Cells Of Proximal Convoluted Tubules Research Articles

74 Unraveling the Complexities of Obesity in Clear Cell Renal Cell Carcinoma Carcinogenesis

Abstract Background Obesity has been identified as an established risk factor in the development of clear cell renal cell carcinoma (ccRCC). ccRCC arises from the proximal convoluted tubule (PCT) of the kidney which is also the site of obesity-associated chronic kidney disease. Obesity can lead to chronic cellular insults which may lead to DNA injury and whether these mechanisms are critical to cancer initiation in the kidney is unclear. One potential way that obesity can lead to cancer is by altering lipid metabolism, including increasing circulating saturated fatty acids. In particular, palmitic acid is known to be lipotoxic to proximal tubules of the kidney. Understanding how free fatty acids initiate cancer is critical to understanding the role of obesity in cancer initiation and development. We hypothesize that chronic injury and repair due to excess FFA exposure could lead to ccRCC carcinogenesis and aim to define the metabolic phenotype of obesity associated ccRCC. Methods Using publicly available data from The KIRC Cancer Genome Atlas Program (TCGA) and clinicopathologic data, we compared the RNAseq profiles of obese (BMI >= 30) to normal weight (BMI =< 25) sample origins. Of the 337, a total of 333 had BMI available for analysis. We evaluated differences in Hallmark and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Using DESeq, we adjusted for age and sex. In vitro, HK PCT cells were cultured in DMEM+10%FBS treated with bovine serum albumin (BSA)-palmitic acid complex for 48 hrs. We measured cell viability using Alamar Blue. Results were normalized to controls with BSA only. We extracted protein and performed western blots to identify DNA damage (pATM, H2AX) and endoplasmic reticulum stress (ATF4, eFI2alpha). Results Most of the patients in our clinical data cohort are male (64.4%), while 45.4% of patients are categorized by BMI as obese and 33.2% are categorized as normal. We utilized 333 individuals from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to link genomic information to pathways associated with fatty acid metabolism as well as other cancer-associated hallmark metabolic pathways. We confirmed that excess free fatty acid supplementation leads to decreased cell viability in renal proximal tubule cells and renal embryonal cells and aim to assess differences in gene expression and metabolomic variation with fatty acid supplementation. Conclusions Through the investigation of these questions, we hope to improve understanding of obesity associated ccRCC and ultimately impact oncological outcomes for patients with this cancer. Understanding changes in lipid metabolism around ccRCC initiation will help identify novel pathways for pharmacologic intervention at earlier stages of the disease. DOD CDMRP Funding: yes

Proximal convoluted tubule cells-derived exosomes attenuate aging-related vascular dysfunction

Aging is an identified risk factor for cardiovascular disease. Aging is associated with increased prevalence of endothelial dysfunction and arterial stiffening. Exosomes are bi-lipid nanoscale vesicles that play important roles in intercellular communications. The purpose of this study was to determine whether administration of renal proximal convoluted tubule cells (PCT)-derived exosomes affects vascular and renal function in aging mice. Aging (24 months old) and young mice (3 months old) were treated with PCT-derived exosomes via non-invasive injection into the kidney 3 times a week for 2 weeks. Administration of PCT-derived exosomes did not affect blood pressure or urinary Na+, K+, and Ca2+ excretions, indicating that PCT-derived exosomes did not affect kidney function. Pulse wave velocity (PWV) was increased in aging mice, indicating arterial stiffening. Interestingly, treatment with PCT-exosomes effectively reduced aging-associated arterial stiffening. In addition, aortic endothelium-dependent relaxing (EDR) responses to acetylcholine (Ach) were reduced in aging mice, indicating endothelial dysfunction. Treatment with PCT-derived exosomes rescued aging-associated aortic endothelial dysfunction. Mechanistically, aortic expression of VCAM1, ICAM1, TNFα, and MCP-1 was upregulated in aging mice, indicating vascular inflammation. Notably, aging-associated vascular inflammation was largely attenuated by delivery of PCT-derived exosomes. Our results suggest that PCT-derived exosomes improves aging-associated arterial stiffening and vascular dysfunction partly via inhibiting vascular inflammation. The findings may help develop novel therapeutic strategies for aging-associated vascular dysfunction using PCT-derived exosomes. This work is supported by NIH research grants R01 HL154147 and AG062375. 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.

XBP1-mediated transcriptional regulation of SLC5A1 in human epithelial cells in disease conditions

BackgroundSodium-Glucose cotransporter 1 and 2 (SGLT1/2) belong to the family of glucose transporters, encoded by SLC5A1 and SLC5A2, respectively. SGLT2 is almost exclusively expressed in the renal proximal convoluted tubule cells. SGLT1 is expressed in the kidneys but also in other organs throughout the body. Many SGLT inhibitor drugs have been developed based on the mechanism of blocking glucose (re)absorption mediated by SGLT1/2, and several have gained major regulatory agencies’ approval for treating diabetes. Intriguingly these drugs are also effective in treating diseases beyond diabetes, for example heart failure and chronic kidney disease. We recently discovered that SGLT1 is upregulated in the airway epithelial cells derived from patients of cystic fibrosis (CF), a devastating genetic disease affecting greater than 70,000 worldwide.ResultsIn the present work, we show that the SGLT1 upregulation is coupled with elevated endoplasmic reticulum (ER) stress response, indicated by activation of the primary ER stress senor inositol-requiring protein 1α (IRE1α) and the ER stress-induced transcription factor X-box binding protein 1 (XBP1), in CF epithelial cells, and in epithelial cells of other stress conditions. Through biochemistry experiments, we demonstrated that the spliced form of XBP1 (XBP1s) acts as a transcription factor for SLC5A1 by directly binding to its promoter region. Targeting this ER stress → SLC5A1 axis by either the ER stress inhibitor Rapamycin or the SGLT1 inhibitor Sotagliflozin was effective in attenuating the ER stress response and reducing the SGLT1 level in these cellular model systems.ConclusionsThe present work establishes a causal relationship between ER stress and SGLT1 upregulation and provides a mechanistic explanation why SGLT inhibitor drugs benefit diseases beyond diabetes.

A comparative modeling study of the mitochondrial function of the proximal tubule and thick ascending limb cells in the rat kidney.

To reabsorb >99% of the glomerular filtrate, the metabolic demand of the kidney is high. Interestingly, renal blood flow distribution exhibits marked inhomogeneity, with typical tissue oxygen tension (Po2) of 50-60 mmHg in the well-perfused cortex and 10-20 mmHg in the inner medulla. Cellular fluid composition and acidity also varies substantially. To understand how different renal epithelial cells adapt to their local environment, we have developed and applied computational models of mitochondrial function of proximal convoluted tubule cell (baseline Po2 = 50 mmHg, cytoplasmic pH = 7.20) and medullary thick ascending limb (mTAL) cell (baseline Po2 = 10 mmHg, cytoplasmic pH = 6.85). The models predict key cellular quantities, including ATP generation, P/O (phosphate/oxygen) ratio, proton motive force, electrical potential gradient, oxygen consumption, the redox state of key electron carriers, and ATP consumption. Model simulations predict that close to their respective baseline conditions, the proximal tubule and mTAL mitochondria exhibit qualitatively similar behaviors. Nonetheless, because the mTAL mitochondrion has adapted to a much lower Po2, it can sustain a sufficiently high ATP production at Po2 as low as 4-5 mmHg, whereas the proximal tubule mitochondria would not. Also, because the mTAL cytosol is already acidic under baseline conditions, the proton motive force (pmf) exhibits higher sensitivity to further acidification. Among the different pathways that lead to oxidative phosphorylation impairment, the models predict that both the proximal tubule and mTAL mitochondria are most sensitive to reductions in Complex III activity.NEW & NOTEWORTHY Tissue fluid composition varies substantially within the mammalian kidney. The renal cortex is well perfused and pH neutral, whereas some medullary regions are hypoxic and acidic. How do these environments affect the mitochondrial function of proximal convoluted tubule and medullary thick ascending limb cells, which reside in the cortex and medulla, respectively? This computational modeling study demonstrates that these mitochondria can adapt to their contrasting environments and exhibit different sensitivities to perturbations to local environments.

Sugarcane ash and sugarcane ash-derived silica nanoparticles alter cellular metabolism in human proximal tubular kidney cells

Multiple epidemics of chronic kidney disease of an unknown etiology (CKDu) have emerged in agricultural communities around the world. Many factors have been posited as potential contributors, but a primary cause has yet to be identified and the disease is considered likely multifactorial. Sugarcane workers are largely impacted by disease leading to the hypothesis that exposure to sugarcane ash produced during the burning and harvest of sugarcane could contribute to CKDu. Estimated exposure levels of particles under 10 μm (PM10) have been found to be exceptionally high during this process, exceeding 100 μg/m3 during sugarcane cutting and averaging ∼1800 μg/m3 during pre-harvest burns. Sugarcane stalks consist of ∼80% amorphous silica and generate nano-sized silica particles (∼200 nm) following burning. A human proximal convoluted tubule (PCT) cell line was subjected to treatments ranging in concentration from 0.025 μg/mL to 25 μg/mL of sugarcane ash, desilicated sugarcane ash, sugarcane ash-derived silica nanoparticles (SAD SiNPs) or manufactured pristine 200 nm silica nanoparticles. The combination of heat stress and sugarcane ash exposure on PCT cell responses was also assessed. Following 6–48 h of exposure, mitochondrial activity and viability were found to be significantly reduced when exposed to SAD SiNPs at concentrations 2.5 μg/mL or higher. Oxygen consumption rate (OCR) and pH changes suggested significant alteration to cellular metabolism across treatments as early as 6 h following exposure. SAD SiNPs were found to inhibit mitochondrial function, reduce ATP generation, increase reliance on glycolysis, and reduce glycolytic reserve. Metabolomic analysis revealed several cellular energetics pathways (e.g., fatty acid metabolism, glycolysis, and TCA cycle) are significantly altered across ash-based treatments. Heat stress did not influence these responses. Such changes indicate that exposure to sugarcane ash and its derivatives can promote mitochondrial dysfunction and disrupt metabolic activity of human PCT cells.

63 A Case for Amorphous Silica Nanoparticle Exposure in the Development of Chronic Kidney Disease of Unknown Etiology

Abstract We have hypothesized that sugarcane burning in developing countries contributes to an endemic kidney pathology in agricultural workers called chronic kidney disease of an unknown etiology (CKDu). Elemental analysis shows that sugarcane stalks contain a high percentage of amorphous silica (SiO2). Therefore, we hypothesized that burning of sugarcane generates silica nanoparticles (SiNPs) that present an inhalation hazard to these agricultural workers. To determine if SiNPs are present in sugarcane ash and kidney biopsies from agricultural workers, we utilized single particle inductively coupled plasma mass spectrometry and found SiNPs, which ranged in size from 190-212 nm, in sugarcane ash. In a small cohort of CKDu patients from El Salvador and Mexico, we found a significant increase in the number of SiNPs in their kidney biopsies within a similar size range as observed in sugarcane ash. Additionally, Wistar rats exposed to sugarcane ash intranasally for 24 weeks developed focal injury in the tubulointerstitium and glomeruli with deposition of SiNPs in the kidney. To further determine the cellular effects of SiNPs, we exposed human proximal convoluted tubule (PCT) cells to sugarcane ash, de-silicated ash, or sugarcane ash derived SiNPs. Despite not being directly cytotoxic to PCT cells at 24 hrs, SiNPs were taken up and generated reactive oxygen species. Vimentin staining confirmed PCT cells underwent epithelial-mesenchymal transition (EMT) following treatment with ash or SiNPs but not with de-silicated ash. These findings suggest SiNPs present in sugarcane ash are capable of driving EMT in PCT cells potentially contributing to a CKDu phenotype.

66 Sugarcane Ash and Sugarcane Ash-Derived Silica Nanoparticles Alter Mitochondrial Function and Metabolic Activity in Human Proximal Tubular Kidney Cells

Abstract Multiple epidemics of chronic kidney disease of an unknown etiology (CKDu), primarily in young healthy agricultural workers, have emerged in agricultural communities around the world. We have hypothesized that the harvest and burning of sugarcane leading to inhalation of sugarcane ash may contribute to development of CKDu. Sugarcane stalks consist of ~80% amorphous silica and we have demonstrated that following burning of sugarcane, nano-sized silica particles (~200nm) are generated. To determine what effect such exposures have on kidney cells, a human proximal convoluted tubule (PCT) cell line (HK-2) was subjected to treatments ranging in concentration from 0.025µg/mL to 25µg/mL of sugarcane ash, desilicated sugarcane ash, sugarcane ash-derived silica nanoparticles (SAD SiNPs), or manufactured pristine 200nm silica nanoparticles. Following 6 to 48 hours of exposure, mitochondrial activity and viability were found to be significantly reduced when exposed to SAD particles at concentrations 2.5µg/mL or higher. Oxygen consumption rate (OCR) and pH were investigated with a Seahorse XF Analyzer, revealing significant changes to cellular metabolism across treatments as early as 6 hours following exposure. While most treatments slightly increased respiration, SAD SiNPs were found to inhibit mitochondrial function, reduce ATP generation, increase reliance on glycolysis, and reduce glycolytic reserve. Metabolomic analysis following 24 hours of exposure found several cellular energetics pathways (e.g., fatty acid metabolism, glycolysis, and TCA cycle) significantly altered across ash-based treatments. Such changes indicate that exposure to sugarcane ash and its derivatives can promote mitochondrial dysfunction and disrupt metabolic activity of human PCT cells.

Reduced methylation correlates with diabetic nephropathy risk in type 1 diabetes.

Diabetic nephropathy (DN) is a polygenic disorder with few risk variants showing robust replication in large-scale genome-wide association studies. To understand the role of DNA methylation, it is important to have the prevailing genomic view to distinguish key sequence elements that influence gene expression. This is particularly challenging for DN because genome-wide methylation patterns are poorly defined. While methylation is known to alter gene expression, the importance of this causal relationship is obscured by array-based technologies since coverage outside promoter regions is low. To overcome these challenges, we performed methylation sequencing using leukocytes derived from participants of the Finnish Diabetic Nephropathy (FinnDiane) type 1 diabetes (T1D) study (n = 39) that was subsequently replicated in a larger validation cohort (n = 296). Gene body-related regions made up more than 60% of the methylation differences and emphasized the importance of methylation sequencing. We observed differentially methylated genes associated with DN in 3 independent T1D registries originating from Denmark (n = 445), Hong Kong (n = 107), and Thailand (n = 130). Reduced DNA methylation at CTCF and Pol2B sites was tightly connected with DN pathways that include insulin signaling, lipid metabolism, and fibrosis. To define the pathophysiological significance of these population findings, methylation indices were assessed in human renal cells such as podocytes and proximal convoluted tubule cells. The expression of core genes was associated with reduced methylation, elevated CTCF and Pol2B binding, and the activation of insulin-signaling phosphoproteins in hyperglycemic cells. These experimental observations also closely parallel methylation-mediated regulation in human macrophages and vascular endothelial cells.

NLRP3 inflammasome in rosmarinic acid-afforded attenuation of acute kidney injury in mice

Cisplatin (CP) is a well-known anticancer drug used to effectively treat various kinds of solid tumors. CP causes acute kidney injury (AKI) and unfortunately, there is no therapeutic approach in hand to prevent AKI. Several signaling pathways are responsible for inducing AKI which leads to inflammation in proximal convoluted tubule cells in the kidney. Furthermore, the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome is involved in the CP-induced AKI. In this study, we investigated therapeutic effects of rosmarinic acid (RA) against inflammation-induced AKI. RA was orally administered at the dose of 100 mg/kg for two consecutive days after 24 h of a single injection of CP at the dose of 20 mg/kg administered intraperitoneally in Swiss albino male mice. Treatment of RA inhibited the activation of NLRP3 signaling pathway by blocking the activated caspase-1 and downstream signal molecules such as IL-1β and IL18. CP activated HMGB1-TLR4/MyD88 axis was also found to be downregulated with the RA treatment. Activation of nuclear factor-κB and elevated protein expression of cyclooxygenase-2 (COX-2) were also found to be downregulated in RA-treated animals. Alteration of early tubular injury biomarker, kidney injury molecule-1 (KIM-1), was found to be subsided in RA-treated mice. RA has been earlier reported for antioxidant and anti-inflammatory properties. Our findings show that blocking a critical step of inflammasome signaling pathway by RA treatment can be a novel and beneficial approach to prevent the CP-induced AKI.

Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2 and -6 in chemoresistant renal cancer

Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC–MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22–C26). Inhibitors of endoplasmic reticulum–associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.

Cell-Type-Specific Profibrotic Scores across Multi-Organ Systems Predict Cancer Prognosis.

Simple SummaryFibrosis is a major player and contributor in the tumor microenvironment. Profibrotic changes precede the early development and establishment of a variety of human diseases, such as fibrosis and cancer. Being able to measure such early signals at the single cell level is critically useful for identifying new mechanisms and potential drug targets for a wide range of diseases. This study was designed to computationally identify profibrotic cell populations using single-cell transcriptomic data and to identify gene signatures that could predict cancer prognosis.Fibrosis is a major cause of mortality. Key profibrotic mechanisms are common pathways involved in tumorigenesis. Characterizing the profibrotic phenotype will help reveal the underlying mechanisms of early development and progression of a variety of human diseases, such as fibrosis and cancer. Fibroblasts have been center stage in response to various stimuli, such as viral infections. However, a comprehensive catalog of cell types involved in this process is currently lacking. Here, we deployed single-cell transcriptomic data across multi-organ systems (i.e., heart, kidney, liver, and lung) to identify novel profibrotic cell populations based on ECM pathway activity at single-cell resolution. In addition to fibroblasts, we also reported that epithelial, endothelial, myeloid, natural killer T, and secretory cells, as well as proximal convoluted tubule cells of the nephron, were significantly actively involved. Cell-type-specific gene signatures were enriched in viral infection pathways, enhanced glycolysis, and carcinogenesis, among others; they were validated using independent datasets in this study. By projecting the signatures into bulk TCGA tumor samples, we could predict prognosis in the patients using profibrotic scores. Our profibrotic cellular phenotype is useful for identifying new mechanisms and potential drug targets at the cell-type level for a wide range of diseases involved in ECM pathway activation.

Mitochondrial Superoxide Dismutase in Cisplatin-Induced Kidney Injury.

Cisplatin is a chemotherapy agent commonly used to treat a wide variety of cancers. Despite the potential for both severe acute and chronic side effects, it remains a preferred therapeutic option for many malignancies due to its potent anti-tumor activity. Common cisplatin-associated side-effects include acute kidney injury (AKI) and chronic kidney disease (CKD). These renal injuries may cause delays and potentially cessation of cisplatin therapy and have long-term effects on renal function reserve. Thus, developing mechanism-based interventional strategies that minimize cisplatin-associated kidney injury without reducing efficacy would be of great benefit. In addition to its action of cross-linking DNA, cisplatin has been shown to affect mitochondrial metabolism, resulting in mitochondrially derived reactive oxygen species (ROS). Increased ROS formation in renal proximal convoluted tubule cells is associated with cisplatin-induced AKI and CKD. We review the mechanisms by which cisplatin may induce AKI and CKD and discuss the potential of mitochondrial superoxide dismutase mimetics to prevent platinum-associated nephrotoxicity.

Single-cell analysis of angiotensin-converting enzyme II expression in human kidneys and bladders reveals a potential route of 2019 novel coronavirus infection.

Background:Since 2019, a novel coronavirus named 2019 novel coronavirus (2019-nCoV) has emerged worldwide. Apart from fever and respiratory complications, acute kidney injury has been observed in a few patients with coronavirus disease 2019. Furthermore, according to recent findings, the virus has been detected in urine. Angiotensin-converting enzyme II (ACE2) has been proposed to serve as the receptor for the entry of 2019-nCoV, which is the same as that for the severe acute respiratory syndrome. This study aimed to investigate the possible cause of kidney damage and the potential route of 2019-nCoV infection in the urinary system.Methods:We used both published kidney and bladder cell atlas data and new independent kidney single-cell RNA sequencing data generated in-house to evaluate ACE2 gene expression in all cell types in healthy kidneys and bladders. The Pearson correlation coefficients between ACE2 and all other genes were first generated. Then, genes with r values larger than 0.1 and P values smaller than 0.01 were deemed significant co-expression genes with ACE2.Results:Our results showed the enriched expression of ACE2 in all subtypes of proximal tubule (PT) cells of the kidney. ACE2 expression was found in 5.12%, 5.80%, and 14.38% of the proximal convoluted tubule cells, PT cells, and proximal straight tubule cells, respectively, in three published kidney cell atlas datasets. In addition, ACE2 expression was also confirmed in 12.05%, 6.80%, and 10.20% of cells of the proximal convoluted tubule, PT, and proximal straight tubule, respectively, in our own two healthy kidney samples. For the analysis of public data from three bladder samples, ACE2 expression was low but detectable in bladder epithelial cells. Only 0.25% and 1.28% of intermediate cells and umbrella cells, respectively, had ACE2 expression.Conclusion:This study has provided bioinformatics evidence of the potential route of 2019-nCoV infection in the urinary system.

Glucose-induced decrease of cystathionine β-synthase mediates renal injuries.

Exogenous hydrogen sulfide (H2 S) protects kidneys from diabetic injuries in animal models. In order to explore the role of endogenous H2 S in diabetic nephropathy, we determined the renal H2S producing enzymes in vivo and in vitro. In diabetic mice, H2 S levels in blood and kidney were decreased while cystathionine β-synthase (CBS), mainly located in mouse renal proximal convoluted tubules (PCT), was reduced selectively. In cultured mouse PCT cells treated with high glucose, CBS protein and activity was reduced while ubiquitinated CBS was increased, which was abolished by a proteasome inhibitor MG132 at 1 hour; high glucose drove CBS colocalized with proteasome 26S subunit ATPase6, indicating an involvement of ubiquitination proteasome degradation. At 48 hours, high glucose also selectively decreased CBS protein, concentration-dependently, but increased the ubiquitination of CBS; silence of CBS by siRNA increased nitrotyrosine, a marker for protein oxidative injury. Nitrotyrosine was also increased by high glucose treatments. The increases of nitrotyrosine either by cbs-siRNA or by glucose were restored by GYY4137, indicating that the H2 S donor may protect kidney from oxidative injury induced by CBS deficiency. In diabetic kidneys, ubiquitinated CBS and nitrotyrosine were increased but restored by GYY4137. The treatment also ameliorated albuminuria and renal morphologic changes in diabetic mice. Our findings suggest that high glucose induces reduction of renal CBS protein and activity in vivo and in vitro that is critical to the pathogenesis of diabetic kidney disease.

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.

Histopathological and Ultrastructural Alterations in The Renal Cortex of Albino Mice Foetuses Induced by Beta-Lactam Antibiotic 'Amoxicillin'

Background: The aim of this investigation was to evaluate the effect of a low and high doses (205 & 820mg/kg body weight, respectively) of the beta-lactam antibiotic amoxicillin on the kidney of maternally treated mice foetuses. Material and Methods: Pregnant mice were allocated into three groups; the first group served as control (injected with the drug solvent) and those of the other two groups were injected intraperitoneally with two doses (205 & 820 mg/kg body weight) of amoxicillin for 8 days from day 7 till day14 during gestation. Results: The histological examination of the renal cortex of maternally treated foetuses showed erosion of the parietal cells of Bowman's capsule, hypoplasia of the mesangial cells of the glomerulus, and erosion of the epithelial cells lining the proximal and distal convoluted tubules. At the electron microscopical level, the alternations of the renal cortex of foetuses maternally treated with amoxicillin were represented by, fusion of the foot processes of the podocytes and partial destruction of the apical brush borders microvilli of the proximal convoluted tubule cells. Also, degeneration of some mitochondria, and fragmentation of the rough endoplasmic reticulum elements of the lining cells of some proximal and distal convoluted tubules were observed. Conclusions: The uses of amoxicillin should be under restricted precautions specially for the pregnant women to avoid the hazardous impact.

Histopathological and Ultrastructural Alterations in The Renal Cortex of Albino Mice Foetuses Induced by Beta-Lactam Antibiotic 'Amoxicillin'

Background: The aim of this investigation was to evaluate the effect of a low and high doses (205 & 820mg/kg body weight, respectively) of the beta-lactam antibiotic amoxicillin on the kidney of maternally treated mice foetuses. Material and Methods: Pregnant mice were allocated into three groups; the first group served as control (injected with the drug solvent) and those of the other two groups were injected intraperitoneally with two doses (205 & 820 mg/kg body weight) of amoxicillin for 8 days from day 7 till day14 during gestation. Results: The histological examination of the renal cortex of maternally treated foetuses showed erosion of the parietal cells of Bowman's capsule, hypoplasia of the mesangial cells of the glomerulus, and erosion of the epithelial cells lining the proximal and distal convoluted tubules. At the electron microscopical level, the alternations of the renal cortex of foetuses maternally treated with amoxicillin were represented by, fusion of the foot processes of the podocytes and partial destruction of the apical brush borders microvilli of the proximal convoluted tubule cells. Also, degeneration of some mitochondria, and fragmentation of the rough endoplasmic reticulum elements of the lining cells of some proximal and distal convoluted tubules were observed. Conclusions: The uses of amoxicillin should be under restricted precautions specially for the pregnant women to avoid the hazardous impact.

Optimizing the reaction temperature to facilitate an efficient osmium maceration procedure

The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20°C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20°C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60°C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40°C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should find wide appeal for the 3D analysis of cellular organelles in various cell types.

Protective Role of Vitamin D in Renal Tubulopathies.

Vitamin D is tightly linked with renal tubular homeostasis: the mitochondria of proximal convoluted tubule cells are the production site of 1α,25-dihydroxyvitamin D3. Patients with renal impairment or tubular injury often suffer from chronic inflammation. This alteration comes from oxidative stress, acidosis, decreased clearance of inflammatory cytokines and stimulation of inflammatory factors. The challenge is to find the right formula for each patient to correctly modulate the landscape of treatment and preserve the essential functions of the organism without perturbating its homeostasis. The complexity of the counter-regulation mechanisms and the different axis involved in the Vitamin D equilibrium pose a major issue on Vitamin D as a potential effective anti-inflammatory drug. The therapeutic use of this compound should be able to inhibit the development of inflammation without interfering with normal homeostasis. Megalin-Cubilin-Amnionless and the FGF23-Klotho axis represent two Vitamin D-linked mechanisms that could modulate and ameliorate the damage response at the renal tubular level, balancing Vitamin D therapy with an effect potent enough to contrast the inflammatory cascades, but which avoids potential severe side effects.

Effect of Neonatal Exposure of Monosodium Glutamate in Kidney of Albino Mice – A Histological Study

Monosodium Glutamate (MSG) is a commonly used food additive. Scientists have found that MSG has toxic effects in several tissues and organs like neurons, liver, testes, ovary, kidneys etc due to oxidative stress both after exposure in neonatal period as well as in adult animal models. Although various reports have suggested that MSG has damaging effect in kidneys only few histological studies are available. This study was done to observe any histological changes in kidneys of albino mice after neonatal exposure with MSG. Study showed significant changes in weight and volume of kidneys in gross morphology. Increased urinary space and dilatation of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) were constant finding in experimental animals. There were loss of luminal microvilli and reduced height of lining cells of both PCT and DCT.