Humanproximaltubularepithelialcells (PTEC) of the kidney are known to respond to and mediate the disease process in a wide range of kidney diseases, yet their exosomal production and exosome molecular cargo remain a mystery. Here we investigate, for the first time, the production and molecular content of exosomes derived from primary human PTEC cultured under normal and diseased conditions representing a spectrum of in vivo disease severity from early inflammation, experienced in multiple initial kidney disease states, through to hypoxia, frequently seen in late stage chronic kidney disease (CKD) due to fibrosis and vascular compro- mise. We demonstrate a rapid reproducible methodology for the purification of PTEC-derived exosomes, identify increased numbers of exosomes from disease-state cultures and identify differential expression levels of both known and unique miRNA and protein species from exo- somes derived from different disease-culture conditions. The validity of our approach is sup- ported by the identification of miRNA, proteins and pathways with known CKD associations, providing a rationale to further evaluate these novel and known pathways as targets for therapeutic intervention.
Previous reports have suggested the production of complement components C4, C2, and factor B by renal tissue. However, the cells involved in production of complement have not been identified. In this study metabolic labeling experiments demonstrated that humanproximaltubularepithelialcells (PTEC) synthesize a 180-kD precursor of C3 that is
According to our results, the resistance shown by C5- def mice to renal I/R injury was probably due to the inability to generate C5b, rather than C5a. A key role for C5 also applies in rat myocardial I/R injury, in which administration of anti-C5 antibody lead to significant reduction of injury (50). However, a limitation in that study, as in the present study of C5-def mice, is that it was unable to distinguish between the possible effects of C5a and MAC, as both routes are defective without C5. Our results in the C6-def mice, however, resolve this issue and clearly emphasize the role of MAC in renal I/R. Tissue injury in the present model was independent of C4, tending to discount a role for classic pathway activa- tion. However, other studies have shown that hypoxia can activate the classic as well as alternative pathways (51, 52). Moreover, classic pathway activation contributes to ischemic damage in mouse muscle and intestine (26, 48). We cannot account for the discrepancy, except to suggest that it may be due to organ-specific differences in anato- my or physiology. For example, humanproximaltubularepithelialcells have deficient expression of the C regula- tory proteins CD55 and CD46 (53) and can activate the alternative pathway spontaneously at low concentrations of serum (28). We have confirmed this observation in cul- tured mouse and humantubularepithelialcells using 5–20% normal serum (unpublished data). In contrast, human endothelial cells are reported not to show spon- taneous activation of complement at this concentration (17), possibly due to differences in the cellular content of proteases that can cleave C3, or in the level of expression of membrane-bound C inhibitors (28).
To investigate the change in expression of MATE1/ SLC47A1, humanproximaltubularepithelialcells were incubated for 24 hours in serum-free renal epithelial cell growth medium (control), cell growth medium with a-HSA (2.5 mg/mL), or AGE-a-HSA (2.5 mg/mL). The cells were fixed by treatment with 90% methanol for 5 minutes at 4 ° C and then incubated with rabbit antihuman MATE1/SLC47A1 polyclonal antibody, or rabbit IgG as an isotype control for 30 minutes at room temperature, following incubation with Alexa Fluor 647™ gout antirabbit IgG polyclonal antibody for 30 minutes at room temperature in the dark. To detect expression changes on cell surfaces, the geometric mean titers of MATE1/SLC47A1 were evaluated by a BD FACSCanto
To our knowledge, HCoV-NL63 has not been reported in natural infections of human kidneys. The ability of HCoV-NL63 to replicate to high titers in primary RPTEC and HRE cells suggests that at least some human kidney cells are fully permissive for the virus. However, we are unable to resolve whether (a) The original batch of contaminated RPTEC were infected (naturally) with the virus prior to harvest, or (b) A worker with a respira- tory infection accidentally contaminated the RPTEC during their initial preparation, or (c) The RPTEC were contaminated in our laboratory. We are unable to re- solve the issue whether the cells were contaminated during preparation for many reasons, foremost being the company that sold the cells was merged with a different entity. It is unlikely that the RPTEC were infected in our laboratory, as we did not have HCoV-NL63 in our laboratory in 2004, and acquired HCoV-NL63 /Amsterdam-1 only recently (Sept. 2012) so that we could compare the biotype of HCoV-NL63/RPTEC with that of Amsterdam-1. Moreover, our laboratory policy dictates that workers refrain from cell culture work when they have a respiratory tract infection. It is plausible (but we lack proof ) that HCoV-NL63 may have been latent in the donor’s kidneys, a possibility consistent with the known biology of various coronaviruses that establish long-term but sub-clinical infections. Noteworthy, SARS-CoV, which shares the same ACE2 receptor as HCoV-NL63, has been associ- ated with kidney disease [61-64]. SARS-CoV causes a systemic infection with viral shedding not only in re- spiratory secretions, but also in stool and urine [63,65,66]. Perhaps HCoV-NL63 is capable of causing systemic infections as well, though the severity is much less than that of SARS-CoV. A parallel to this notion is the finding that HCoV-NL63 replicates to high titers in CaCo-2 cells , which are derived from a human colon carcinoma. In April of 2012, a new coronavirus capable of causing severe acute respiratory infections of humans emerged in Jordan. The same coronavirus was isolated in the summer of 2012 from a patient with acute pneumonia and renal failure in Saudi Arabia
Interestingly, although renal EMT-related effects were reached in our model only with very high concentration of this drug, we can not exclude that other different cells (including pneumocytes, bronchial epithelium cells) or pa- tients with a genetic predisposition could present this con- dition after exposure to lower or therapeutic dose of EVE. This assumption is in line with a recent work published by Xu X et al. describing a pro-fibrotic effect of mTOR in- hibitors in lung epithelialcells . However, our hypoth- esis, although suggestive, need to be better addressed and validated in future in vivo studies.
and are not the result of autolysis. Although ATN is frequently seen in post mortem examinations of neonates, children and adults, mainly in the context of hypoxic-ischemic conditions [7, 8], we have been unable to identify any previous ob- servation of the occurrence of tubular cell ne- crosis in the non-viable human fetus. This is mainly due to the rapid autolysis of tubularepithelialcells after death . In our study, all cases were non-macerated in order to avoid false positive or false negative results.
Isolation and culture of human primary tubularcells Segments of macroscopically and histologically normal renal cortex were obtained under aseptic conditions from patients undergoing nephrectomy for small (<6 cm) tumors in the Department of Urology, Chinese PLA General Hospital. Patients were accepted into the study if they had no history of renal or systemic disease associated with tubulointerstitial pathology. Tubular fragments were derived from the segments of renal cor- tex by collagenase digestion and were isolated by centri- fugation in 45 % Percoll (Pharmacia, Uppsala, Sweden). The PTECs were re-suspended in a 1:1 (vol/vol) mixture of Dulbecco’s modified Eagle’s (GIBCO TM
Tubularepithelialcells are the natural in vivo targets of TGF-β1, a compound that plays a critical role in renal fibrosis in the 5/6-nephrectomy rat model . Based on this finding, the expression of human CIP4 (hCIP4) in proximalepithelialcells (HK-2 cells) treated with TGF-β1 in vitro were exam- ined. After stimulation with 10 g/L of TGF-β1, the mRNA and protein levels of hCIP4 in HK-2 cells in- creased gradually from 24 h to 72 h (Figure 3A and 3B). Confocal fluorescent microscopy revealed that hCIP4 exhibited punctate localization throughout the cytosol, with the highest levels localized in the peri- nuclear region of HK-2 cells. After stimulation with TGF-β1, hCIP4 levels increased, and the compound was recruited into cluster formations located adjacent to the cell periphery. Gradually, these clusters were observed to be redistributed into the cytoplasm (Fig- ure 3C). This data suggest that upregulation of hCIP4 is closely related to the development of EMT in HK-2 cells exposed to TGF-β1.
The TGF-β1/Smad signalling pathway is well characterised as central to the induction and perpetuation of fibrosis in naturally occurring human CKD, as well as experimental models of renal injury . Recently, an association between urinary active TGF-β1 excretion and sever- ity of renal interstitial fibrosis has also been reported in cats . The present study is the first to show that increased extracellular concentrations of TGF-β1 induce pro-fibrotic alterations in feline FPTEC which are comparable to those documented in human cell types. Tubular epi- thelial cells exposed to TGF-β1, and other injurious factors, are believed to play a causal role in the development of tubulointerstitial fibrosis through paracrine cross-talk with the intersti- tium, resulting in myofibroblast activation and proliferation . This pro-inflammatory, pro- fibrotic phenotype is associated with a loss of epithelial phenotype and a gain of mesenchymal markers through a process known as partial EMT or epithelial plasticity . In the present study, cell morphology and gene expression in FPTEC, HREC and HK-2 were assessed as a measure of EMT in response to incubation with human recombinant TGF-β1. All three cell types exhibited an apparent concentration-dependent loss of epithelial morphology and devel- opment of a hypertrophic, fusiform phenotype in response to TGF-β1 stimulation. These mor- phological changes were accompanied by a decrease in the epithelial marker E-cadherin, and an increase in the mesenchymal markers N-cadherin, collagen type 1α1 and fibronectin. These changes are typical of EMT and are consistent with previous studies utilising HK-2 cells [49, 50] and human PTEC [46, 49, 51]. In the present study, there was no significant change in expression of α-SMA, a myofibroblast marker, in the FPTEC. Although α-SMA has been used previously as a marker of EMT , its suitability has been questioned by recent work suggest- ing that epithelialcells do not migrate into the interstitium and transdifferentiate into myofi- broblasts .
hybridization with 35S-labeled antisense riboprobes of HB-EGF indicated that compared with control, there was increased HB-EGF mRNA expression in the 6 h post-ischemic kidney in the inner cortex and outer medulla in a patchy distribution, with the greatest expression in the inner stripe of the outer medulla. Expression occurred primarily in tubularepithelialcells. Recombinant human HB-EGF stimulated [3H]-thymidine incorporation in both primary cultures of rabbit proximal tubule cells and NRK 52E normal rat kidney epithelialcells, with potency similar to that of EGF. Induction of HB-EGF mRNA was
oxidative stress condition, Nrf2 is translocated from cytoplasm into the nucleus to initiate the expression of cytoprotective proteins. Liu et al.  reported that Nrf2 was protective during ischemic and nephrotoxic AKI in mice. Mean- while, several clinical drug targets have focused on the activation of Nrf2 and Nrf2 related path- way [34, 35]. While the expression content of Nrf2 is limited under untreated condition. As shown in our study, HBSP treatment resulted in the enhanced level of nuclear translocation, antioxidant response element, and transcrip- tional activity of Nrf2 via PI3K/Akt pathway, and further induced the enhancement of down- stream antioxidative protein expression. Activation of Nrf2 contributes to the induction of many target antioxidative proteins, such as Heme oxygenase-1 (HO-1) and NADH quinone oxidoreductase 1 (NQO1). HO-1 and NQO1 are important antioxidant enzymes that exert cyto- protective effects against oxidative stress. Numerous studies have suggested the protec- tive effects of HO-1 on a variety of pathologies, including sepsis and renal injury [36, 37]. NQO1 is also considered as a desirable therapeutic strategy to protect cells from oxidative damage . In our study, Nrf2 siRNA suppressed the HBSP-induced activation of Nrf2, and resulted in the decreased expression of HO-1 and NQO1, indicating that the expression of HO-1 and NQO1 enhanced by HBSP was regulated by Nrf2. These findings were consistent with previ- ous study , in which NQO1 and HO-1 were significantly lower in Nrf2-knockout mice, sug- gesting that Nrf2 regulated the induction of antioxidative proteins in response to hypero- xia.
Reagents. Medium, reagents for cell culture, Ab’s for cell characterization, PI3K inhibitors (wortmannin and LY294002), PKC inhibitors (staurosporin and GF109203X), and general chemicals were purchased from Sigma-Aldrich Ltd. Co. (Paisley, United Kingdom). HSA was obtained from CSL Laboratory (CSL Limited, Parkville, Victoria, Australia). Other brands of HSA were obtained from Calbiochem-Novabiochem Corp. (San Diego, California, USA) and Sigma-Aldrich. The endo- toxin level in all albumin preparations were < 10 EU/ml, as determined using the QCL-1000 limulus amebocyte lysate kit (BioWhittaker Inc., Walkersville, Maryland, USA). Antibiotics, sera, agarose, and DNA size markers were obtained from Invitrogen Corporation (Carlsbad, California, USA). Reagents for cDNA synthesis were obtained from Life Technologies Inc. (Paisley, United Kingdom) and Promega Corp. (Madison, Wisconsin, USA), and those for PCR and cycle sequencing were from Perkin Elmer Life Sciences Inc. (Boston, Massachusetts, USA). The enzyme immunoassay kit for detection of IL-8 was purchased from Bender MedSystems (Vienna, Aus- tria). Ab’s for detection of DNA-bound NF- κ B by flow cytometry and cell membrane-permeable inhibitory pep- tide or mutant peptide for NF- κ B were from Biomol Research Laboratories (Plymouth Meeting, Pennsylvania, USA) and Dakopatts (Glostrup, Denmark). The fluores- cence probe for intracellular reactive oxygen species detec- tion was from Molecular Probes Inc. (Eugene, Oregon, USA). Reagents and Ab’s for in situ hybridization were from Boehringer Mannheim GmbH (Mannheim, Ger- many). Anti–Tamm-Horsfall glycoprotein was from Chemicon International (Temecula, California, USA). All other Ab’s were from DAKO A/S (Glostrup, Denmark). Cell culture. Human colorectal epithelial cell line (HT-29) and human lung type 2 epithelial cell line (A549) were obtained from American Type Culture Collection (Rockville, Maryland, USA). Human prox- imal tubularepithelialcells (PTECs) were isolated
The present study correlates altered connexin expression to altered cell function in proximaltubularepithelialcells of the diabetic kidney. Novel histological evidence, suggests that expression of both CX26 and CX43 are significantly upregulated in tubules from renal biopsy material isolated from patients with diabetic nephropathy. These findings are further supported by observations that CX26 and CX43 exhibit increased expression in both our cell model and human primary proximal tubule cells at 7days incubation with TGF-β1. Interestingly, in the present study, the effect of TGF-β1 on connexin expression appears temporally dependent. Acute 48h incubation with glucose/TGF-β1 significantly reduced Smad2/3-dependent CX26 and CX43 expression in HK2 cells. However, chronic incubation with the same stimuli for 7days increased CX26 and CX43, a trend consistent with data from renal biopsy material isolated from patients with DN and with recent reports that CXs appear to be upregulated in chronic kidney disease [20, 21, 49]. To delineate the functional consequences of this biphasic response, we used paired whole cell patch clamp recordings to directly measure how TGF-β1 affected GJIC. At both 48h and 7day incubation with the cytokine, junctional conductance was significantly reduced as compared to control. This counterintuitive finding can be explained by considering the relationship between cell-cell coupling and the ability of connexons to align and form functional gap-junctions [4, 5, 50]. Following acute challenge with high glucose and TGF-β1, connexin expression decreases providing less protein to form connexins and ultimately gap junctions. As expected, GJIC decreased. Contrary to this, chronic 7day incubation in high glucose/TGF-β1 increased the expression of both CX26 and CX43. This shift in expression should mean that there is more protein to form connexons and thus
BKV entered HRPTEC through structures in the cell mem- brane that are enriched in cholesterol, such as caveolae, and these agents had no effect upon the clathrin-mediated pathway. Damm et al. (5) and Pelkmans et al. (31) reported that a combination of Nys and progesterone blocked SV40 infection of a human hepatoma cell line (HuH7 cells) and CV-1 cells. Gilbert et al. reported that MBCD or Nys inhibited SV40 infection of murine fibroblasts (NIH 3T3 cells) and primary baby mouse kidney epithelialcells, and also reported that MBCD or Nys inhibited mPy infection against GD1a-supple- mented rat glioma cells (C6 cells) (16). These results implied that SV40 and mPy entered cells through the caveolae. More- over, BKV infection of monkey-derived Vero cells was re- ported to be inhibited by MBCD (12), providing additional support for our conclusion. Thus, our experiments strongly support a role for cholesterol in BKV internalization into cells. Caveolins are scaffolding domains of caveolae. Three caveo- lins exist: caveolin-1, caveolin-2, and caveolin-3. Caveolin-1 and -2 are coexpressed in most cells; caveolin-3 is expressed in muscle-specific cells, such as skeletal, cardiac, and smooth muscle cells. Caveolin-1 expression can induce the formation of caveolae, but caveolin-2 cannot. Therefore, disruption of the caveolin-1 gene results in the abolishment of caveolae (22, 34, 36). Our results showed that siRNA-mediated Cav-1 knockout caused inhibition of BKV infection in HRPTEC, but clathrin knockout did not. It seems likely that BKV enters HRPTEC through caveolae and not through the clathrin-mediated path- way. Eash et al. reported that BKV infection of Vero cells was inhibited by the expression of a dominant-negative Cav-1 mu- tant compared to the infection of Vero cells expressing wild- type Cav-1. Despite the different host cells, our data are in good concordance with their results (12). We also investigated the dynamics of the colocalization of labeled BKV with Cav-1. Labeled BKV particles were colocalized with Cav-1, but not with clathrin, and the colocalization rate of labeled BKV with Cav-1 peaked at 4 h after incubation. Our data suggest that BKV entered HRPTEC through caveolae and that, at about 4 h after infection, BKV particles were mostly trapped in the caveolae. Pelkmans et al. (30) and Gilbert and Benjamin (15) reported colocalization of polyomaviruses SV40 and mPy with Cav-1. These viruses were frequently trapped in caveolae until 1 h after incubation. Our data suggest that the internalization of BKV proceeds relatively slowly.
Although a conclusive demonstration of EMT in vivo in patients with kidney diseases remains a challenge, it has emerged as an important pathway leading to kidney fibrosis in diseased kidneys in recent years. The process of EMT is characterized by a loss of epithelial proteins, such as E-cadherin and cytokeratin, and the acquisition of new mesenchymal markers, including vimentin, α-SMA, fibroblast-specific protein-1 (FSP1), interstitial matrix components type I collagen, and fibronectin [21,22]. These alterations in protein expression are usually accompanied by a change in morphology to a fibroblastoid appearance and an enhanced migratory capacity. It has been proven that there is a correlation between the severity of tubular injury and urine protein quality in various renal diseases . Additionally, Sajni Josson et al.  have found that β2-M, which is an important component of urine protein, induces epithelial to mesenchymal transition and confers cancer lethality and bone metastasis in human cancer cells. However, the molecular biological mechanism of β2-M in the damage of renal tubularepithelialcells is far from clear. In our study, β2-M induced tubularcells to undergo sig- nificant morphologic changes to a fibroblastoid appearance and promoted the expression of EMT-related proteins in these cells. These results indicate that this protein may also be an important factor in the peritubular microenviron- ment that induces or promotes EMT. Although we did not find any new molecular mechanism by which β2-M induces EMT, our results support the function of the β2-M/HFE complex as a mediator participating in β2- M-induced EMT in both renal proximal tubule epithelialcells and cancer cells. Further, this mechanism may play an important role in tumor-associated nephropathy because the concentrations of serum and urine β2-M have been shown to be increased in many types of cancer [13-17].
Our in-vitro studies of melamine crystals interaction with renal cells is modeled after our earlier work with calcium oxalate cell culture studies . The study of crystal-cell interaction and its subsequent cell-damage and response is an active area of research in urolithiasis. Melamine cyanurate crystals mainly interacted with the cells physically whether it was 1:1 or 99:1 (clinical relevant). Pet food and milk product were mainly contaminated with melamine, and cyanurate, was a by-product contaminant with less than 1%  and this ratio of 99:1 is taken to be clinical relevant in our studies. Unlike CaOx crystals which have been shown to be actively taken up by renal tubularcells [12,14], melamine crystals caused physical damage to the cells and did not have any appearance of adherence to the cell surface - a necessary event before endocytosis occurs. LDH is a classic marker for cell-damage and subsequent oxidative stress on cells can be seen with melamine cyanurate present at 1:1 only and not at clinical relevant 99:1 ratios. Such cellular damage was also seen on human erythrocytes . Oxidative stress is a cellular response to signal the immune system and cells in the vicinity of immediate danger and while our results does not show such stress at 99:1 melamine ratio, it does however predict that cells are either damaged due to high load of melamine, such as those infants continuously taking melamine-containing milk for prolonged periods. It is also suggested that low concentrations or acute dose of melamine would not damage the cells, however this needs further elucidation.
The cells were plated on 24-well plates and cultured in DMEM supplemented with 10% FBS for 24 hours. After the cells were treated with GNPs of determined concentra- tions for another 24 hours, they were washed three times with ice-cold PBS and lysed with radio immunoprecipitation assay buffer containing protease inhibitors. After 15 min- utes of incubation on ice to ensure complete lysis, the cell lysates were centrifuged at 13,000 × g for 10 minutes at 4 ° C. The supernatant containing the cytoplasmic protein fraction was transferred to a new tube. The protein concentrations of the lysates were examined using the Bradford protein assay kit. The cell lysates were boiled and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane via semidry transfer (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membranes were washed in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), blocked with 5%
we found kidney tissue NKA expression was decreased in LN patients compared with con- trols, which indicated NKA downregulation might be involved in the etiology of LN. We found ouabain promoted HK-2 cells apoptosis while it inhibits proliferation in a dose-depen- dent way; moreover, ouabain decreased NKA expression and activated pSrc, pERK, pAkt, pS6K and caspase 3 expression. These find- ings implied ouabain may contribute to LN development and progression by modulating NKA, pSrc, pERK, pAkt, pS6K and caspase 3.
Transforming growth factor- (TGF- ) has been shown to play an important role in renal fibrosis, due to its ability to induce extracellular matrix (ECM) accumulation by stimulating the expression of ECM proteins such as connective tissue growth factor (CTGF) and up-regulating synthesis of proteinase inhibitors such as tissue inhibitor of metalloproteinase 1 (TIMP-1). Furthermore, it has been reported that TGF- inhibition seems to be a promising therapeutic strategy for prevention of renal fibrosis. The present work demonstrates that Nitric Oxide (NO) has the ability to induce a rapid Smad signaling cascade in human renal proximaltubularcells (HK-2 cells) which is abrogated by the addition of neutralizing TGF- -antibody indicating that Smad signaling induced by NO is TGF- dependent. Furthermore, addition of TGF- receptor I kinase inhibitor totally prevented NO-dependent Smad-2 phosphorylation indicating that TGF- receptor I kinase is involved in NO-triggered Smad signaling. Interestingly, activation of TGF- /Smad signaling cascade by NO is accompanied by an increase in TGF- -dependent expression of CTGF and TIMP-1. Collectively, these data demonstrate that NO causes a rapid activation of Smad signaling cascade in HK-2 cells via activation of latent TGF- that is sufficient to elicit fibrogenic cell responses as indicated by an increase in the expression of the fibrogenic genes CTGF and TIMP-1 which play an important role in renal fibrosis.