Human Kidney Cultures Research Articles

Insulin receptor orchestrates kidney antibacterial defenses

Urinary tract infection (UTI) commonly afflicts people with diabetes. This augmented infection risk is partly due to deregulated insulin receptor (IR) signaling in the kidney collecting duct. The collecting duct is composed of intercalated cells (ICs) and principal cells (PCs). Evidence suggests that ICs contribute to UTI defenses. Here, we interrogate how IR deletion in ICs impacts antibacterial defenses against uropathogenic Escherichia coli. We also explore how IR deletion affects immune responses in neighboring PCs with intact IR expression. To accomplish this objective, we profile the transcriptomes of IC and PC populations enriched from kidneys of wild-type and IC-specific IR knock-out mice that have increased UTI susceptibility. Transcriptomic analysis demonstrates that IR deletion suppresses IC-integrated stress responses and innate immune defenses. To define how IR shapes these immune defenses, we employ murine and human kidney cultures. When challenged with bacteria, murine ICs and human kidney cells with deregulated IR signaling cannot engage central components of the integrated stress response-including activating transcriptional factor 4 (ATF4). Silencing ATF4 impairs NFkB activation and promotes infection. In turn, NFkB silencing augments infection and suppresses antimicrobial peptide expression. In diabetic mice and people with diabetes, collecting duct cells show reduced IR expression, impaired integrated stress response engagement, and compromised immunity. Collectively, these translational data illustrate how IR orchestrates collecting duct antibacterial responses and the communication between ICs and PCs.

IL11 Stimulates IL33 Expression and Proinflammatory Fibroblast Activation across Tissues.

Interleukin 11 (IL11) is upregulated in inflammatory conditions, where it is mostly believed to have anti-inflammatory activity. However, recent studies suggest instead that IL11 promotes inflammation by activating fibroblasts. Here, we assessed whether IL11 is pro- or anti-inflammatory in fibroblasts. Primary cultures of human kidney, lung or skin fibroblasts were stimulated with IL11 that resulted in the transient phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the sustained activation of extracellular signal-regulated protein kinases (ERK). RNA sequencing over a time course of IL11 stimulation revealed a robust but short-lived transcriptional response that was enriched for gene set hallmarks of inflammation and characterized by the upregulation of SERPINB2, TNFRSF18, Interleukin 33 (IL33), CCL20, IL1RL1, CXCL3/5/8, ICAM1 and IL11 itself. IL33 was the most upregulated signaling factor (38-fold, p = 9.8 × 10−5), and IL1RL1, its cognate receptor, was similarly increased (18-fold, p = 1.1 × 10−34). In proteomic studies, IL11 triggered a proinflammatory secretome with the notable upregulation of IL8, IL6, MCP1, CCL20 and CXCL1/5/6, which are important chemotaxins for neutrophils, monocytes, and lymphocytes. IL11 induced IL33 expression across fibroblast types, and the inhibition of STAT3 but not of MEK/ERK prevented this. These data establish IL11 as pro-inflammatory with specific importance for priming the IL33 alarmin response in inflammatory fibroblasts across tissues.

Renal proximal tubular epithelial cells: review of isolation, characterization, and culturing techniques.

The kidney is a complex organ, comprised primarily of glomerular, tubular, mesangial, and endothelial cells, and podocytes. The fact that renal cells are terminally differentiated at 34weeks of gestation is the main obstacle in regeneration and treatment of acute kidney injury or chronic kidney disease. Furthermore, the number of chronic kidney disease patients is ever increasing and with it the medical community should aim to improve existing and develop new methods of renal replacement therapy. On the other hand, as polypharmacy is on the rise, thought should be given into developing new ways of testing drug safety. A possible way to tackle these issues is with isolation and culture of renal cells. Several protocols are currently described to isolate the desired cells, of which the most isolated are the proximal tubular epithelial cells. They play a major role in water homeostasis, acid-base control, reabsorption of compounds, and secretion of xenobiotics and endogenous metabolites. When exposed to ischemic, toxic, septic, or obstructive conditions their death results in what we clinically perceive as acute kidney injury. Additionally, due to renal cells' limited regenerative potential, the profibrotic environment inevitably leads to chronic kidney disease. In this review we will focus on human proximal tubular epithelial cells. We will cover human kidney culture models, cell sources, isolation, culture, immortalization, and characterization subdivided into morphological, phenotypical, and functional characterization.

Circumvention of multi-drug resistance in human kidney and kidney carcinoma in vitro.

This report investigated whether the calmodulin inhibitor, trifluoperazine, can circumvent multi-drug resistance both in primary tissue cultures of human kidney and kidney carcinoma. For detection of inherent multi-drug resistance, the expression of P-glycoprotein was determined by immunofluorescence and immunocytochemistry using the monoclonal antibody C219. For detection of doxorubicin resistance and reversal of this resistance by trifluoperazine, the incorporation of nucleic acid precursor was measured after addition of doxorubicin and trifluoperazine, respectively. Both P-glycoprotein expressing resistant normal and malignant kidney tissue cultures could be modified by trifluoperazine. However, sensitive normal kidney and kidney carcinoma cultures were little affected by trifluoperazine. Thus, circumvention of primary resistance to doxorubicin is not limited to tumor cells. This might have important implications for the use of resistance modifiers in the clinical setting.

Presence of oestrogen receptors on target cells and antiproliferative activity of estramustine phosphate: Positive correlation for human tumours in vitro

Incubation with estramustine phosphate for 24 h inhibited DNA, RNA and protein synthesis in primary cultures of human kidney, mammary, prostatic, cervical and endometrial carcinoma. Not only the presence, but also the concentration of oestrogen receptors correlated with estramustine phosphate effects on tumour cell proliferation.

Establishment of immortalized primate epithelial cells with sub‐genomic EBV DNA

The genetic information in a sub-fragment of EBV DNA, designated p31 (containing less than a quarter of the viral genome and derived from a recombinant DNA cosmid library) allows epithelial cells from primary monkey and human kidney cultures to escape senescence under standard tissue culture conditions. A number of epithelial cell lines, designated M1/31, 483/31, 199/31 and HK/31, have been established and characterized following transfection of primary cells with p31 DNA. They share many properties, although morphologically they are not all identical. The cultures are immortalized but not fully transformed or tumorigenic. They appear to be phenotypically stable, although DNA hybridization studies indicate that genotypic alterations, including amplification, occur subsequent to transfection with p31 DNA and the establishment of a continuously proliferating epithelium. All cell lines consistently express high levels of cytokeratin 18 and varying amounts of cytokeratin 7, demonstrating their epithelial origin. From a single marmoset kidney (designated 199) a series of related immortalized cells, with subtle phenotypic differences, have been generated by p31 or sub-fragments of it. Although hallmarks of a "hit-and-run" mechanism are apparent in all of our studies, 2 different techniques (in situ hybridization or selection for cell survival in semi-solid media, followed by nucleic acid hybridization) show that, in late-passaged cultures, a small proportion of the cells still contain some viral DNA. The studies focus on genetic information within the BamHI A and I regions as being relevant to immortalization. The role of the EBV DNA fragment in the genesis of epithelial cell lines is considered.

Comparison of Tissue Culture Cells and Histological Sections for Use in Screening Monoclonal Antibodies

AbstractMonoclonal antibodies have been screened using either tissue culture cells or histologic sections as a source of antigen. We evaluated and compared these two methods using six murine monoclonal antibodies on human kidney cultures and histologic sections of human normal kidney and renal carcinoma. Antibody binding demonstrated shared antigens on kidney culture cells, renal tubular cells and renal carcinoma cells. The patterns of antibody binding were generally similar with either cultured cells or histologic sections. However, certain antibodies demonstrated significant differences in antibody binding depending on the method used for screening.

Renotropic growth factor found in cancer patient sera after removal of cancer-bearing kidney

We have used two sets of primary human kidney culture systems for assaying human renotropic growth factor in day 5 postnephrectomized human sera. The first set of primary kidney cell cultures was prepared from one hydronephrotic kidney and used for assaying the renotropic growth stimulating activity of six kidney cancer patients sera simultaneously. The level of renotropic growth-stimulating activity varies from patient to patient. The postnephrectomized sera of three patients showed rather small (27 to 68%) increase over their prenephrectomized sera. The postnephrectomized sera of the remaining three patients showed a much greater (2.69- to 3.19-fold) increase over their prenephrectomized sera. Variation in the growth-stimulating activities produced in these uninephrectomized kidney cancer patients is most probably due to different levels of renal function loss by removal of the cancer-bearing kidney. Each cancer-bearing kidney has a different proportion of healthy kidney region to diseased (nonfunctional) region. The second set of primary kidney cell cultures was prepared from a healthy noncancerous region of a cancer-bearing kidney and used to assay for renotropic growth-stimulating activities in two different unilaterally nephrectomized kidney transplantation donor sera. Increased levels of growth-stimulating activity are quite similar and high (4.19- to 4.79-fold increase above their prenephrectomized sera). These high and similar assay values of the renotropic growth factor produced in two healthy humans suggest the sensitivity and reliability of the assay method with the use of the noncancerous cells from the cancer-bearing kidney.

The in situ determination of melting-solidification points of lipid inclusions in fixed cultured cells.

Melting-solidification points of microscopic lipid inclusions in fetal human kidney cultures were determined visually by gradual heating of formol-fixed cell monolayers while immersed in dilute aqueous solutions of Nile blue A. Intracellular lipid inclusions stained red when their melting point was reached and reverted to blue color at the solidification temperature. The melting-solidification points of intracellular lipid inclusions in kidney cells were determined to fall within 19-25, 40-50, and 60-65 degrees C range.

A cytotoxic serum factor in polyarteritis nodosa and related conditions

A cytotoxic factor is described in the serum of nine patients suffering from polyarteritis nodosa and certain related conditions and in the serum of some of their asymptomatic relatives. The factor was found in the IgG fraction and is active against human leukocytes and isolated human kidney culture cells. A similar but not identical factor was found in the serum of eleven patients with systemic lupus erythematosus. These findings seem to support the view that this group of diseases has an immunologic and hereditary basis.

Synthesis of deoxyribonucleic acid in human and hamster kidney cells infected with human adenovirus types 5 and 12

The incorporation of 3H-labeled thymidine ( 3H-dT) into deoxyribonucleic acid (DNA) has been studied in human embryonic kidney and hamster kidney cells infected with human adenovirus types 5 and 12 (Ad5 and Ad12). The Ad5-infected human and hamster kidney cultures incorporated about 5–10 times as much 3H-dT into DNA as did uninfected cultures. The Ad12-infected human and hamster kidney cultures incorporated about 4–6 times and 3–5 times as much 3H-dT into DNA as did uninfected cultures. DNA-DNA hybridization experiments using a membrane filter technique were performed to elucidate the kind of DNA that was pulse-labeled with 3H-dT. In human and hamster kidney cells infected with Ad5, there was a progressive increase in the incorporation of 3H-dT into viral DNA. Incorporation of 3H-dT into cellular DNA was not immediately shut off. An increased incorporation during the early stage of infection (16–22 hours post infection) was followed by a decreased incorporation at later stages. The shutoff of incorporation into cellular DNA was found to occur gradually in hamster kidney cultures and rather rapidly in human kidney cultures. In human embryonic kidney cells infected with Ad12, incorporation of 3H-dT into viral DNA increased progressively until about 32 hours PI. Incorporation of 3H-dT into cellular DNA was stimulated somewhat at the early stage of infection, and was not shut off either during the period of viral DNA synthesis or for at least 20 hours after virus maturation had begun. In abortive infection of hamster kidney cells with Ad12, incorporation of 3H-dT into cellular DNA was stimulated. A small amount of incorporation of 3H-dT into viral DNA was detected at an early stage of infection. From these results, it can be concluded that in productive infection of human and hamster kidney cells by Ad5 or of human kidney cells by Ad12, cellular DNA synthesis is not immediately shut off. In abortive infection of hamster kidney cells by Ad12, cellular DNA synthesis is stimulated, whereas viral DNA is synthesized in small amounts at an early stage of infection.

CHAPTER 3: Cytotoxicity tests with cultures of human kidney as target

CHAPTER 3: Cytotoxicity tests with cultures of human kidney as target

Detection of Tissue Isoantigens on Primary and Serial Heteroploid Cell Lines of Human Origin by the Mixed Agglutination Technique

Summary The technique of mixed agglutination has been used to detect tissue isoantigens on human cells grown in tissue culture. Both primary human kidney cultures and heteroploid serial cell lines were shown to possess the isoantigens. The antisera used in these studies were from multiparous women and immunized chimpanzees. The sera were known to contain antibodies which reacted with human isoantigens by leukocyte agglutination. Absorption experiments showed the isoantigens detected on the serial heteroploid cultures were also present on human leukocytes and platelets.

The freezing of human kidney cell suspensions

The availability of a continuous supply of human kidney cultures is necessary for the efficient operation of virus screening and production programs. This report describes procedures for the processing of human kidneys and the freezing, storage, and culture of human kidney cells. The total cell yield, infant age, and postmortem age are listed for 89 cases. The results obtained from over 100 shipments of frozen cell suspensions are also listed. Results of this study indicate that safe and reliable repositories of frozen kidney cell suspensions can be successfully maintained for subsequent recovery and use as cell cultures.

Phlebotomus (sandfly) fever viruses in tissue culture.

Summary Propagation of the Naples and Sicilian types of Phlebotomus (sandfly) fever viruses in primary human and mouse kidney tissue cultures has been described. Plaques in human kidney cultures were delayed but were produced consistently by both Naples and Sicilian types. Cytopathogenic effects as a result of multiplication by either virus type, could be demonstrated in infant kidney cultures only after an adaptation period. In contrast, cytopathic changes could be seen in the first and subsequent virus passages when mouse kidney cultures were inoculated with either the Naples or Sicilian virus types. Mouse kidney cultures were found to be more susceptible to virus infection than human kidney monolayers.

Use of human kidney cultures in the study of enteroviruses.

Summary1) Human kidney cultures were prepared successfully from tissues obtained following surgical removal or at autopsy from individuals of all ages, when specimens were collected promptly. 2) Baby kidney cells were superior to adult both for culture growth and for isolation and propagation of enteroviruses. 3) Poliovirus types 1, 2 and 3 (virulent and attenuated strains) grew well in both adult and baby HK cultures. In contrast, Coxsackie A9, B 1-5 and all ECHO viruses tested (except types 7 and 10) induced distinct cellular degeneration only in baby HK cultures, while incomplete destruction with low virus titers were obtained in cultures derived from certain adult human kidneys. 4) The advantages of using baby HK cultures for isolation of certain ECHO viruses are discussed.