Proximal Tubule Cells In Primary Culture Research Articles

NHERF1 loss results in metabolic stress and increased susceptibility to cisplatin‐induced acute kidney injury

BackgroundAcute kidney injury (AKI) is associated with a high mortality rate and develops in 30% of patients who receive cisplatin. The sodium hydrogen regulatory factor isoform 1 (NHERF1) is a scaffolding protein that anchors multiple membrane proteins, in renal proximal tubules. We have recently demonstrated that NHERF1 loss results in changes in mitochondrial protein abundance and function.HypothesisWe hypothesize that NHERF1 loss increases susceptibility to AKI through an underlying metabolic stress.Materials and MethodsWe treated 2 month old male and female wild type (WT) C57BL/6 and NHERF1 knock out (KO) mice with vehicle (VEH) or cisplatin (CIS) (20 mg/kg dose IP) and euthanized after 72 hours. Blood was collected for blood urea nitrogen (BUN) levels. Urine was collected for NGAL. Kidneys were harvested for histology, TUNEL assay, and Western Blot for cleaved caspase 3. Proteomic analysis was conducted on kidney brush border membrane (BBM) of WT and KO kidneys. Metabolic measurements were conducted via the XF Flux analyzer on non‐treated primary proximal tubule cell cultures. Three‐way ANOVA was used to compare the different treatment groups. Ordinal regression was performed for the semi‐quantitative scoring. All statistical analysis was conducted with SPSS software. P values of <0.05 were considered statistically significant.ResultsCIS caused significantly greater severity of kidney injury in KO compared to WT mice by semi‐quantitative injury score (WT: 1.89, KO: 2.8), BUN levels (WT: 97.8 mg/dL +/− 10.1, KO: 151.8 mg/dL +/− 17.2) and NGAL protein (WT: 2.7 μg/mL +/− 0.53, KO: 55.6 μg/mL +/−21.3). Apoptosis markers were significantly increased in CIS treated KO and WT mice compared to respective controls. Proteomic comparison of BBM protein expression in WT and KO showed marked changes in the expression of mitochondrial proteins in KO mice, suggesting alterations in cell metabolism. KO proximal tubule cells also exhibited decreases of 35% in glycolytic reserve capacity, 40% in baseline mitochondrial respiration rate, 37% in ATP‐linked mitochondrial respiration rate, 40% in mitochondrial reserve capacity, and 40% in maximum mitochondrial capacity.ConclusionsWe conclude that the KO mice have increased susceptibility to CIS‐induced AKI, which may be due to altered metabolism and/or mitochondrial dysfunction.Support or Funding InformationFunding for this research was provided by VA and UofL School of Medicine (to EDL).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Rab11a Positive Compartments in Endocytic Traffic in Primary Proximal Tubule Cells

The proximal tubule (PT) reabsorbs ~ 70% of sodium, bicarbonate, chloride ions, phosphate, glucose, water, and the majority of the plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell function, the organization of the endocytic pathway in these cells remains poorly understood. We have used live‐cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PT cells isolated from mice. Cells from the S1 segment could be distinguished from more distal segments by their robust uptake of albumin and low expression of γ‐glutamyltranspeptidase. Rab11a in these cells is localized to numerous dynamic spherical compartments that resemble the apical vacuoles found in electron microscopy analysis of cultured PT cells. These structures are highly dynamic and receive both membrane and fluid phase cargo. In contrast, Rab11a in immortalized kidney cell lines is localized to a condensed and largely tubular apical recycling compartment through which recycling membrane proteins (but not fluid phase cargoes) transit. The unusual morphology of Rab11a‐positive compartments in primary PT cells may reflect the demands of handling a high endocytic load and suggests a possible role for Rab11a in early sorting steps of fluid and membrane proteins in these cells. This research was supported by NIH R01 DK54407 and P30 DK079307.

Tranilast attenuates connective tissue growth factor-induced extracellular matrix accumulation in renal cells

Tranilast (N-[3,4-dimethoxycinnamoyl]anthranilic acid) is a synthetic compound that we have recently reported to inhibit transforming growth factor-beta1 (TGF-beta1)-induced tubulointerstitial fibrosis in the kidney. Connective tissue growth factor (CTGF) is recognized as a potent downstream mediator of TGF-beta1. Both proximal tubule cells (PTCs) and cortical fibroblasts (CFs) are considered to be responsible for the production of tubulointerstitial extracellular matrix (ECM). These studies were undertaken to assess the profibrotic effects of CTGF in an in vitro model of the human PTCs and CFs, and to determine whether tranilast is effective in limiting the in vitro matrix responses induced by CTGF. Primary cultures of PTCs and CFs were exposed to CTGF (20 ng/ml)+/-tranilast (100 microM). Cell hypertrophy and the secretion of the ECM proteins fibronectin and collagen IV were determined. The effects of tranilast on TGF-beta1-induced CTGF mRNA expression and on phosphorylation of Smad2 were determined. CTGF significantly induced cell hypertrophy, increased fibronectin, and collagen IV secretion in PTCs and CFs. In all cases, the CTGF-induced increase in ECM protein was inhibited in the presence of tranilast. Tranilast reduced CTGF mRNA and phosphorylation of Smad2, which were induced by TGF-beta1 in PTCs and CFs. These results suggest that tranilast is a potential effective antifibrotic compound in the kidney, exerting its effects via inhibition of TGF-beta1-induced CTGF expression and downstream activation of the Smad2 pathway in both PTCs and CFs.

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca2+ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca2+ ([Ca2+]i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca2+]i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D 600) and felodipine on [Ca2+]i during anoxia. A method was developed whereby [Ca2+]i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca2+]i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca2+]i were 118 +/- 2 (n = 98) and 662 +/- 22 (n = 160) nM, respectively. D 600 (1 microM), but not felodipine (10 microM), significantly reduced basal [Ca2+]i in normoxic incubations. During anoxia 1 microM and 100 microM D 600 significantly decreased anoxic [Ca2+]i levels by 22 and 63% respectively. Felodipine at 10 microM was as effective as 1 microM D 600. Removal of extracellular Ca2+ and addition of 0.1 mM La3+ completely abolished anoxia-induced increases in [Ca2+]i. We conclude that anoxia induces increases in [Ca2+]i in rabbit PT cells in primary culture, which results from Ca2+ influx. Since this Ca2+ influx is partially inhibited by low doses of CCBs, L-type Ca2+ channels may be involved.

Dramatic changes of sulfated proteoglycans composition in a tumorigenic SV-40-transformed renal proximal-tubule cell line.

To characterize the sulfated proteoglycans (PGs) alterations associated with malignant transformation of epithelial cells in vitro, the localization, charge, size, and composition of cell-associated and secreted sulfated PGs have been compared in rabbit renal proximal-tubule cells in primary culture (Ronco et al., 1990) and in a derived SV-40 transformed cell line (RC.SV1) exhibiting a proximal phenotype and high tumor-inducing ability (Vandewalle et al., 1989). Both normal and transformed cells incorporated PGs into a thick basement membrane layer as shown by ruthenium red staining and immunodetection with a monoclonal antibody raised against the core protein of the bovine basement membrane heparan sulfate-PG (HS-PG). In primary cultures of normal cells, cell-associated PGs were almost identical to those extracted from renal tubule fractions in vivo by their size (Kav = 0.27 vs. 0.26 on Sepharose CL-6B) and composition characterized by the exclusive presence of heparan sulfate glycosaminoglycan (HS-GAG) chains. In addition, the cells secreted a HS-PG with similar biochemical characteristics (Kav = 0.29; 100% HS-GAG chains). The SV-40-transformed RC.SV1 cells also synthesized and secreted a unique PG with the same charge and Kav values and apparently the same core protein (35 kDa) as in nontransformed cells, but three major differences were observed: (i) an increased proportion of PG-associated [35S]sulfate radioactivity released into the culture medium (36 vs. 21%), (ii) the emergence of free GAG chains unincorporated into PGs and detected only in the cell-associated fraction, and (iii) a dramatic change in the composition of GAG chains in which chondroitin sulfate replaced heparan-sulfate. The latter finding is in keeping with the known chondroitin sulfate increase and heparan-sulfate decrease in epithelial tumors. The alterations of PGs observed in this study may play a role in the acquisition and/or maintenance of the malignant phenotype.

Streptomycin toxicity in primary cultures of flounder renal proximal tubule cells

The aminoglycoside antibiotic streptomycin is a known nephrotoxin in vivo and a common component of cell culture media. The effects of streptomycin (100 micrograms/ml) on transepithelial electrical properties, glucose transport, glycolytic metabolism, and morphology were examined in primary proximal tubule cell cultures from winter flounder (Pseudopleuronectes americanus) kidney. Streptomycin treatment on either Days 2 to 12 or Days 8 to 13 abolished the transepithelial potential difference and short-circuit current across the monolayer but had no effect on transepithelial resistance in confluent 12 to 13-d cultures, suggesting the loss of active transepithelial transport. Consistent with these findings, mucosal-to-serosal glucose fluxes were greatly reduced in streptomycin-treated cultures and insensitive to the transport inhibitor phlorizin, indicating the absence of the apical Na-dependent glucose transport system associated with net glucose reabsorption. In addition to transport processes, antibiotic treatment also interfered with cellular energy metabolism as judged by the rapid reduction in glycolytic lactate production observed in the presence of streptomycin. Scanning and transmission electron microscopy revealed that streptomycin-treated cultures were composed of cuboidal-to-columnar shaped cells which maintained intact tight junctions similar to control cultures. However, apical microvilli, the presumed sites of mucosal transport systems, were severely reduced in number in streptomycin-treated cultures. We concluded that streptomycin, at a dose commonly used in cell culture, inhibited the expression of differentiated function by flounder proximal tubule cell cultures. These cell cultures may provide a suitable model system for examination of the mechanisms of aminoglycoside nephrotoxicity.