Distribution of the cellular uptake of phosphorothioate oligodeoxynucleotides in the rat kidney in vivo.

Previous animal studies have demonstrated that following systemic administration phosphorothioate oligodeoxynucleotides (S-ODNs) are primarily excreted by the kidneys and that renal tissue levels of S-ODNs exceed that of other organs. Thus, the kidney may be an ideal target organ for application of antisense S-ODNs in vivo. We examined which cells within the rat kidney have uptake of radiolabeled S-ODNs following intravenous infusion. A 20-base 35S-ODN was infused into 6 adult male Wistar rats. Three animals each were sacrificed 30 min and 4 h after infusion. The kidneys were then removed, fixed, and tissue autoradiography was performed. Similar results were obtained in both groups. The highest level of radioactivity was seen within the proximal tubules. Lower levels of activity were seen within the glomerulus, the parietal epithelial cells of Bowman's space, and distal tubular cells. Very weak activity was also detected within the cells of the loop of Henle and the medullary collecting ducts. These results demonstrated that within the kidney S-ODNs were taken up primarily by proximal tubular cells, with much lower uptake by cells in other segments of the nephron.

Effect of glycine on cisplatin nephrotoxicity and heat-shock protein 70 expression in the rat kidney.

Glycine has been shown to protect against cisplatin (CP) nephrotoxicity in rats and to enhance the in vitro expression of heat-shock protein (hsp) 70 in renal epithelial cells following sublethal heat shock. We hypothesized that the protective effect of glycine against CP nephrotoxicity may be due to an up-regulation of Hsp 70 protein expression. Male Sprague-Dawley rats were divided into 4 treatment groups based upon infusion of glycine and injection of CP or their respective vehicles. At 5 days after treatment animals administered CP alone demonstrated a significant decrease in creatinine clearance compared to baseline (0.77 +/- 0.32 mL/min vs. 3.90 +/- 0.87 mL/min, p < 0.05). Treatment with glycine and CP attenuated this response, with no significant decline seen in creatinine clearance at day 5 compared to baseline (2.25 +/- 0.31 mL/min vs. 3.40 +/- 0.86 mL/min). Semiquantitative histological study revealed a marked decrease in proximal tubular injury at the juxtamedullary and outer medullary regions among animals treated with glycine and CP compared to those animals treated with CP alone. There were no differences in renal cortical and medullary Hsp 70 levels by Western immunoblotting between animals treated with glycine and CP compared to CP alone at 4 h and 5 days after treatment. Immunohistochemical studies of animals treated with CP alone revealed the diffuse presence of Hsp 70 in the cytoplasm of injured and necrotic proximal tubular cells 5 days after treatment. Animals receiving CP and glycine demonstrated a more focal presence of Hsp 70 restricted to injured proximal tubular cells, with no staining of uninjured cells. The protective effect of glycine in CP-induced acute and renal failure in the rat does not appear to be associated with enhancement of Hsp 70 expression.

Sublethal heat shock and cyclosporine exposure produce tolerance against subsequent cyclosporine toxicity.

Sublethal heat shock has been shown to produce tolerance in cells and tissues subsequently exposed to heat or ischemia/ATP depletion. We tested whether heating LLC-PK1 cells for 2 h at 42 degrees C induced heat shock protein-70 (HSP-70) gene expression and conferred tolerance against subsequent cyclosporine A (CyA) toxicity. HSP-70 mRNA was increased immediately after heat shock, returning to baseline by 4 h. HSP-70 protein increased by 1 h after heat shock and declined thereafter, approaching baseline after 72 h. Cells heat shocked at 4 and 24 h prior to CyA exposure were significantly more viable than controls, at CyA concentrations near the median lethal dose (LD50). Cytoprotection declined with time after heat shock, concurrent with declining HSP-70 protein levels. Sublethal CyA exposure (50 micrograms/ml) for 24 h produced upregulation of HSP-70 mRNA and protein. Pretreatment with 50 micrograms/ml CyA for 24 h followed by exposure to a toxic concentration of CyA (200 micrograms/ml) produced significant cytoprotection compared with untreated controls. In conclusion, HSP-70 protein induction by sublethal heat shock or CyA exposure was associated with tolerance against subsequent lethal CyA exposure.

Overexpression of transforming growth factor-beta 1 mRNA is associated with up-regulation of glomerular tenascin and laminin gene expression in nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop immune-mediated insulin-dependent diabetes mellitus and nephropathy, providing an opportunity to study the early molecular events in a model of diabetic glomerulosclerosis. The expression of several genes coding for growth factors and extracellular matrix was examined in microdissected glomeruli, by the use of reverse transcription-competitive polymerase chain reaction, in diabetic NOD mice (mean duration of diabetes, 28.5 +/- 7 days) and age-matched nondiabetic NOD mice with normal glucose tolerance. The levels of mRNA coding for transforming growth factor-beta 1, tenascin, and laminin B1 increased 1.9-, 2.0-, and 1.7-fold, respectively, whereas platelet-derived growth factor (PDGF)-B, alpha 1(IV) collagen, 72-kd collagenase, alpha-smooth muscle actin, and beta-actin mRNA remained stable in the diabetic mice. The kidney advanced glycosylation end-products levels increased 2.1-fold in the diabetic mice, and the diabetic glomeruli showed an accumulation of tenascin and laminin but not of type IV collagen by immunofluorescence microscopy. There was no increase in cell number per glomerulus after the onset of diabetes, a finding consistent with stable PDGF-B and alpha-smooth muscle actin mRNA levels. These findings provide evidence that increased glomerular transforming growth factor-beta 1, but not PDGF-B, mRNA is associated with the up-regulation of tenascin and laminin expression after advanced glycosylation endproduct accumulation, early after the onset of diabetes.

Assessment of 72-kilodalton gelatinase and TIMP-1 gene expression in normal and sclerotic murine glomeruli.

Mice transgenic for bovine growth hormone (bGH) develop progressive diffuse glomerulosclerosis. Because murine mesangial cells in vitro were found to express the genes for 72-kd gelatinase and the metalloproteinase inhibitor TIMP-1, the expression of these genes in vivo in isolated whole glomeruli from bGH mice and normal control littermates was examined. Intact glomeruli were isolated by microdissection and subjected to reverse transcription. TIMP-1 cDNA was not detected by standard polymerase chain reaction (PCR) in glomeruli from bGH or control mice. In contrast, cDNA for 72-kd gelatinase was detected by standard PCR in both bGH and control mice, and the level was subsequently measured by quantitative competitive PCR. The gelatinase cDNA level was 14.7 +/- 2.8 x 10(-4) attomoles/glomerulus in 2- to 3-month-old control mice and was unchanged in 6-month-old controls. The bGH mice had 3.5-fold and 4.5-fold higher cDNA levels at 2 to 3 months and 6 months of age, respectively. Finally, zymography of glomerular extracts revealed increased levels of 72-kd and 96- to 100-kd gelatinase activity in bGH glomeruli in comparison to that in controls. In summary, whereas the genes for both TIMP-1 and 72-kd gelatinase are expressed in vitro in cultured mesangial cells, only the gelatinase gene appeared to be expressed in vivo in intact glomeruli. In addition, there was an up-regulation in the glomerular expression of the 72-kd gelatinase in bGH mice, a murine model of glomerulosclerosis.

Human glomeruli express TIMP-1 mRNA and TIMP-2 protein and mRNA.

Alterations in the balance between synthesis and degradation of extracellular matrix may result in glomerulosclerosis. The interaction between metalloproteinases and their inhibitors presumably modulates the rate of glomerular matrix degradation. We examined the gene expression of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in human glomeruli and TIMP-2 protein in tissue sections. Kidney tissue was obtained from adults undergoing nephrectomy for renal tumor (n = 9) or biopsy for nephrosis and renal failure (n = 1). Glomeruli were microdissected and subjected to reverse transcription. TIMP cDNAs were quantitated by competitive polymerase chain reaction assays. Five nephrectomy specimens had normal glomeruli and four had diffuse glomerulosclerosis. TIMP-1 and TIMP-2 cDNA levels, detected in glomeruli from all patients, were increased fourfold and threefold, respectively, in patients with glomerulosclerosis. The elevated TIMP cDNA levels could not be attributed to an increased number of glomerular cells. TIMP-2 protein was detected within normal and sclerotic glomeruli. In conclusion, both TIMP genes were expressed in normal glomeruli, and their level of expression was increased in glomerulosclerosis associated with renal carcinoma, suggesting that expression of these inhibitors may correlate with the development of sclerosis.

Proteinuria.

Proteinuria is an important finding in patients because it may signify either a benign disorder or renal disease. A systematic approach to evaluation, which incorporates a careful clinical evaluation coupled with a few simple laboratory tests, is required to determine whether the patient can be followed, or whether a more invasive study, such as renal biopsy, is indicated. Although the causes of proteinuria are diverse, a structured approach to the patient with proteinuria provides maximal information with minimal expense and risk.

The contribution of increased collagen synthesis to human glomerulosclerosis: a quantitative analysis of alpha 2IV collagen mRNA expression by competitive polymerase chain reaction.

We previously reported that one of the main components of the sclerotic material in human glomerular diseases was type IV collagen. In this study we examined the contribution of increased synthesis to this process at the gene expression level. Sufficient material has not been available to study type IV collagen synthesis by normal or sclerotic glomeruli in humans. We took advantage of the availability of nephrectomy specimens from patients with renal carcinoma, and of the observation that approximately 50% of these patients develop varying degrees of glomerulosclerosis. We microdissected glomeruli from 10 patients and analyzed them using in situ reverse transcription coupled with polymerase chain reaction (PCR) analyses (in situ RT-PCR). alpha 2IV collagen mRNA, after reverse transcription into cDNA, was detected in all patients and appeared to be increased in those with glomerulosclerosis (n = 5). A competitive PCR assay was developed to quantitate this change. There was an average 3.7-fold increase in glomerular type IV collagen cDNA in patients with significant sclerosis. This change was not due to an increased number of glomerular cells. Thus, glomerulosclerosis in humans is associated with an elevation of glomerular type IV collagen gene expression, suggesting that increased synthesis of type IV collagen may represent one component of this process.

Oxygen consumption and ventilation during normal labor.

Oxygen consumption (VO2) and minute ventilation (VE) were measured breath-by-breath for 10 min periods in the third trimester of pregnancy in 16 healthy women. These measurements were repeated during the first stage of labor in eight of the women. The 10-min mean VO2 was 3.56 ml/kg/min (+/- 0.82 SD) at term and 4.28 ml/kg/min (+/- 0.93) during labor, for an average increase of 23 percent (+/- 28 percent, p = 0.04) from third trimester to labor. The mean VE was 0.15 L/kg/min (+/- 0.03) at term and increased significantly (p = 0.05) to 0.24 L/kg/min (+/- 0.11) during labor for an average increase in VE of 65 percent (+/- 78 percent). Peak VO2 and VE occurred during contractions with five-breath average peak VO2 being 86 percent (+/- 53%) above the 10-min mean value at term and VE increasing 167 percent (+/- 154 percent) from third trimester to peak values during labor. These data may be useful in identifying patients at risk for developing respiratory insufficiency during labor. We propose an algorithm for approaching the obstetric patient with respiratory disease.

Nephrotic syndrome in adults. A diagnostic and management challenge.

The adult with nephrotic syndrome presents diagnostic and treatment challenges for the primary care physician. Early consultation with a nephrologist is advisable to assist in choosing between empirical therapy or renal biopsy to identify the specific causative lesion. Some patients respond to treatment of either the specific disorder or the underlying cause. All patients should be afforded specific therapy, where available, and nonspecific therapy to minimize the severity of the nephrosis and attenuate the incidence and severity of complications. Although the cause of most disorders resulting in nephrotic syndrome remains unclear, active research into the syndrome's pathogenesis and treatment options should prove fruitful.