Henle's Loop Cells Research Articles

#2485 CellMatchR—RNA-seq based matching of kidney cell lines to kidney reference cell types

Abstract Background and Aims Cultured kidney cell lines are broadly used to model the physiology and pathophysiology of the kidney. Due to immortalization, passaging and culture conditions, a cell line's gene expression profile might differ greatly from the primary cells it was originally derived from. This might be further enhanced by experimental treatments with compounds or genetic modifications. It is therefore a relevant question if a given cell culture system is still the appropriate model for a certain disease or scientific investigations. Reasoning that RNA sequencing (RNA-seq) based transcriptomes are generated as part of many experimental setups and hence may be used to address this question, we developed and tested an RNA-seq based approach, CellMatchR. This approach matches kidney cell lines, primary cells and even tissue specimen to known kidney reference cell types to determine which primary kidney cell type is the most similar and to what degree global gene expression or the expression of selected marker genes differs. Method CellMatchR uses published murine and human kidney single cell- and tubule-level transcriptomic datasets from healthy mice and human donors as references. Single cell datasets were further processed to pseudobulk references for each of the contained cell types. Then RNA-seq data from cell lines or tissues of interest (test data) was compared to the reference (pseudo)bulk data with Spearman correlations of gene counts-per-million values (CPM) or Euclidean distance using log-transformed CPM values. Both approaches were systematically tested for various combinations of test and reference datasets across different species and using global gene expression (i.e. all genes measured in reference and test datasets) and a set of 315 manually curated, kidney cell type marker genes. Results We sequentially used different kidney tissue types, primary cells and cell lines as positive controls and matched them to our reference datasets. Spearman correlations of gene expression rankings showed the highest correlation coefficients with biologically correct reference cell types (examples in Fig. 1A-C). We found Spearman correlations to be superior to Euclidean distances as method of comparison. Analyses based on global gene expression compared to our curated set of 315 kidney cell type marker genes yielded similar results. Notably, correlation coefficients were higher and showed less variation between reference cell types when using the global gene set. Matchings across species, i.e. using murine test and human reference data or vice versa still yielded mostly correct results. However, correlation coefficients were generally lower (i.e. rho = 0.9 vs. 0.6) and varied more across reference datasets if comparisons were performed across species. Using published RNA-seq data for different cell lines of the proximal tubule (e.g. human HK-2 and opossum OKH cells) we observed low similarities with proximal tubule cells (Fig. 1D), whereas two tested cell lines of the collecting duct (mIMCD-3 and mpkCCD cells) plausibly showed the highest similarity to medullary cell types like collecting duct and Loop of Henle cells. The most similar reference cell types did not change in mIMCD-3 cells when kept in 2-dimensional vs. 3-dimensional culture conditions, and in mpkCCD cells when the osmolality of the culture medium was changed from 300 to 600 mosmol/kg. Also, a Pkd1 knockout in mIMCD-3 cells did not change the most similar reference cell type in our analyses (Fig. 1E and F). Conclusion Our CellMatchR approach uses publicly available kidney single cell and bulk RNA-seq datasets and combines these with simple, computationally fast yet effective statistical methods to determine similarities of kidney cell lines and tissue samples to reference cell types of interest. It can easily be implemented by trained users or integrated into online resources for usage with a visual interface. It relies on RNA-seq data from the cell lines of interest and hence presents a feasible complementary method to check the general similarity of cell culture models to kidney cell types and assess their stability across experimental conditions.

No Amelioration of Uromodulin Maturation and Trafficking Defect by Sodium 4-Phenylbutyrate in Vivo: STUDIES IN MOUSE MODELS OF UROMODULIN-ASSOCIATED KIDNEY DISEASE

Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium 4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as a potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IκB kinase α/β, an indirect activator of NF-κB. Furthermore, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52, and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention.

Calreticulin is crucial for calcium homeostasis mediated adaptation and survival of thick ascending limb of Henle's loop cells under osmotic stress

The thick ascending limb of Henle's loop (TALH) is normally exposed to variable and often very high osmotic stress and involves different mechanisms to counteract this stress. ER resident calcium binding proteins especially calreticulin (CALR) play an important role in different stress balance mechanisms. To investigate the role of CALR in renal epithelial cells adaptation and survival under osmotic stress, two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry and functional proteomics were performed. CALR expression was significantly altered in TALH cells exposed to osmotic stress, whereas renal inner medullary collecting duct cells and interstitial cells exposed to hyperosmotic stress showed no significant changes in CALR expression. Moreover, a time dependent downregulation of CALR was accompanied with continuous change in the level of free intracellular calcium. Inhibition of the calcium release, through IP3R antagonist, prevented CALR expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. Overexpression of wild type CALR in TALH cells resulted in significant decrease in cell viability under hyperosmotic stress. In contrast, the hyperosmotic stress did not have any effect on cells overexpressing the CALR mutant, lacking the calcium-binding domain. Silencing CALR with siRNA significantly improved the cell survival under osmotic stress conditions. Taken together, our data clearly highlight the crucial role of CALR and its calcium-binding role in TALH adaptation and survival under osmotic stress.

Secreted Wnt antagonist Dickkopf-1 controls kidney papilla development coordinated by Wnt-7b signalling

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.

Mutant Tamm-Horsfall Glycoprotein Accumulation in Endoplasmic Reticulum Induces Apoptosis Reversed by Colchicine and Sodium 4-Phenylbutyrate

As a consequence of uromodulin gene mutations, individuals develop precocious hyperuricemia, gout, and progressive renal failure. In vitro studies suggest that pathologic accumulation of uromodulin/Tamm-Horsfall glycoprotein (THP) occurs in the endoplasmic reticulum (ER), but the pathophysiology of renal damage is unclear. It was hypothesized that programmed cell death triggered by accumulation of misfolded THP in the ER causes progressive renal disease. Stably transfected human embryonic kidney 293 cells and immortalized thick ascending limb of Henle's loop cells with wild-type and mutated uromodulin cDNA were evaluated to test this hypothesis. Immunocytochemistry, ELISA, and deglycosylation studies indicated that accumulation of mutant THP occurred in the ER. FACS analyses showed a significant increase in early apoptosis signal in human embryonic kidney 293 and thick ascending limb of Henle's loop cells that were transfected with mutant uromodulin constructs. Colchicine and sodium 4-phenylbutyrate treatment increased secretion of THP from the ER to the cell membrane and into the culture media and significantly improved cell viability. These findings indicate that intracellular accumulation of THP facilitates apoptosis and that this may provide the pathologic mechanism responsible for the progressive renal damage associated with uromodulin gene mutations. Colchicine and sodium 4-phenylbutyrate reverse these processes and could potentially be beneficial in ameliorating the progressive renal damage in uromodulin-associated kidney diseases.

Localization and regulation of a functional GHRH receptor in the rat renal medulla.

To provide information about the kidney GHRH receptor (GHRH-R), we assessed its tissue and cellular localization, defined its pattern of expression in developing and aging rats, and studied the effects of GHRH on the regulation of GHRH-R mRNA levels and receptor internalization. In situ hybridization and ribonuclease protection assay demonstrated that GHRH-R mRNA is restricted to the Henle's loop (HL). GHRH-R mRNA levels were low in the medulla from 3- and 12-d-old male rats, increased significantly in that from 30- to 70-d-old rats, and decreased in that from 12- and 18-month-old animals. Compared with the GHRH-R mRNA profile obtained in the pituitary, these data support the concept of a tissue-specific regulation of GHRH-R. In HL cell cultures from 70-d-old rats, a 4-h incubation with 1-100 nM rat GHRH-(1-29)NH(2) reduced GHRH-R mRNA levels significantly. As anti-GHRH-R- (392-404) immunoreactivity was demonstrated in HL cells, internalization of [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2),Ala(8), Ala(15),Lys(22)]hGHRH-(1-29)NH(2) in a time- and temperature-dependent manner and inhibition of this process by phenyl arsine oxide indicate that desensitization to GHRH involves both GHRH-R internalization and down-regulation of GHRH-R mRNA levels. Localization of a functional GHRH-R in HL and its regulation during development and aging suggest roles associated with cellular proliferation, differentiation, and/or water/electrolyte transport.

Altered expression of prostacyclin synthase in a subset of the thick ascending limb cells and mesangial cells in 5/6-nephrectomized rats.

The aim of this study was to evaluate the possible role of prostacyclin (PGl2) in the onset and development of hypertension and chronic renal failure in 5/6-nephrectomized rats (5/6NX). We measured the systolic blood pressure, 24-h urinary excretion levels of 6-keto-PGF1alpha, which was a stable metabolite of PGI2, and levels of PGI2 synthase (PCS) mRNA in the kidneys. Immunostaining for PCS in the kidneys was also evaluated. Systolic blood pressure was higher in 5/6NX than in sham-operated rats. The 24-h urinary excretion levels of 6-keto-PGF1alpha in 5/6NX at 1 week postsurgery were lower than in sham-operated rats. In renal morphology, tubulointerstitial injury was observed at 2 weeks postsurgery, and glomerulosclerosis at 4 weeks. Levels of PCS mRNA in 5/6NX decreased significantly at 1 and 2 weeks postsurgery compared with those in sham-operated rats, but at 8 weeks these levels showed a tendency to increase. Immunostaining for PCS was positive in a subset of the cortical thick ascending limb of Henle's loop cells, including macula densa in both groups. Moreover, in 5/6NX at 8 weeks postsurgery, mesangial cells also stained positive for PCS. In conclusion, our findings suggest that PCS might play an important role in mitigating glomerular hemodynamic changes associated with reduction of renal mass.

Carbonic anhydrase IV is expressed in H(+)-secreting cells of rabbit kidney.

Carbonic anhydrase (CA) IV is a membrane-bound enzyme that catalyzes the dehydration of carbonic acid to CO(2) and water. Using peptides from each end of the deduced rabbit CA IV amino acid sequence, we generated a goat anti-rabbit CA IV antibody, which was used for immunoblotting and immunohistochemical analysis. CA IV was expressed in a variety of organs including spleen, heart, lung, skeletal muscle, colon, and kidney. Rabbit kidney CA IV had two N-glycosylation sites and was sialated, the apparent molecular mass increasing by at least 11 to approximately 45 kDa in the cortex. Medullary CA IV was much more heavily glycosylated than CA IV from cortex or any other organ, such modifications increasing the molecular mass by at least 20 kDa. CA IV was expressed on the apical and basolateral membranes of proximal tubules with expression levels on the order of S2 > S1 > S3 = 0. Because CA IV is believed to be anchored to the apical membrane by glycosylphosphatidylinositol, the presence of basolateral CA IV suggests an alternative mechanism. CA IV was localized on the apical membranes of outer medullary collecting duct cells of the inner stripe and inner medullary collecting duct cells, as well as on alpha-intercalated cells. However, CA IV was not expressed by beta-intercalated cells, glomeruli, distal tubule, or Henle's loop cells. Thus CA IV was expressed by H(+)-secreting cells of the rabbit kidney, suggesting an important role for CA IV in urinary acidification.

Cytokines affect ion transport in primary cultured thick ascending limb of Henle's loop cells.

Tumor necrosis factor-alpha (TNF) and interleukin-1 (IL-1) affect epithelial cell ion transport. However, the site of action along the nephron has not been elucidated fully for these cytokines. Thus, the effect of TNF and IL-1 on the ion transport function of primary cultured medullary thick ascending limb of Henle's loop (mTALH) cells was determined by measuring rubidium (86Rb) uptake. TNF, IL-1, and lipopolysaccharide (LPS), a known activator of cytokine production, inhibited 86Rb uptake by cultured mTALH cells after a 24-h incubation period but had no effect when incubated with the cells for 1 or 4 h. Furthermore, mTALH cells produced biologically active TNF after stimulation with LPS for 24 h, and the LPS-induced inhibition of 86Rb uptake was abolished in the presence of an anti-TNF antibody, suggesting that TNF produced by the mTALH cells acted in an autocrine manner to inhibit 86Rb uptake. The effects of LPS on 86Rb uptake also were inhibited by the cyclooxygenase inhibitor, indomethacin. As TNF increased prostaglandin E2 synthesis by cultured mTALH cells and as prostaglandin E2 also inhibited 86Rb uptake, LPS presumably inhibited 86Rb uptake by inducing a TNF-mediated increase in prostaglandin synthesis. These data demonstrate that a prostanoid produced by mTALH cells mediates the inhibitory effect of LPS and TNF on 86Rb uptake and imply that endogenous TNF inhibits ion fluxes in the mTALH via a prostaglandin-dependent mechanism.

Dibutyryl cyclic AMP inhibits transport dependent QO2 in cells isolated from the rabbit medullary ascending limb.

Because the medullary thick ascending limb of the loop of Henle is the target of several polypeptide hormones that stimulate adenyl cyclase in this nephron segment, we examined the effects of cyclic AMP on thick ascending limb of the loop of Henle cells isolated by enzymatic digestion and density gradient centrifugation from the outer medulla of the rabbit kidney. The functional parameter that was measured was transport dependent oxygen consumption. Oxygen consumption was measured using a polarographic oxygen electrode in a constant temperature chamber. We found that dibutyryl cyclic AMP inhibited oxygen consumption in a dose dependent way. Maximal inhibition was observed at a concentration of 10(-5) M. The effect of dibutyryl cyclic AMP was not present in the absence of either sodium, chloride or both implying that its effect is restricted to the sodium and chloride dependent oxygen consumption. The effect of dibutyryl cyclic AMP was additive to that of furosemide 10(-4) M while that of furosemide was not additive to that of cyclic AMP suggesting that the site of action of cyclic AMP is distal to that of furosemide. The effect of dibutyryl cyclic AMP was not additive to that of ouabain and was absent in cells where oxygen consumption was stimulated with amphotericin B in the absence of chloride indicating that it has no effect on Na-K-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture.

Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin greater than isoproterenol greater than calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin greater than vasopressin = PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin = calcitonin. Neither isoproterenol nor PTH had an effect. These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.

Nephron epithelia in culture: Growth of loop of Henle cells in hormonally defined serum-free media

Nephron epithelia in culture: Growth of loop of Henle cells in hormonally defined serum-free media

Klossiella equi Baumann, 1946 (Sporozoa: Eucoccidia: Adeleina) from Equids

Kidneys from 5 of 40 ponies (Equus caballus) and from 3 of 14 burrows (Equus asinus) were found infected with Klossiella equi. In addition to previously reported sporogonous stages in epithelial cells of Henle's loop, schizogonic stages in endothelial cells of Bowman's capsule and epithelial cells of the proximal convoluted tubules are described. The association of macroand microgametocytes in syzygy is discounted, and a microgametocyte with 8 to 10 microgametes is characterized. Microgametes in the process of migrating to macrogametes are reported. A life cycle for this parasite is proposed. Baumann (1946) first described Klossiella equi from the kidney of a horse in Hungary. He suggested that (1) macrogametes and microgametes develop in syzygy with the formation of four microgametes and (2) after syngamy, the zygote grows and produces, by multiple nuclear fission followed by budding from a large central residual mass, a large number of sporoblasts that develop into sporocysts. Akcay and Urman (1954) found the parasite in kidneys of 72 donkeys in Turkey incidental to experimental work on equine infectious anemia. The species was also described from a burro (Equus asinus) in the United States by Seibold and Thorson (1955a). These workers observed four distinct developmental stages in kidney tubule cells and proposed the following descriptive terms: (1) uninuclear, (2) multinuclear, (3) budding, and (4) thin-walled sac with sporocysts enclosed. In the fourth stage, they counted as many as 12 sporozoites in the sporocysts. They later (1955b) concluded that their parasite was identical to Baumann's K. equi after examining his slides. Newberne, Robinson, and Bowen (1958) found K. equi in the kidney of a zebra from Africa that died in a Florida zoo. Four different sporogonic stages were found in the tubule cells of the medulla. One to four small eosinophilic masses, which were present within the vacuoles of the uninuclear form, were thought to be the microgametocytes suggested by Baumann (1946). Sasmore (1959) found four morphological stages of the parasite Received for publication 28 September 1971. 589 in 28 burros (E. asinus) and one horse (E. caballus) in Tennessee. He proposed the following terms: (1) trophozoite, (2) preschizont, (3) schizont, and (4) sporocyst. He also speculated upon a possible interrelationship of K. equi with skin and intestinal globidial cysts. Hartman (1961) also found K. equi in eight burros. He followed the terminology proposed by Seibold and Thorson (1955a), described the sporogonic phases of the life cycle, and mentioned that the small bodies in the uninuclear phase may be macroand microgametocytes. Klossiella equi has been found in eight equids used in piroplasmosis research during the past 3 years at this laboratory. This report describes the endogenous stages that were found and presents our version of the life cycle of the parasite. MATERIALS AND METHODS Kidney tissues, taken from ponies (Equus caballus) and burros (Equus asinus) that died or were killed during studies of piroplasmosis, were either quick-frozen and sectioned in a cryostat or fixed with Helly's fluid, infiltrated with paraffin, and sectioned. Sections were stained with either Heidenhain's iron hematoxlyin with no counterstain (Lillie, 1965), Himes and Moriber's trichrome (Himes and Moriber, 1956), or Whipfs polychrome stain (Vetterling and Thompson, in press). Sections were examined at either 400 or 800 X with light-field or dark-field fluorescent micros-