Proximal Tubular Epithelium Research Articles

Tumour necrosis factor receptor-associated factor-1 (TRAF-1) expression is increased in renal cell carcinoma patient serum but decreased in cancer tissue compared with normal: potential biomarker significance

Renal cell carcinoma (RCC) generally has a poor prognosis because of late diagnosis and metastasis. We have previously described decreased tumour necrosis factor receptor-associated factor-1 (TRAF-1) in RCC compared with paired normal kidney in a patient cohort in Australia. In the present study, TRAF-1 expression in clear cell RCC (ccRCC) and normal kidney was again compared, but in a cohort from University Malaya Medical Centre. Serum TRAF-1 was also evaluated in RCC and normal samples.Immunohistochemistry with automated batch staining and Aperio ImageScope morphometry was used to compare TRAF-1 in 61 ccRCC with paired normal kidney tissue. Serum from 15 newly diagnosed and untreated ccRCC and 15 healthy people was tested for TRAF-1 using ELISA.In this cohort, TRAF-1 was highly expressed in proximal tubular epithelium of normal kidney, and significantly decreased in ccRCC tissue (p < 0.001). Conversely, TRAF-1 in serum from ccRCC patients was significantly increased over control serum (132 ± 30 versus 54 ± 14 pg/mL, respectively; p = 0.013).Decreased TRAF-1 in RCC tissue, reported previously, was confirmed. This, along with significantly increased serum TRAF-1 may indicate the protein is actively secreted during development and progression of ccRCC. Therefore, the increased serum TRAF-1 may be a useful non-invasive indicator of RCC development.

Oxidative stress-induced alterations in PPAR-γ and associated mitochondrial destabilization contribute to kidney cell apoptosis.

The mechanism(s) underlying renoprotection by peroxisome proliferator-activated receptor (PPAR)-γ agonists in diabetic and nondiabetic kidney disease are not well understood. Mitochondrial dysfunction and oxidative stress contribute to kidney disease. PPAR-γ upregulates proteins required for mitochondrial biogenesis. Our aim was to determine whether PPAR-γ has a role in protecting the kidney proximal tubular epithelium (PTE) against mitochondrial destabilisation and oxidative stress. HK-2 PTE cells were subjected to oxidative stress (0.2-1.0 mM H₂O₂) for 2 and 18 h and compared with untreated cells for apoptosis, mitosis (morphology/biomarkers), cell viability (MTT), superoxide (dihydroethidium), mitochondrial function (MitoTracker red and JC-1), ATP (luminescence), and mitochondrial ultrastructure. PPAR-γ, phospho-PPAR-γ, PPAR-γ coactivator (PGC)-1α, Parkin (Park2), p62, and light chain (LC)3β were investigated using Western blots. PPAR-γ was modulated using the agonists rosiglitazone, pioglitazone, and troglitazone. Mitochondrial destabilization increased with H₂O₂concentration, ATP decreased (2 and 18 h; P < 0.05), Mitotracker red and JC-1 fluorescence indicated loss of mitochondrial membrane potential, and superoxide increased (18 h, P < 0.05). Electron microscopy indicated sparse mitochondria, with disrupted cristae. Mitophagy was evident at 2 h (Park2 and LC3β increased; p62 decreased). Impaired mitophagy was indicated by p62 accumulation at 18 h (P < 0.05). PPAR-γ expression decreased, phospho-PPAR-γ increased, and PGC-1α decreased (2 h), indicating aberrant PPAR-γ activation and reduced mitochondrial biogenesis. Cell viability decreased (2 and 18 h, P < 0.05). PPAR-γ agonists promoted further apoptosis. In summary, oxidative stress promoted mitochondrial destabilisation in kidney PTE, in association with increased PPAR-γ phosphorylation. PPAR-γ agonists failed to protect PTE. Despite positive effects in other tissues, PPAR-γ activation appears to be detrimental to kidney PTE health when oxidative stress induces damage.

Comparison of Renal Biomarkers with Glomerular Filtration Rate in Susceptibility to the Detection of Gentamicin-Induced Acute Kidney Injury in Dogs

Fourteen renal biomarkers were compared with measurement of glomerular filtration rate (GFR) in detecting acute kidney injury (AKI) in beagle dogs given gentamicin (40 mg/kg/day by subcutaneous injection) for 7 consecutive days. Serum and urinary biomarkers were measured before administration of gentamicin and then on days 4 and 8 after starting administration. GFR was derived by use of a simplified equation. Increased urinary cystatin C and decreased GFR occurred from day 4 and were detected before increases in blood urea nitrogen (BUN) and serum creatinine concentrations and changes in other urinary parameters. The closest correlation was between urinary cystatin C and GFR. At termination, microscopical examination revealed extensive necrosis of proximal tubular epithelium with hyaline casts in the kidney of treated dogs. These data indicate that urinary cystatin C is the most sensitive index of kidney injury and GFR reflects the kidney functional mass.

Changes in Viral and Human miRNA Expression Associated With Polyomavirus BK (BKV) Infection of Proximal Tubular Epithelium.

Changes in Viral and Human miRNA Expression Associated With Polyomavirus BK (BKV) Infection of Proximal Tubular Epithelium.

V-ATPase/mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis

mTOR kinase is a master growth regulator that can be stimulated by multiple signals, including amino acids and the lysosomal small GTPase Rheb. Recent studies have proposed an important role for the V-ATPase in the sensing of amino acids in the lysosomal lumen. Using the Drosophila wing as a model epithelium, we show here that the V-ATPase is required for Rheb-dependent epithelial growth. We further uncover a positive feedback loop for the control of apical protein uptake that depends on V-ATPase/mTOR signaling. This feedback loop includes Rheb-dependent transcriptional regulation of the multiligand receptor Megalin, which itself is required for Rheb-induced endocytosis. In addition, we provide evidence that long-term mTOR inhibition with rapamycin in mice causes reduction of Megalin levels and proteinuria in the proximal tubular epithelium of the kidney. Thus, our findings unravel a homeostatic mechanism that allows epithelial cells to promote protein uptake under normal conditions and to prevent uptake in lysosomal stress conditions.

Blockade of cysteine-rich protein 61 attenuates renal inflammation and fibrosis after ischemic kidney injury.

Emerging data have suggested that acute kidney injury (AKI) is often incompletely repaired and can lead to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. However, the underlying mechanisms linking AKI to CKD remain obscure. The present study aimed to investigate the role of cysteine-rich protein 61 (Cyr61) after unilateral kidney ischemia-reperfusion injury (IRI) in mice. After IRI, increased expression of Cyr61 was detected, predominately in the proximal tubular epithelium. This was confirmed by in vitro experiments, which showed that hypoxia stimulated Cyr61 expression in cultured proximal tubular epithelial cells. The proinflammatory property of Cyr61 was indicated by its ability to upregulate monocyte chemoattractant protein-1 and IL-6. Additionally, we found elevated urinary Cyr61 excretion in patients with AKI. Notably, treatment of mice with an anti-Cyr61 antibody attenuated the upregulation of kidney monocyte chemoattractant protein-1, IL-6, IL-1β, and macrophage inflammatory protein-2 and reduced the infiltration of F4/80-positive macrophages on days 7 and 14 after IRI. In addition, blockade of Cyr61 reduced the mRNA expression of collagen, transforming growth factor-β, and plasminogen activator inhibitor-I as well as the degree of collagen fibril accumulation, as evaluated by picrosirius red staining, and levels of α-smooth muscle actin proteins by day 14. Concurrently, in the treated group, peritubular microvascular density was more preserved on day 14. We conclude that Cyr61 blockade inhibits the triad of inflammation, interstitial fibrosis, and capillary rarefaction after severe ischemic AKI. The results of this study expand the knowledge of the mechanisms underlying the AKI-to-CKD transition and suggest that Cyr61 is a potential therapeutic target.

Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway.

In rabbit proximal tubular cells, ANG II type 2-receptor (AT2)-induced arachidonic acid release is PLA2 coupled and dependent of G protein βγ (Gβγ) subunits. Moreover, ANG II activates ERK1/2 and transactivates EGFR via a c-Src-dependent mechanism. Arachidonic acid has been shown to mimic this effect, at least in part, by an undetermined mechanism. In this study, we determined the effects of ANG II on fibronectin expression in cultured rabbit proximal tubule cells and elucidated the signaling pathways associated with such expression. We found that ANG II and transfection of Gβγ subunits directly increased fibronectin protein expression, and this increase was inhibited by overexpression of β-adrenergic receptor kinase (βARK)-ct or DN-Src. Moreover, ANG II-induced fibronectin protein expression was significantly abrogated by the AT2 receptor antagonist PD123319. In addition, inhibition of cystolic PLA2 diminished ANG II-induced fibronectin expression. Endogenous arachidonic acid mimicked ANG II-induced fibronectin expression. We also found that overexpression of Gβγ subunits induced c-Src, ERK1/2, and EGFR tyrosine phosphorylation, which can be inhibited by overexpression of βARK-ct or DN-Src. Gβγ also induced c-Src SH2 domain association with the EGFR. Supporting these findings, in rabbit proximal tubular epithelium, immunoblot analysis indicated that βγ expression was significant. Interestingly, arachidonic acid- and eicosatetraenoic acid-induced responses were preserved in the presence of βARK-ct. This is the first report demonstrating the regulation of EGFR, ERK1/2, c-Src, and fibronectin by Gβγ subunits in renal epithelial cells. Moreover, this work demonstrates a role for Gβγ heterotrimeric proteins in ANG II, but not arachidonic acid, signaling in renal epithelial cells.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

Histological Peculiarities Of The Renal Cortical Substance In The Dynamics Of Fever Development

Under conditions of aseptic fever development and decreased sodium dietary intake in the experiment on 60 non-linear white male rats with 0.160.20 kg bodyweight the following was found: during the first stage (rise of temperature) vacuolar degeneration of the proximal tubular epithelium and microfocal character of changes in protein properties with the shift of stain to red color, during the second stage (high fever) dilation of Shumlianskiy-Bowman’s capsule lumen and dystrophic changes of the distal tubular epithelium, during the third stage (decrease of temperature) moderate dilation of ShumlianskiiBowman's capsule lumen and insignificant dystrophic changes of the distal tubular epithelium. Morphological changes in the dynamics of fever development reflect the character of kidney function changes.

The emerging role of the inflammasome in kidney diseases

To focus on the latest data that elucidate the role of the NLRP3 inflammasome in kidney diseases. The NLRP3 inflammasome is not limited by the traditional microbial stimuli of innate immunity and its connection with autophagy, apoptosis, fibrosis, and pro-inflammatory cytokines has broader implications for a variety of kidney diseases. In a wide spectrum of glomerular and tubulointerstitial diseases, the NLRP3 inflammasome is upregulated in both classical immune cells such as infiltrating macrophages and resident dendritic cells as well as in renal tubular epithelial cells, and even podocytes. Inhibition of the NLRP3 inflammasome ameliorates renal injury in a variety of animal models. Interestingly, this extends to models of proteinuria, which suggests that the deleterious effect of albuminuria on the proximal tubular epithelium and podocytes is, in part, mediated by inflammasome activation. Recent studies in animal models, and limited studies in humans, suggest a broad role for inflammasome activation in renal disease. Surprisingly, individual components of the inflammasome, independent of inflammasome activation, may also contribute to progressive renal injury. Additional, studies are needed to define the relative importance of the inflammasome in specific diseases and the therapeutic opportunities afforded by targeting the inflammasome.

Loss of alpha(E)‐catenin increases cisplatin nephrotoxicity (691.2)

Cisplatin is one of the most potent and widely used antineoplastic drugs. However, the use of cisplatin is dramatically limited by its side effect, nephrotoxicity. Evidence has shown that there is an increased incidence and severity of acute kidney injury (AKI) in the elderly. We previously reported a dramatic decrease of α‐catenin expression in proximal tubular epithelium of aged kidney. We hypothesize that loss of α(E)‐catenin increases susceptibility to cisplatin. To study the effects of reduced α‐catenin, shRNA knock‐down of α(E)‐catenin (C2) was generated in NRK‐52E cells; NT3 is the non‐targeted control. Confluent cultures of cells were challenged with cisplatin (50, 100, 150 μM) for 24 hr and C2 cells exhibited a significant loss of viability at all concentrations as compared with NT3 cells. Interestingly, the expression of α(E)‐catenin was further decreased in both cell lines. Importantly, higher caspase 3/7 activity was observed in C2 cells than NT3 cells after cisplatin treatment indicating the increased susceptibility may be due to apoptosis. To elucidate the mechanism of increased apoptosis in C2 cells, we performed RNA sequencing and identified that CYFIP2, a pro‐apoptotic gene, had 6.44 fold higher expression in C2 cells than NT3 cells. This finding was confirmed by qPCR. Taken together, these results suggest that loss of α(E)‐catenin may increase cisplatin‐induced nephrotoxicity via apoptosis.Grant Funding Source: Supported by NIH RO1AAG034154

The role of IgA in proximal tubular cell activation and tubulointerstitial scarring in IgA nephropathy

BackgroundIgA nephropathy (IgAN) is the commonest form of glomerulonephritis worldwide, and around 30% of patients progress to end-stage renal failure within 20 years. One of the most powerful prognostic factors for progression in IgAN is the development of tubulointerstitial inflammation and fibrosis. The extent of tubulointerstitial damage in IgAN does not however correlate with the degree of mesangial IgA deposition. Changes in glomerular permeability in IgAN result in exposure of the proximal tubular epithelium to IgA. It is therefore possible that a pathogenic interaction between filtered IgA and proximal tubular cells drives tubulointerstitial scarring in IgAN, independent of the effect of albuminuria. We aimed to assess the interaction between IgA and proximal tubular cells to establish whether IgA could independently generate a proinflammatory and profibrotic phenotypic transformation in proximal tubular cells thereby favouring the development of renal fibrosis. MethodsIgA1 was purified from serum from patients with IgAN and healthy individuals by jacalin-agarose affinity chromatography. Human proximal tubular epithelial HK2 cells (PTEC) were exposed for 24 h to IgA1 (100 μg/mL) from patients with IgAN or healthy individuals, or to control conditions IgM (100 μg/mL), IgG (100 μg/mL), albumin (100 μg/mL or 5 mg/mL), or medium alone, and then subjected to protein extraction, with supernatants assayed by sandwich ELISA. FindingsIgA1 from patients with IgAN activated extracellular signal-regulated protein kinase in PTEC to a greater extent than did IgM or IgA1 from healthy individuals. IgA1 from patients with IgAN also stimulated PPAR-response-element-driven luciferase expression, indicating PPAR activation. PTEC synthesis of transforming growth factor β, fibronectin, and interleukin 6 was substantially increased by IgA1 (100 μg/mL) compared with IgM, IgG, and albumin at concentrations up to 50 times higher (5 mg/mL). PTEC production of complement component C3 and activation of the complement cascade with generation of the chemotactic component C5a were induced to a greater extent by IgA1 compared with the other conditions. Finally, IgA1 induced phosphorylation of a megalin cytoplasmic tail plus glutathione-s-transferase fusion protein in HK2 cells suggesting that IgA1 binding to PTEC can trigger the same intracellular signalling pathways as those activated after exposure to albumin. InterpretationWe provide evidence that IgA1 in IgAN triggers a proinflammatory and profibrotic phenotypic transformation of PTECs. Interaction between filtered IgA1 and PTEC might be a key factor in driving tubulointerstitial damage and progression of renal fibrosis in IgAN. Further elucidation of the mechanism of this interaction might reveal novel therapeutic targets relevant in slowing progression of IgA nephropathy. FundingUK Medical Research Council.

Evidence for a morphologically distinct and functionally robust cell type in the proximal tubules of human kidney

Acute tubular necrosis (ATN), elicited by ischemia and/or toxicity, is a potentially life-threatening condition. Histologically, ATN corresponds to necrosis and detachment of renal tubular epithelial cells. However, the tubules possess a considerable regenerative capacity and may be restored. We have previously identified a scattered population of progenitor-like cells within the proximal tubules, sharing marker expression with the parietal epithelial cells of Bowman's capsule as well as with renal tubules regenerating after ATN. In the present analysis, we use transmission electron microscopy, immunoelectron microscopy and immunofluorescence of human kidney cortex to further explore these cells. We demonstrate that the cells are smaller and have drastically fewer mitochondria than the surrounding proximal tubule cells. They also display strong expression of several structural proteins such as vimentin, collagen-7A1 and the tight junction protein claudin-1. To functionally assess these cells, we also developed a novel human kidney explant model of ATN demonstrating that the cells are more resilient to injury than the surrounding proximal tubular cells. Taken together the results suggest a novel robust cell type with a contrasting biological role to that of the bulk of proximal tubular epithelium.

Proximal Tubular Hypertrophy and Enlarged Glomerular and Proximal Tubular Urinary Space in Obese Subjects with Proteinuria

BackgroundObesity is associated with glomerular hyperfiltration, increased proximal tubular sodium reabsorption, glomerular enlargement and renal hypertrophy. A single experimental study reported an increased glomerular urinary space in obese dogs. Whether proximal tubular volume is increased in obese subjects and whether their glomerular and tubular urinary spaces are enlarged is unknown.ObjectiveTo determine whether proximal tubules and glomerular and tubular urinary space are enlarged in obese subjects with proteinuria and glomerular hyperfiltration.MethodsKidney biopsies from 11 non-diabetic obese with proteinuria and 14 non-diabetic lean patients with a creatinine clearance above 50 ml/min and with mild or no interstitial fibrosis were retrospectively analyzed using morphometric methods. The cross-sectional area of the proximal tubular epithelium and lumen, the volume of the glomerular tuft and of Bowman’s space and the nuclei number per tubular profile were estimated.ResultsCreatinine clearance was higher in the obese than in the lean group (P=0.03). Proteinuria was similarly increased in both groups. Compared to the lean group, the obese group displayed a 104% higher glomerular tuft volume (P=0.001), a 94% higher Bowman’s space volume (P=0.003), a 33% higher cross-sectional area of the proximal tubular epithelium (P=0.02) and a 54% higher cross-sectional area of the proximal tubular lumen (P=0.01). The nuclei number per proximal tubular profile was similar in both groups, suggesting that the increase in tubular volume is due to hypertrophy and not to hyperplasia.ConclusionsObesity-related glomerular hyperfiltration is associated with proximal tubular epithelial hypertrophy and increased glomerular and tubular urinary space volume in subjects with proteinuria. The expanded glomerular and urinary space is probably a direct consequence of glomerular hyperfiltration. These effects may be involved in the pathogenesis of obesity-related renal disease.

Loss of Villin Immunoexpression in Colorectal Carcinoma Is Associated with Poor Differentiation and Survival

Background and Aims. Villin is a highly specialised protein and is expressed in intestinal and renal proximal tubular epithelium. It was detected in colorectal carcinomas (CRC) and other nongastrointestinal tumours. The aim of the current study is to investigate the immunohistochemical expression of villin in a subset of primary CRC and determine its relation to tumour differentiation, invasion, nodal metastasis, recurrence, and disease-free survival. Patients and Methods. Paraffin blocks of 93 cases of CRC were retrieved constituting 93 primary CRC and 58 adjacent normal mucosa. Immunohistochemistry was performed using antivillin antibody. The extent (%) of villin immunoexpression was categorised for statistical analysis. Statistical tests were used to determine the association of villin with clinicopathological characteristics: age, sex, tumour location, tumour size, depth of invasion, tumour grade, nodal metastasis, lymphovascular invasion, margin status, recurrence, and survival. Results. Villin immunostaining results showed that villin is downregulated in CRC. Villin has no association with age, sex, tumour location, depth of invasion, nodal metastasis, lymphovascular invasion, margin status, and recurrence. However, villin is expressed in higher rate in CRC less than 5 cm, well- and moderately differentiated CRC. Poor survival was associated with tumour with low villin immunoexpression. Conclusion. Villin was downregulated in CRC. Villin immunoexpression in CRC is associated with better survival, well-differentiated tumours, and small-sized tumours. Villin has no significant association with disease recurrence or nodal metastasis. More in vivo and in vitro studies are required for further elucidation of how villin may be involved in CRC.

CD133 Staining Detects Acute Kidney Injury and Differentiates Clear Cell Papillary Renal Cell Carcinoma from Other Renal Tumors

CD133 has recently been characterized as a progenitor cell marker in the kidney. However, the expression of this marker has not been thoroughly investigated in kidney injury and variants of renal tumors for pathology practice. We quantified CD133 expression in kidney biopsies from patients with acute renal failure and compared staining intensity with serum creatinine levels. CD133 expression levels were also evaluated in several subtypes of renal neoplasms. Normal adult renal parenchyma showed CD133 expression in parietal epithelium and in less than 5% of the epithelial cells in proximal and distal nephron tubules. However, CD133 was diffusely upregulated in the injured proximal and distal tubular epithelium and the CD133 expression scores in renal tubules were significantly correlated with serum creatinine levels. Amongst the renal tumors, CD133 was diffusely expressed in clear cell papillary renal cell carcinoma but was only focally present in other types of renal tumors. In summary, CD133 is a useful marker to detect renal tubular injury and to differentiate clear cell papillary renal cell carcinoma from other tumor types.

Diclofenac Mediated Demodulation of Alkaline Phosphatase and Renal Cortical Damage in Experimental Albino Mice

Diclofenac sodium is known to interfere with renal physiology by inhibiting prostaglandins. Previous studies indicate that various nephrotoxins damage proximal renal tubules by altering alkaline phosphatase (APase) activity. APase has been reported to be a function related marker in renal proximal tubular epithelia where it is highly expressed. Present investigation deals with toxicity caused in mice kidney at histological and biochemical levels after diclofenac administration. Diclofenac toxicity was assessed by localizing APase in kidney histochemically and biochemically. Intramuscular diclofenac administration (10 mg/kg/body wt) for 30 days exhibited substantial degeneration in kidney. A marked change in APase activity was observed in histochemical and biochemical studies. A change was noticed in specific activity of APase at different periods of diclofenac treatment. Decrease in specific activity of APase after 10 days (18.41 %) and 30 days (55.3 %) of diclofenac exposure was observed. However, an insignificant hike in APase was observed after 20 days of drug therapy. Similar trends in APase activity were evidenced by the electrophoretic analysis. Histological and ultrastructural observations also corroborated above mentioned findings. Present investigation gives an insight into probable mechanism of renal pathology caused by diclofenac administration in mice.

Cisplatin nephrotoxicity involves mitochondrial injury with impaired tubular mitochondrial enzyme activity

Cisplatin is a commonly used chemotherapeutic drug, the clinical use of which is limited by the development of dose‐dependent nephrotoxicity. Recent studies have suggested that mitochondrial dysfunction in tubular epithelium contributes to cisplatin‐induced nephrotoxicity (PMID: 22120494). Here we extend those findings by describing the role of an important electron transport chain enzyme, cytochrome c oxidase (COX). Immunohistochemistry for COX 1 protein demonstrated that, in response to cisplatin, expression was mostly maintained in focally damaged tubular epithelium. In contrast, COX enzyme activity in proximal tubules (by light microscopy) was decreased. Ultrastructural analysis of the cortex and outer stripe of the outer medulla (OSOM) showed decreased mitochondrial mass, disruption of cristae and extensive mitochondrial swelling in proximal tubular epithelium. Functional electron microscopy (EM) showed that COX enzyme activity was decreased in the remaining mitochondria in the proximal tubules, but maintained in distal tubules. A single systemic dose of mitochondrially targeted anti‐oxidant prevented mitochondrial injury and dysfunction. In summary, cisplatin induced nephrotoxicity is associated with structural and functional damage to the mitochondria. Mitochondrially targeted antioxidants may offer a novel therapeutic approach to prevent cisplatin nephrotoxicity.

Loss of Alpha(E)‐Catenin: Potential Role in the Renal Disrepair Following Injury in the Aging Kidney

As the kidney ages, morphological and functional changes lead to renal dysfunction. Clinical data indicates decreased ability to repair following acute insult which leads to increased susceptibility and severity of acute kidney injury (AKI). We have previously shown the aging kidney has a marked loss of α‐catenin expression in the proximal tubular epithelium. We hypothesize that the loss of this protein may be involved in the decreased repair of the aging kidney. To study the effects of reduced α‐catenin, shRNA knock‐down of α‐catenin was generated in NRK‐52E cells. Single cell clones were selected for a non‐targeted (NT3) control and a targeted knock‐down (C2). The 75% reduction of α‐catenin mRNA and protein levels was stable over several passages compared to the NT3 control. Interestingly, the catenin knockdown affected expression of other components of the cadherin/catenin complex. Most notably, mRNA expression of N‐cadherin was reduced &gt;;99% and protein was undetectable in C2. This was paralleled by a decrease in cadherin/catenin function as assessed by aggregate formation, as well as inhibition of wound healing as determined by a scratch assay. NextGen RNA sequencing revealed alterations in several genes which are implicated in renal repair, including proliferation/growth factor signaling, and cytoskeletal organization. This work was supported by NIH AG034154.

The HDAC inhibitor, vorinastat, prevents TGF‐β1 induced EMT and apoptosis in human renal proximal tubular cells

Renal interstitial fibrosis is characterized by renal fibroblast proliferation. This process involves renal tubular epithelial cells adopting a fibroblast morphology through epithelial‐to‐mesenchymal transition (EMT). EMT produces phenotypic changes such as epithelial cell adherins junction loss and de novo α‐smooth muscle actin (SMA) expression. TGF‐ß1 has been shown to stimulate EMT. Identifying agents that inhibit TGF‐ß1 induced EMT may prevent patient progression towards chronic renal failure (CRF). Previous work from our laboratory has shown that 4‐phenylbuterate (4‐PBA), a histone deacetylase inhibitor (HDACi), inhibited EMT. In this study, we hypothesized that a broad spectrum HDACi, vorinastat, would inhibit TGF‐ß1 induced EMT and apoptosis in human proximal tubular epithelium (hPTE). We tested vorinastat and 4‐PBA on TGF‐ß1 induced EMT in hPTE. TGF‐ß1 induced EMT was inhibited by both 1mM 4‐PBA and 5μM vorinastat as shown by cadherin junction preservation and reduced de novo α‐SMA expression. Vorinastat was also shown to prevent TGF‐ß1‐induced decrease of E‐cadherin and increase of type I collagen transcript levels. Further, 4‐PBA and vorinastat inhibited TGF‐ß1 induced apoptosis in hPTE. These findings suggest that HDACi prevent EMT and apoptosis induced by TGFß1 and may reduce the progression of patients with chronic kidney disease towards CRF. Funding, CIHR OSO‐115895.