renal biopsy for investigation of a possible relapse of disease as indicated by increased serum creatinine and positive urinary Bence-Jones protein containing increased kappa light chain. Paraprotein-related renal disease was not evident by light microscopy or immunofluorescence microscopy however electron microscopy demonstrated a proximal tubulopathy with intracytoplasmic non-crystalline inclusions. The ultrastructural findings suggested possible end-organ involvement by the disease and follow-up studies subsequently revealed a relapsed multiple myeloma in the patient. The case exemplifies the usefulness of electron microscopy in detecting paraproteins that, in some instances, may be difficult to demonstrate by other techniques.

Nephron-specific knockout of TMEM16A leads to reduced number of glomeruli and albuminuria.

TMEM16A is a transmembrane protein from a conserved family of calcium-activated proteins that is highly expressed in the kidney. TMEM16A confers calcium-activated chloride channel activity, which is of importance for various cellular functions in secretory epithelia and involved in secretion-dependent renal cyst growth. However, its specific function in renal physiology has remained elusive so far. Therefore, we generated conditional nephron-specific TMEM16A-knockout mice and found that these animals suffered from albuminuria. Kidney histology demonstrated an intact corticomedullary differentiation and absence of cysts. Electron microscopy showed a normal slit diaphragm. However, the total number of glomeruli and total nephron count was decreased in TMEM16A-knockout animals. At the same time, glomerular diameter was increased, presumably as a result of the hyperfiltration in the remaining glomeruli. TUNEL and PCNA stainings showed increased cell death and increased proliferation. Proximal tubular cilia were intact in young animals, but the number of properly ciliated cells was decreased in older, albuminuric animals. Taken together, our data suggest that TMEM16A may be involved in ureteric bud branching and proper nephron endowment. Loss of TMEM16A resulted in reduced nephron number and, subsequently, albuminuria and tubular damage.

Reabsorption in the proximal tubuli-ultrastructural evidence for a novel aspect of renal VEGF trafficking.

Many clinical and experimental studies have revealed VEGF as an important factor in the pathophysiology of renal damage during diabetes mellitus (DM). Anti-VEGF therapy is in clinical use for treatment of DM and other diabetes-related (and unrelated) diseases. Nevertheless, little is known about the metabolism of VEGF in the kidneys. In order to determine the ultrastructural localization of VEGF in the kidney, we study the distribution of VEGF in the kidney of rats by using immunogold immunohistochemistry. Our light-microscopic data showed remarkable re-distribution of VEGF in proximal tubular cells (PTCs) during prolonged hyperglycemia, a DM type 2 model (DM2), which was confirmed by transmission electron microscopy (TEM) findings. TEM findings revealed an initial presence of VEGF in the vesicular transport apparatus of PTCs in healthy rats and its gradual translocation to the apical membrane of PTCs after renal damage caused by high sucrose treatment. The presented data add to our understanding of kidney VEGF trafficking, providing novel insight into the renal metabolism and pharmacodynamics of the cytokine. This could have a high impact on the use of VEGF and anti-VEGF therapy in different renal diseases.

Mitochondria-mediated disturbance of fatty acid metabolism in proximal tubule epithelial cells leads to renal interstitial fibrosis.

OBJECTIVE: To investigate the role of mitochondria-mediated fatty acid metabolism in proximal tubule cells in renal interstitial fibrosis. MATERIALS AND METHODS: Intraperitoneal injection of folate was performed to induce renal interstitial fibrosis in mice. Polymerase chain reaction (PCR) was used to detect the expression of cytochrome c oxidase subunit IV (COX4IL) and phosphoenolpyruvate carboxykinase 1 (PCK1) in samples. Electron microscope was used to detect the activity of mitochondria. Serum creatinine and urea nitrogen were chosen as evaluation criteria for renal function. Western-blotting was used to detect protein expression of cells. Immunohistochemistry was used to test renal structure and deposition of collagen. RESULTS: In renal interstitial fibrosis, mitochondria mediated the dysfunction and the promotion of tubulointerstitial fatty acid metabolism. Besides, it could also reduce renal interstitial fibrosis and alleviate the fatty acid metabolism of tubulointerstitial fibrosis. CONCLUSIONS: Mitochondrial dysfunction induced fatty acid metabolism is an important factor to promote the progress of renal interstitial fibrosis. Intervention of related targets of fatty acid metabolism is expected to become a new treatment for renal interstitial fibrosis.

Injury induced expression of caveolar proteins in human kidney tubules - role of megakaryoblastic leukemia 1.

BACKGROUND: Caveolae are membrane invaginations measuring 50-100 nm. These organelles, composed of caveolin and cavin proteins, are important for cellular signaling and survival. Caveolae play incompletely defined roles in human kidneys. Induction of caveolin-1/CAV1 in diseased tubules has been described previously, but the responsible mechanism remains to be defined. METHODS: Healthy and atrophying human kidneys were stained for caveolar proteins, (caveolin 1-3 and cavin 1-4) and examined by electron microscopy. Induction of caveolar proteins was studied in isolated proximal tubules and primary renal epithelial cells. These cells were challenged with hypoxia or H2O2. Primary tubular cells were also subjected to viral overexpression of megakaryoblastic leukemia 1 (MKL1) and MKL1 inhibition by the MKL1 inhibitor CCG-1423. Putative coregulators of MKL1 activity were investigated by Western blotting for suppressor of cancer cell invasion (SCAI) and filamin A (FLNA). Finally, correlative bioinformatic studies of mRNA expression of caveolar proteins and MKL1 were performed. RESULTS: In healthy kidneys, caveolar proteins were expressed by the parietal epithelial cells (PECs) of Bowman's capsule, endothelial cells and vascular smooth muscle. Electron microscopy confirmed caveolae in the PECs. No expression was seen in proximal tubules. In contrast, caveolar proteins were expressed in proximal tubules undergoing atrophy. Caveolar proteins were also induced in cultures of primary epithelial tubular cells. Expression was not enhanced by hypoxia or free radical stress (H2O2), but proved sensitive to inhibition of MKL1. Viral overexpression of MKL1 induced caveolin-1/CAV1, caveolin-2/CAV2 and SDPR/CAVIN2. In kidney tissue, the mRNA level of MKL1 correlated with the mRNA levels for caveolin-1/CAV1, caveolin-2/CAV2 and the archetypal MKL1 target tenascin C (TNC), as did the MKL1 coactivator FLNA. Costaining for TNC as readout for MKL1 activity demonstrated overlap with caveolin-1/CAV1 expression in PECs as well as in atrophic segments of proximal tubules. CONCLUSIONS: Our findings support the view that MKL1 contributes to the expression of caveolar proteins in healthy kidneys and orchestrates the induction of tubular caveolar proteins in renal injury.

The lipid 5-phoshatase SHIP2 controls renal brush border ultrastructure and function by regulating the activation of ERM proteins.

The microvillus brush border on the renal proximal tubule epithelium allows the controlled reabsorption of solutes that are filtered through the glomerulus and thus participates in general body homeostasis. Here, using the lipid 5-phosphatase Ship2 global knockout mice, proximal tubule-specific Ship2 knockout mice, and a proximal tubule cell model in which SHIP2 is inactivated, we show that SHIP2 is a negative regulator of microvilli formation, thereby controlling solute reabsorption by the proximal tubule. We found increased PtdIns(4,5)P2 substrate and decreased PtdIns4P product when SHIP2 was inactivated, associated with hyperactivated ezrin/radixin/moesin proteins and increased Rho-GTP. Thus, inactivation of SHIP2 leads to increased microvilli formation and solute reabsorption by the renal proximal tubule. This may represent an innovative therapeutic target for renal Fanconi syndrome characterized by decreased reabsorption of solutes by this nephron segment.

Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells.

Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity.

Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction.

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

Puerarin protects against cadmium-induced proximal tubular cell apoptosis by restoring mitochondrial function.

Puerarin (PU) is a potent free radical scavenger with a protective effect in nephrotoxin-mediated oxidative damage. Here, we show a novel molecular mechanism by which PU exerts its anti-apoptotic effects in cadmium (Cd)-exposed primary rat proximal tubular (rPT) cells. Morphological assessment and flow cytometric analysis revealed that PU significantly decreased Cd-induced apoptotic cell death of rPT cells. Administration of PU protected cells against Cd-induced depletion of mitochondrial membrane potential (ΔΨm) and lipid peroxidation. Cd-mediated mitochondrial permeability transition pore (MPTP) opening, disruption of mitochondrial ultrastructure, mitochondrial cytochrome c (cyt-c) release, caspase-3 activation and subsequently poly ADP-ribose polymerase (PARP) cleavage could be effectively blocked by the addition of PU. Moreover, up-regulation of Bcl-2 and down-regulation of Bax and hence increased Bcl-2/Bax ratio were observed with the PU administration. In addition, PU reversed Cd-induced ATP depletion by restoring ΔΨm to affect ATP production and by regulating expression levels of ANT-1 and ANT-2 to improve ATP transport. In summary, PU inhibited Cd-induced apoptosis in rPT cells by ameliorating the mitochondrial dysfunction.