Thick ascending tubular cells in the loop of Henle: regulation of electrolyte homeostasis

Renal medullary tubular cells in the loop of Henle have crucial importance for the regulation of homeostasis of the extracellular fluid. These cells receive limited amount of blood and oxygen, and are also constantly challenged by the hypertonic environment. The medullary tubular cells in the last part of the loop of Henle have one of the highest known contents of mitochondria of all mammalian cells, reflecting their need for oxidative metabolism in order to sustain high ATP production for active transepithelial electrolyte transport. The commonly used diureticum furosemide targets one of the transporters present in these tubular cells with resulting diuresis. Several pathological states are associated with altered function of the medullary tubular cells, and the nephrotoxic substances tacrolimus and cyclosporine act on these cells. The specific Tamm-Horsfall glycoprotein is produced by medullary tubular cells. Alteration in the urinary excretion of this protein is used as marker of tubular damage.     

A possible correlation between ultrastructure and function in the thin descending and ascending limbs of the loop of Henle of rabbit kidney

Summary An ultrastructural study of the thin loops of Henle has been made in the renal papilla of the rabbit. Animals in different states of water balance were used but no morphological difference was observed in the loops obtained from animals in different experimental groupings. The cytoplasm of the squamous cells lining the limbs was characterised by a paucity of organelles. Descending and ascending limbs were distinguishable. A distinct morphological difference was seen in the junctional regions of cell processes of the descending and ascending thin limbs of the loop. The ascending limb processes were joined by continuous tight junctions whereas the descending limb junctional regions invariably showed a space of at least 70 Å between adjacent processes. It is suggested that there may be a correlation between the structure of these junctional regions and the different permeability characteristics of the two limbs. The thin ascending limb must, on physiological evidence, be relatively impermeable with reference to the thin descending limb.The author wishes to thank Professor F. R. Johnson for his advice and assistance, and Mr. R. F. Birchenough, Mr. P. L. Hyam and Mr. J. Manston for valuable technical assistance.     

Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney

The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N-linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina.     

Hypovolemic models of acute tubular necrosis in the rat kidney.

Studies were undertaken to determine whether a hypotensive episode under variable conditions is capable of inducing experimental acute renal failure in rats. Animals were subjected to hypovolemic shock by withdrawing volumes of blood necessary to maintain a systolic pressure of 30-40 mm Hg for 105-110 min. The blood was then reinfused and the animal was allowed to recover for 48 h prior to sacrifice. In an attempt to increase the injury, a second group of animals was salt-depleted prior to injury, a third group was volume-depleted by being deprived of H2O for 72 h prior of injury, a fourth group received 7.5 mg/kg indomethacin 30 min prior to injury, and a fifth group had 30% of the blood which was removed to produce shock hemolyzed and returned following the injury. In all groups examined, light microscopy revealed a moderate to severe acute tubular necrosis localized mainly in the outer stripe of the outer zone as defined by Peter (1909). Tubular damage was confined to the medullary pars recta of the proximal tubule and only in the most severe cases did injury involve the cortical pars recta and pars convoluta. Casts were present in the distal tubules and collecting ducts. Despite these significant histologic alterations, BUN values from all experimental groups remained within control levels. These studies clearly show that extensive necrosis of the medullary pars recta can be dissociated from the development of acute renal failure.     

ERK-mediated suppression of cilia in cisplatin-induced tubular cell apoptosis and acute kidney injury

In kidneys, each tubular epithelial cell contains a primary cilium that protrudes from the apical surface. Ciliary dysfunction was recently linked to acute kidney injury (AKI) following renal ischemia–reperfusion. Whether ciliary regulation is a general pathogenic mechanism in AKI remains unclear. Moreover, the ciliary change during AKI and its underlying mechanism are largely unknown. Here we examined the change of primary cilium and its role in tubular cell apoptosis and AKI induced by cisplatin, a chemotherapy agent with notable nephrotoxicity. In cultured human proximal tubular HK-2 epithelial cells, cilia became shorter during cisplatin treatment, followed by apoptosis. Knockdown of Kif3a or Polaris (cilia maintenance proteins) reduced cilia and increased apoptosis during cisplatin treatment. We further subcloned HK-2 cells and found that the clones with shorter cilia were more sensitive to cisplatin-induced apoptosis. Mechanistically, cilia-suppressed cells showed hyperphosphorylation or activation of ERK. Inhibition of ERK by U0126 preserved cilia during cisplatin treatment and protected against apoptosis in HK-2 cells. In C57BL/6 mice, U0126 prevented the loss of cilia from proximal tubules during cisplatin treatment and protected against AKI. U0126 up-regulated Polaris, but not Kif3a, in kidney tissues. It is suggested that ciliary regulation by ERK plays a role in cisplatin-induced tubular apoptosis and AKI.     

Immunolocalization of the HNK-1 epitope in the autonomic innervation to the liver and upper digestive tract of the developing rat embryo

The immunohistochemical analysis of the HNK-1 epitope presence in the liver and upper digestive tract nerves was carried out in 12- to 18-day-old rat embryos embedded in acrylamide–agarose and observed with laser scanning confocal microscopy. The vagus and sympathetic trunk were intensely immunostained at all ages; branches of both structures were also HNK-1 positive, and ramified ventrocaudally following the course of the thoracic and abdominal aorta, caval vein, portal vein and ductus venosus. As early as day 12, some immunostained cells were seen in the mesentery that formed the enteric nervous system. Clearly immunostained HNK-1-immunoreactive fibres were detected innervating the digestive wall after day 14, forming both myenteric and submucosal plexuses. After day 16, the Glisson sheath showed streams of HNK-1-positive fibres coming from dorsal areas, lining the peritoneal surface of the diaphragm, invading the capsule, and ramifying superficially around the lobes of the liver. We saw no immunoreactive structures pervading the hepatic lobes at all ages studied, with the exception of occasional HNK-1-positive cells in the superficial parenchyma, which were visualized after 16 days of gestation. Our findings can help to understand the development of the gastrointestinal and liver innervation in the rat.     

Colloidal gold particles as a new in vivo marker of early acute lung injury

Permeability of the endothelial barrier to large molecules plays a pivotal role in the manifestation of early acute lung injury. We present a novel and sensitive technique that brings microanatomical visualization and quantification of microvascular permeability in line. White New Zealand rabbits were anesthetized and ventilated mechanically. Rabbit serum albumin (RSA) was labeled with colloidal gold particles. We quantified macromolecular leakage of gold-labeled RSA and thickening of the gas exchange distance by electron microscopy, taking into account morphology of microvessels. The control group receiving a saline solution represented a normal gas exchange barrier without extravasation of gold-labeled albumin. Infusion of lipopolysaccharide (LPS) resulted in a significant displacement of gold-labeled albumin into pulmonary cells, the lung interstitium, and even the alveolar space. Correspondingly, intravital fluorescence microscopy and digital image analysis indicated thickening of width of alveolar septa. The findings were accompanied by a deterioration of alveolo-arterial oxygen difference, whereas wet/dry ratio and albumin concentration in the bronchoalveolar lavage fluid failed to detect that early stage of pulmonary edema. Inhibition of the nuclear enzyme poly(ADP-ribose) synthetase by 3-aminobenzamide prevented LPS-induced microvascular injury. To summarize: colloidal gold particles visualized by standard electron microscopy are a new and very sensitive in vivo marker of microvascular permeability in early acute lung injury. This technique enabling detailed microanatomical and quantitative pathophysiological characterization of edema formation can form the basis for evaluating novel treatment strategies against acute lung injury.     

Molecular cloning and expression of a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope

A cDNA encoding a novel glucuronyltransferase was cloned from a rat brain cDNA library. The cDNA sequence contained an open reading frame encoding 324 amino acids, with type II transmembrane topology. The amino acid sequence revealed 49% homology to rat GlcAT-P, a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope of glycoproteins, [Terayama et al. (1997) Proc. Natl. Acad. Sci. USA 94, 6093-6098] and the highest sequence homology was found in the catalytic region. Northern blot analysis indicated that this newly cloned glucuronyltransferase is expressed in the nervous system, consistent with the selective localization of the HNK-1 carbohydrate epitope in the nervous system. Transfection of this cDNA into COS-1 cells induced the expression of the HNK-1 carbohydrate epitope on cell surfaces, and induced the morphological changes in these cells. These results indicated that this newly cloned cDNA is a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope.     

The protective role of autophagy against aging and acute ischemic injury in kidney proximal tubular cells

In kidney, proximal tubules consume a large amount of energy in the process of electrolyte reabsorption. These tubules contain large quantities of mitochondria which provide the energy for this reabsorption. Proximal tubules are susceptible to many kinds of insults such as ischemia-reperfusion injury and nephrotoxic substrates, but little is known of the factors that counteract cellular stress signaling pathways. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. We demonstrated the critical role of autophagy in normal proximal tubule function and protection against acute tubular injury.     

The protective role of autophagy against aging and acute ischemic injury in kidney proximal tubular cells

In kidney, proximal tubules consume a large amount of energy in the process of electrolyte reabsorption. These tubules contain large quantities of mitochondria which provide the energy for this reabsorption. Proximal tubules are susceptible to many kinds of insults such as ischemia-reperfusion injury and nephrotoxic substrates, but little is known of the factors that counteract cellular stress signaling pathways. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. We demonstrated the critical role of autophagy in normal proximal tubule function and protection against acute tubular injury.     

Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line.

Background  

Aquaporin-1 (AQP1) channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM) was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex.     

Production of monoclonal antibodies against a new carcinoma-associated marker in view of developing a serological test.

By immunizing a mouse with human metastatic breast tumor cells from patient effusions and infiltrated lymph nodes, a monoclonal antibody (MLuC2), which identifies a new carcinoma-associated marker, was raised. The reactivity of this reagent was studied by immunohistochemistry on live and fixed cells from tumor cell lines and on frozen sections from surgical specimens. Besides reacting with 73% of breast carcinomas, MLuC2 also reacted with 93% of non-small cell lung carcinoma (NSCLC) and with a few normal tissues. The MLuC2-recognized molecule (CaMLuC2), whose MW was 90 KDa according to immunoblotting experiments, was found to be detectable in the serum and could therefore be of particular interest for serological diagnostic applications. Since the CaMLuC2 epitope was not polyexpressed on the bearing molecule, we produced a new generation of MAbs in order to define epitopes coexpressed with CaMLuC2 on the same 90 kDa molecule, and which are therefore suitable to develop a double-determinant immunoradiometric assay (DDIRMA) for the detection of this marker in the sera of lung carcinoma patients. Different analyses by immunohistochemistry, binding inhibition tests and DDIRMA, proved that the two new reagents developed, MLuC8 and MLuC9, recognize the same or closely related epitopes, which are however different from CaMLuC2, but which are all present on the same molecule. Preliminary immunoradiometric tests performed on sera from lung cancer and control patients showed a good specificity but a low sensitivity. In fact, only 42% of the 28 tested sera samples from NSCLC patients scored positive despite the fact that more than 90% of the NSCLC expressed the relevant antigen.     

Comparison of the course of biomarker changes and kidney injury in a rat model of drug-induced acute kidney injury

Objective: To aid in evaluating the performance of biomarkers, we measured kidney injury biomarkers in rat models of drug-induced acute kidney injury. Methods and results: Rats were treated with site-specific nephrotoxins, puromycin, gentamicin, cisplatin, or 2-bromoethylamine. Fifteen biomarkers (β-2-microglobulin, calbindin, clusterin, cystatin-C, KIM-1, GST-α, GST-μ, NGAL, osteopontin, EGF, TIMP-1, VEGF, albumin, RPA-1, and urinary total protein) were examined in comparison with BUN, serum creatinine, and NAG. Some biomarkers, which were different depending in each nephrotoxin, showed ability to detect the prodromal stage of drug-induced kidney injury. Characteristic changing patterns of biomarkers were also found depending on the specific lesion site in the kidney. Conclusion: These data suggested that establishment of a suitable biomarker panel would facilitate detection of site-specific kidney injury with high sensitivity.     

Comparison of the course of biomarker changes and kidney injury in a rat model of drug-induced acute kidney injury

Objective: To aid in evaluating the performance of biomarkers, we measured kidney injury biomarkers in rat models of drug-induced acute kidney injury.

Methods and results: Rats were treated with site-specific nephrotoxins, puromycin, gentamicin, cisplatin, or 2-bromoethylamine. Fifteen biomarkers (β-2-microglobulin, calbindin, clusterin, cystatin-C, KIM-1, GST-α, GST-μ, NGAL, osteopontin, EGF, TIMP-1, VEGF, albumin, RPA-1, and urinary total protein) were examined in comparison with BUN, serum creatinine, and NAG. Some biomarkers, which were different depending in each nephrotoxin, showed ability to detect the prodromal stage of drug-induced kidney injury. Characteristic changing patterns of biomarkers were also found depending on the specific lesion site in the kidney.

Conclusion: These data suggested that establishment of a suitable biomarker panel would facilitate detection of site-specific kidney injury with high sensitivity.     

Protein kinase C: a new linkage marker for growth hormone and for COL1A1

An expanded linkage group on the long arm of human chromosome 17 is reported. Using the CEPH panel of DNAs and restriction fragment length polymorphism (RFLP) markers for the centromere locus (D17Z1), growth hormone (GH1), collagen type I alpha 1 (COL1A1), and protein kinase C-alpha polypeptide (PKCA) loci, theta values of 0.03, 0.11, and 0.23 were found between PKCA and GH1, PKCA and COL1A1, and PKCA and D17Z1, respectively. The theta values calculated for GH1 versus COL1A1 or D17Z1 were 0.11 and 0.23, respectively. Sex-specific recombination rates were calculated for the best likelihood order and demonstrate female recombination greater than male recombination. Therefore, the loci studied span a map region of approximately 30 cm between 17cen and 17q24, with the most likely gene order being D17Z1-COL1A1-PKCA-GH1.     

The impact of Caspase-1 deletion on apoptosis and acute kidney injury in a murine transplant model

BackgroundCaspase-1 knockout mice (Casp1KO) are protected from Acute Kidney Injury (AKI) after warm ischemia/reperfusion injury in non-transplant models. Since Caspase-1 plays a central role as an inflammatory response initiator, we hypothesized that Casp1KO mice would be protected from AKI following transplant.MethodsRenal tubular cells (RTECs) were subjected to cold storage and rewarming (CS/REW). C57Bl/6 J wild type or Casp1KO kidneys were subjected to CI for 30 min and then transplanted into wild type recipients (CI + Txp). The recipients underwent bilateral native nephrectomy at the time of transplant. Serum creatinine (sCr) was measured 24 h after native nephrectomy to assess transplant function.ResultsWe found that RTECs subjected to CS/REW had significantly increased expression of the Caspase-1 and inflammasome protein NLRP1. Wild type kidneys subjected to CI + Txp into wild type recipients also demonstrated significantly increased Caspase-1 and NLRP1 protein expression compared to kidneys transplanted from Casp1KO donors into wild type recipients. Caspase-1 deletion results in significantly decreased RTEC apoptosis in transplanted Casp1KO vs WT kidneys. Surprisingly, however, renal function, ATN scores including brush border injury, cast formation and tubular simplification were similar in both groups and not significantly different.ConclusionsOur data suggest that other triggers of inflammation and programmed necrosis may need to be inhibited in addition to attenuating Caspase-1 to fully prevent AKI after kidney transplant. Importantly, requirements may be distinct for AKI induced by transplantation as opposed to other transient models such as the clamp model of AKI.