Studies of organic anion and cation transport in isolated segments of proximal tubules

The transport of organic anions and cations has interested physiologists for many years despite the generally held view that these transport mechanisms are involved principally in the elimination of foreign substances and metabolites from the body. The clearance and excretion of para-aminohippurate (PAH) has been used an an index of renal plasma flow, and the maximal rate of PAH secretion has been assumed to reflect the mass of functioning renal tissue in patients [1]. Clinicians also utilize the fractional excretion of urate as a guide to volume sufficiency of the extracellular fluid compartment and quantify the excretion rate of this organic solute in the evaluation of gout and urolithiasis [2]. The renal excretion of organic cations has been of principal interest to renal pharmacologists, although there is increased awareness among clinicians that this system is important in the regulation of endogenous levels of organic bases [1]

Preparation of renal papillary collecting duct cells for studyin vitro

The mammalian kidney is comprised of a heterogeneous array of tubules and blood vessels. Over the years the function of the individual nephron segments in the production of urine has been reasonably well defined by micropuncture andin vitro microperfusion techniques. Beyond this descriptive step, however, to clarify the mechanisms of solute and water transport, it is necessary to study cellular compartments, specifically. Several techniques are used currently for cell studies, but in each the usefulness is limited. For example, thin slices of kidney tissue contain a variety of cell types as do suspensions of kidney cortex [1]. By dissectionin vitro it is possible to isolate specific segments of nephron, but the mass of tissue is insufficient for the limits of resolution of many analytical techniques [2]. The purpose of this report is to describe a method for collecting milligram quantities of viable cells from the papillary collecting duct of the rabbit kidney

Localization of overexpressed c-myc mRNA in polycystic kidneys of the cpk mouse

Localization of overexpressed c-myc mRNA in polycystic kidneys of the cpk mouse. The C57BL/6J-cpk mouse has a form of autosomal-recessive polycystic kidney disease characterized by the rapid growth of large collecting duct cysts and the development of severe renal failure usually by three to four weeks of age. Previous studies had shown higher steady-state levels of proto-oncogene mRNA in these cystic kidneys. It is now shown using nuclear run-on transcription that the c-fos and c-myc proto-oncogenes are transcribed at higher rates in cystic kidneys, and thus that increased transcription, in part, may account for the increased mRNA levels. c-myc mRNA was detected by in situ hybridization in nephron anlagen and elongating tubules of normal and cystic kidneys during late fetal and early neonatal kidney development. Localization of c-myc expression in the normal kidney decreased with age over the three-week postnatal period. By contrast, c-myc mRNA was found in cysts as early as three days of age, with increased levels at two and three weeks, c-myc expression was also elevated in apparently normal, non-dividing proximal tubules in three-week-old cystic animals. On the basis of these findings, we suggest that c-myc expression is linked to the proliferation of cells engaged in the primary cystogenic process, and that expression of this gene in proximal tubule cells of severely azotemic animals reflects the compensatory response of residual tubular epithelial cells to progressive renal dysfunction

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys.BackgroundThe proliferation of mural epithelial cells is a major cause of progressive cyst enlargement in autosomal-dominant polycystic kidney disease (ADPKD). Adenosine 3′, 5′ cyclic monophosphate (cAMP) stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney cortex (HKC), through the activation of protein kinase A (PKA), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK/MAPK). In the current study, we examined the signaling pathway between PKA and MEK in ADPKD and HKC cells.MethodsPrimary cultures of human ADPKD and HKC cells were prepared from nephrectomy specimens. We determined the effects of cAMP and epidermal growth factor (EGF) on the activation of ERK, B-Raf and Raf-1 in ADPKD and HKC cells by immune kinase assay and Western blot.Results8-Br-cAMP increased phosphorylated ERK (2.7- ± 0.6-fold, N = 7), and B-Raf kinase activity (3.6- ± 1.1-fold, N = 5) in cells from ADPKD kidneys; levels of phosphorylated Raf-1 were not changed. Inhibition of PKA by H89 strikingly decreased cAMP-stimulated phosphorylation of ERK and B-Raf, and MAPK inhibition by PD98059 blocked the effect of the nucleotide to activate ERK. By contrast, in HKC cells 8-Br-cAMP did not activate B-Raf and ERK. EGF stimulated the phosphorylation of ERK and Raf-1 in both ADPKD and HKC cells, but had no effect on B-Raf. 8-Br-cAMP and EGF conjointly increased ERK activation above that of either agonist alone in ADPKD cells, and this combined effect was abolished by PD98059, indicating that ERK was activated by EGF- and cAMP-responsive cascades that converge at MAPK.ConclusioncAMP activates ERK and increases proliferation of ADPKD epithelial cells, but not cells from normal human kidney cortex, through the sequential phosphorylation of PKA, B-Raf and MAPK in a pathway separate from, but complementary to, the classical receptor tyrosine kinase cascade. Consequently, cAMP and EGF have great potential to accelerate the progressive enlargement of renal cysts

Identification of a Forskolin-Like Molecule in Human Renal Cysts

Renal cyst enlargement is increased by adenosine cAMP, which is produced within mural epithelial cells. In a search for modulators of cAMP synthesis cyst fluids from 18 patients with autosomal dominant or recessive polycystic kidney disease (PKD) were analyzed, and in 15 of them, a stable lipophilic molecule that increased cAMP levels, stimulated transepithelial chloride and fluid secretion, and promoted the proliferation of human cyst epithelial cells was characterized. With the use of HPLC-mass spectrometry, a bioactive lipid with the same mass spectral fingerprint, the same chromatographic retention time, and the same biologic properties as forskolin, a widely known, potent adenylyl cyclase agonist, has been isolated and identified within the cyst fluid. Forskolin is synthesized by the plant Coleus forskohlii, but its appearance or compounds like it have not been reported in animals. The origin of forskolin in patients with PKD was not revealed by this study. Synthesis by mural cyst epithelial cells or an exogenous source are the most likely possibilities. Forskolin is sold for weight management and as a cardiovascular tonic in health stores and through the Worldwide Web. It is concluded that forskolin may have a role in promoting the enlargement of cysts in autosomal dominant PKD and recommended that patients avoid oral and parenteral preparations that contain this compound

Experiences and Perspectives of Polycystic Kidney Disease Patients following a Diet of Reduced Osmoles, Protein, and Acid Precursors Supplemented with Water:A Qualitative Study

Background Salt, protein, acid precursors, and fluid intake have been identified as factors that influence cyst growth in ADPKD. Unfortunately, the feasibility of following these dietary restrictions/enhancements from a patient's point-of-view has yet to be studied. The purpose of this study is to understand better the experiences of patients following a relatively complex dietary prescription targeting these factors. Methods Twelve adults with ADPKD and kidney function >30ml/min/1.73m(2) were recruited from the University of Kansas Medical Center Polycystic Kidney Disease clinic. In a qualitative design, semi-structured interviews of participants were conducted following a four week dietary intervention (experimental diet lower in sodium, protein, and acid precursors, and supplemented with water) either face-to-face or by telephone. All interviews were recorded, transcribed verbatim, and checked for accuracy. Transcripts were analyzed thematically for emerging themes. Results Participants reported that eating less meat and more fruits and vegetables were the easiest components of the diet, whereas reaching the daily goal amount of fruits and vegetables and tracking the diet constantly were the most difficult components. Participants had little difficulty with fluid intake and reported the prescribed fluid goal as achievable. The tracking system for fruits and vegetables and protein was reported to be both helpful and intuitive, but tracking their intake on paper was tedious. Eating out was the most significant barrier to following the diet with some individuals avoiding restaurants in order to comply with the dietary prescription. Conclusion Participants on the experimental diet heightened their awareness of the consumption of dietary salt, protein, acid precursors, and fluid intake. Additionally, most participants believed adherence to the prescribed diet was feasible. However, participants wanted less cumbersome ways to track and monitor the diet, especially given that the prescribed diet is designed for lifelong adherence. Future studies should focus on targeting these specific dietary factors in larger groups of more ethnically and culturally diverse populations to help inform clinicians and how best to help diverse populations adhere to the dietary intervention