Papillary Interstitial Cells Research Articles

Expanding roles of the hypoxia-response network in chronic kidney disease.

Studies over the last two decades have established tubulointerstitial hypoxia as a final common pathway leading to end-stage kidney disease (ESKD). Chronic kidney disease (CKD) is frequently associated with various degrees of hypoxic injury in distinct tubular segments, depending on the etiology and pathological stages, which constitutes an intricate link among inflammation, oxidative stress and fibrosis. Resident cells in the kidney are equipped with mechanisms through which they cope with hypoxia. Here, transcription of genes by hypoxia-inducible factors (HIFs) plays a central role. In the ischemic kidney, HIF-1 is expressed in tubular and glomerular epithelial cells and in papillary interstitial cells, whereas HIF-2 is expressed in endothelial cells and interstitial fibroblasts. There is ample evidence that HIF protects the kidney from acute ischemic damage. In CKD, studies suggest that the function of HIF may be suppressed because of factors, such as oxidative stress and uremia, which may underlie the pathogenesis of both CKD and co-existing problems, such as renal anemia. Based on these observations, efforts are in progress to test whether restoration and activation of HIF might protect the kidney from CKD. Initial studies using non-specific or supraphysiological HIF activation suggested that the role of HIF may be multifactorial and depend on pathological context. On the other hand, specific HIF stabilizers, such as prolyl hydroxylase (PHD) inhibitors, are being developed for the treatment of renal anemia. Application of these compounds in experimental CKD may override those previous findings and provide deeper insight into the roles of hypoxia and oxygen-sensing pathways.

Activation of hypoxia-inducible factors ameliorates hypoxic distal tubular injury in the isolated perfused rat kidney

Preconditional activation of HIF with specific prolyl-hydroxylase inhibitors (PHD-I) attenuates proximal tubular injury, induced by warm ischaemia/ reperfusion (Bernhardt, JASN, 2006). Distal tubular damage occurs in humans with acute kidney injury (AKI), in experimental contrast media-induced nephropathy (CIN), as well as in cell-free isolated perfused kidneys (IPKs). Since in the IPK distal tubular damage inversely correlates with HIF activation (Rosenberger, KI, 2005), we explored the potential of PHD-I to improve morpho-functional outcome in this model. Male SD rats were randomly given the synthetic PHD-inhibitor FG-4497 (FibroGen, 50 mg/kg IV) or its vehicle (CTR, n = 10 per group). Six hours later, the right kidney was perfused for 90 min with cell-free oxygenated medium and subsequently perfusion-fixed for morphologic assessment. The left kidney was used for HIF immunostaining. As compared with CTR kidneys, at 6 h after FG-4497 HIF-alpha isoforms were markedly up-regulated in all renal zones: HIF-1alpha in tubules and in papillary interstitial cells (IC), HIF-2alpha in IC and vascular endothelial cells. FG-4497 treatment resulted in a higher perfusate flow rate (P < 0.04, ANOVA). Tubular injury to medullary thick ascending limbs (mTALs) was significantly attenuated in the treatment versus control group (38.9 +/- 7.4% versus 62.7 +/- 4.9% of mTALs in the mid-inner stripe (P < 0.02); 23.8 +/- 6.8% versus 45.6 +/- 7.4% in the innermost zone of the inner stripe (P < 0.05). These findings illustrate that PHD-I preconditioning attenuates hypoxic distal tubular injury produced in the IPK in the same fashion in which it protects proximal tubules. mTAL conservation may be related to the stabilization of cellular HIF, as well as to preserved endothelial function and microcirculation.

Differential expression of heat shock protein 27 and 70 in renal papillary collecting duct and interstitial cells – implications for urea resistance

The adaptation of renal medullary cells to their hyperosmotic environment involves the accumulation of compatible organic osmolytes and the enhanced synthesis of heat shock proteins (HSP) 27 and 70. While the mechanisms leading to osmolyte accumulation are similar in papillary collecting duct (PCD) and papillary interstitial (PI) cells, the present data demonstrate that HSP27 and HSP70 are expressed differentially in these cells both in vivo and in vitro. HSP70 is abundant in PCD, but not expressed in PI cells in the papilla in situ, while HSP27 is expressed in both PCD and PI cells. These observations could be reproduced by non-permeant solutes in cultured cells. Osmotic stress strongly induced HSP70 in MDCK cells (as a model for PCD cells), but not in PI cells, while HSP27 was constitutively expressed in MDCK cells and was up-regulated in PI cells. Since prior hypertonic stress (NaCl addition) protects MDCK against subsequent exposure to high urea concentrations, this effect was also assessed in PI cells. In both cell lines, hypertonic pretreatment prior to urea exposure (400 mm) strongly attenuated caspase-3 activation. Inhibition of HSP27 expression by antisense transfection diminished the protective effect of hypertonic preconditioning in PI cells, while attenuation of HSP70 expression in MDCK cells diminished the protective effect of hypertonic preconditioning in these cells. These observations indicate that PCD and PI cells employ cell-specific mechanisms for protection against high urea concentrations as present in the renal papilla during antidiuresis.

Interspecies Differences in the Nephrotoxic Response to Cyclooxygenase Inhibition

In contrast to cyclooxygenase‐1 (COX‐1), the basal expression of renal cyclooxygenase‐2 (COX‐2) varies among species. High basal levels of COX‐2 in the renal cortex and papilla in dogs compared with monkeys suggest that COX‐2 inhibition may lead to distinct nephrotoxic responses. In this study, we compared the renal effects of COX inhibition between dogs and cynomolgus monkeys (n = 6/group) following the administration of naproxen sodium, a non‐selective COX‐1/COX‐2 inhibitor. Dogs and monkeys were treated with 50 or 150 mg/kg/day naproxen sodium, respectively, for 2 to 6 weeks. Naproxen doses used in this study resulted in equivalent inhibition of COX activity in both species as measured by reductions in urinary prostaglandin E2 (PGE2) and 6‐keto‐PGF1‐α levels. There was prominent reduction in renal blood flow (43%) and urinary sodium excretion (62%) in dogs but no alterations in renal blood flow and only minimal change (19%) in urinary sodium excretion in monkeys. The canine but not monkey kidney showed prominent COX‐2 expression in the macula densa, thick ascending limb of Henle and papillary interstitial cells by immunohistochemistry. After treatment, the canine but not monkey kidneys had mild to moderate renal tubular atrophy and interstitial fibrosis and renal papillary necrosis. Obstructive nephropathy secondary to intra‐tubular drug accumulation was seen in monkeys but not in dogs. Collectively, these data demonstrate species differences in the renal response to COX inhibition. The nature of functional and morphologic changes suggests a more prominent role of COX‐2 in renal hemodynamics and natriuresis in dogs than in monkeys.

Osmoregulation of aldose reductase and sorbitol dehydrogenase in cultivated interstitial cells of rat renal inner medulla.

Little is known about sorbitol metabolism in renal papillary interstitial cells. For characterization we studied regulation of sorbitol synthesis by aldose reductase (AR) and degradation by sorbitol dehydrogenase (SDH) in papillary interstitial cells. Interstitial cells were isolated from rat renal inner medulla to a pure cell fraction. mRNA was isolated from cultivated cells and sorbitol, AR and SDH activity were determined enzymatically in homogenates. Sorbitol concentration in these cells at 300 mosmol/l was 4.4+/-0.3 vs 78+/-3.6 micro mol/g protein at 600 mosmol/l. At steady-state conditions at 300 mosmol/l, AR activity was nearly the same as SDH activity (15.1+/-1.6 vs 16.6+/-2.0 U/g protein). At 600 mosmol/l, AR activity increased to 82.5+/-11.4 U/g protein and SDH activity to 31.5+/-6.0 U/g protein. Studying the time course of enzyme activity after changing osmolarity from 300 to 600 mosmol/l, we found half maximal stimulation after 2-3 (AR) or 3 (SDH) days. The amount of AR-mRNA preceded the rise of enzyme activity, whereas SDH-mRNA was not significantly influenced. Lowering osmolarity from 600 to 300 mosmol/l, enzyme activity decreased to less than half within 2 (AR) or 1 (SDH) day(s). The results suggest that sorbitol metabolism contributes to handling of osmotic stress in rat renal papillary interstitial cells.

Hypertonicity-induced accumulation of organic osmolytes in papillary interstitial cells

Medullary cells of the concentrating kidney are exposed to high extracellular solute concentrations. It is well established that epithelial cells in this kidney region adapt osmotically to hypertonic stress by accumulating organic osmolytes. Little is known, however, of the adaptive mechanisms of a further medullary cell type, the papillary interstitial cell [renal papillary fibroblast (RPF)]. We therefore compared the responses of primary cultures of RPFs and papillary collecting duct (PCD) cells exposed to hypertonic medium. In RPFs and PCD cells, organic osmolytes were determined by high-performance liquid chromatography; mRNA expression for organic osmolyte transporters [Na+/Cl(-)-dependent betaine transporter (BGT), Na(+)-dependent myo-inositol transporter (SMIT)], and the sorbitol synthetic and degrading enzymes [aldose reductase (AR) and sorbitol dehydrogenase (SDH), respectively] was determined by Northern blot analysis. Exposure to hypertonic medium (600 mOsm/kg by NaCl addition) caused intracellular contents of glycerophosphorylcholine, betaine, myo-inositol, and sorbitol, but not free amino acids, to increase significantly in both RPFs and PCD cells. The rise in intracellular contents of these organic osmolytes was accompanied by enhanced expression of mRNAs coding for BGT, SMIT, and AR in both RPFs and PCD cells. SDH mRNA abundance, however, was unchanged. Nonradioactive in situ hybridization studies on sections from formalin-fixed and paraffin-embedded, normally concentrating kidneys showed strong expression of BGT, SMIT, and AR mRNAs in interstitial and collecting duct cells of the papilla, whereas expression of SDH mRNA was much weaker in both cell types. These results suggest that both RPFs and PCD cells use similar strategies to adapt osmotically to the high interstitial NaCl concentrations characteristic for the inner medulla and papilla of the concentrating kidney.

Differential expression and cellular distribution of mRNAs encoding alpha- and beta-isoforms of Na(+)-K(+)-ATPase in rat kidney

We have used in situ hybridization histochemistry with isoform-specific, digoxigenin-labeled cRNA probes to characterize systematically the cellular distribution of mRNAs encoding alpha- and beta-subunit isoforms of the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the normal rat kidney. Transcripts encoding the alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-subunits were detected in virtually all of the nephron segments, with prominent hybridization signal in the S3 segment of the proximal tubule, the cortical and medullary thick ascending limb of Henle's loop, the distal convoluted tubule, the cortical collecting duct along its entire length, and the renal pelvic epithelium. Several differences in the cell-specific pattern of expression of the various isoforms were observed. Among the alpha-isoforms, the alpha 3-subunit appeared to be preferentially expressed in the glomerular podocytes and mesangial cells, papillary interstitial cells, and renal pelvic epithelium. The beta-isoforms also differed in their distribution pattern, with the beta 1-subunit expressed to a greater degree in the glomerulus and renal pelvic epithelium and the beta 2-subunit preferentially expressed in the papillary interstitial cells and papillary surface epithelium. The detection and expression pattern of alpha- and beta-subunit mRNAs in structures throughout the kidney is compatible with the possibility of six structurally unique Na(+)-K(+)-ATPase isozymes and suggests a potentially greater role for isozymes comprised of the alpha 2-, alpha 3-, and beta 2-subunits in renal sodium and potassium transport.

Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney

The localization of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) was determined in the rabbit kidney by immunohistochemistry with the use of a monoclonal, anti-GR antibody and a monoclonal, anti-idiotypic, anti-MR antibody. Immunostaining was performed on serial histological sections from normal and adrenalectomized rabbits. The specificity of immunostaining was assessed for MR by in situ competition studies with steroids and for GR by presaturation of the antibody with GR preparation. Immunostaining by both the anti-MR and the anti-GR antibodies was present in all parts of the distal nephron (beyond proximal tubule) and absent in the glomerulus and proximal tubule. The absence of staining by the anti-GR antibody in the proximal tubule suggests that the effects of glucocorticoids in this structure involve either a GR different from that of distal structures or a non-receptor mediated mechanism of action. MR immunostaining predominates in the distal and all along the collecting tubule in its cortical, medullary, and papillary portions. GR immunostaining was most abundant in the medullary ascending limb and distal tubule. Immunostaining by both antibodies was present in papillary interstitial cells and cells of the epithelium lining the papilla. Fifteen to twenty percent of the cells of the cortical collecting tubule, presumably intercalated cells, were devoid of MR and GR immunostaining. Immunostaining was present in both nuclear and cytoplasmic cell compartments. No clear difference was observed between normal and adrenalectomized rabbits. This study is the first report on renal immunolocalization of GR compared with MR. In addition, we show evidence for new targets for corticosteroid hormones such as papillary interstitial cells and papillary epithelium.

Spontaneous papillary necrosis in the heterozygous Gunn rat

Spontaneous papillary necrosis develops in aging heterozygous non-jaundiced Gunn rats. The lesion is situated in the subapical or mid papilla and in its earliest stages is manifest by the appearance of amorphous material in the interstitial space. This is seen in plastic-embedded sections taken from rats 6 months old. In its later stages, the accumulation of amorphous material is accompanied by loss of interstitial cells and cyst formation, but there is no associated inflammatory reaction. The largest lesions are found in the oldest rats, but even in these animals the macroscopic appearance of the papilla is normal. Similar papillary changes were not found in albino or homozygous Gunn rats, but in aging albino rats there was loss of papillary interstitial cells without accumulation of amorphous material.

Ultrastructural appearances of acute renal papillary lesions induced by aspirin

Renal papillary necrosis develops 16 hr after intravenous administration of aspirin to the uninephrectomised homozygous Gunn rat. Ultrastructural studies show the papillary interstitial cells to be most severely affected, the first changes being visible at 1 hr. Changes in capillaries are late in onset, and this suggests that the lesions are due to a direct toxic effect rather than to ischaemia.

Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. III. Elemental content of cells in the rat renal papillary tip.

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections. Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40 degrees C. Frozen 0.5-micrometer sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at -175 degrees C during analysis. The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments. Tissue mass (solutes + water) was determined by continuum radiation from regions of interest. After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, Cl, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured. Cells had lower water content (mean 58.7%) than interstitium (77.5%). Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight); P was markedly higher in cells than in interstitium. S was the same in all compartments. Intracellular Na levels were extremely high (CDC, 344 +/- 127 SD mm/kg wet weight; PEC, 287 +/- 105; IC, 898 +/- 194). Mean interstitial Na was 590 +/- 119 mm/kg wet weight. CI values paralleled those for Na. If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.

In vitro and in vivo lipogenesis of the rat renal papillae from glucose

The rate of [ 14C] glucose incorporation into rat renal papillary glycerolipids has been studied. Glucose is incorporated mainly into the glycerol moiety of these lipids. 74 and 93% of the activity incorporated into triacylglycerols in vitro and in vivo, respectively, was present in the glycerol backbone. In phospholipids it was 82 and 96%, respectively. The fatty acid synthesis from [ 14C]-glucose was similar to that from [ 14C]acetate. In accordance with previous experiments with [ 14C]acetate the papillary tissue was found to have a high capacity for chain elongation of preexisting fatty acids with the major product being cis-7,10,13,16-docosatetraenoic acid (chain elongated arachidonic acid). The in vitro and in vivo turnover rates of papillary glycerolipids can be calculated. A half-life of 25 h can be estimated for membrane phospholipids and less than 11 h for lipid droplet triacylglycerols. These appreciably high rates of turnover are discussed in relation to membrane turnover in renal papillae from rats in different physiological states and to variation in number of lipid droplets of papillary interstitial cells reported by others.

Histochemistry and ultrastructure of the interstitium of the renal papilla in rats with hereditary diabetes insipidus (Brattleboro strain).

The Brattleboro strain of Long-Evans hooded rats has hereditary hypothalamic diabetes insipidus due to the inability to produce antidiuretic hormone. Animals homozygous for this autosomal recessive trait have extreme polyuria and polydipsia, whereas heterozygotes are less severely affected. Light and electron microscopy were used to study the interstitial tissue of the renal papilla of Brattleboro rats and normal Long-Evans rats. Staining with alcian blue or colloidal iron revealed that homozygous Brattleboro rats (DI) have greatly reduced quantities of glycosaminoglycans in the papillary interstitium. Heterozygotes showed staining similar but not identical to that of normal rats. The papillary interstitial cells of DI rats lacked the cytoplasmic processes seen in normal rats, and the normal relationship of these cells to the tubular elements of the papilla was absent. Electron microscopy revealed that the papillary interstitial cells of DI rats appeared less active than those of heterozygous or normal rats. In DI rats these cells displayed reduced numbers of lipid droplets and mitochondria, and the Golgi apparatus and rough endoplasmic reticulum were poorly developed. The altered ultrastructure of the papillary interstitial cells may be responsible for the reduction of interstitial glycosaminoglycans in DI rats. Glycosaminoglycans possess properties which may contribute to urinary concentration, It is suggested that the interstitial tissue of the renal papilla is influenced by antidiuretic hormone.

Relationship of cytoplasmic granules in renal papillary interstitial cells to "postsalt" hypertension.

Relationship of cytoplasmic granules in renal papillary interstitial cells to "postsalt" hypertension.