Myofibroblasts and the progression of diabetic nephropathy

Background. The cellular mediators of progressive renal fibrosis in diabetic nephropathy remain unknown. Myofibroblasts have been implicated in the pathogenesis of experimental and clinical renal fibrosis. Their role in the progression of diabetic nephropathy is the subject of this study.Subjects and methods. We have studied by immunohistochemistry the expression of cytoskeletal proteins associated with the activation of myofibroblasts; &agr;-smooth-muscle actin (&agr;-SMA), vimentin (Vi) and desmin (D), in the kidneys of 25 patients with diabetic nephropathy (5 patients with diabetic nephropathy (5 patients had a superimposed glomerulonephritis). Comparisons were made with normal tissue for three kidneys removed for renal-cell carcinoma. Correlations were studied between clinical and biochemical parameters with the expression renal cytoskeletal proteins. Results. In normal kidneys, cells expressing &agr;-SMA were confined to the vascular media and adventia while immunoreactive Vi was detected in glomerular epithelial cells. In diabetic kidneys, cells expressing &agr;-SMA were detected primarily in the renal interstitium and to a lesser extent in some glomeruli in association with mesangial proliferation. Vimentin immunostain decreased in glomeruli displaying diabetic hyalinosis and sclerosis. By contrast, strong Vi immunoreactivity was noted in atrophic diabetic tubules and to a lesser extent in the interstitium. Desmin was not detected in either normal or diabetic kidneys. Close correlations were observed between the expression of renal cytoskeletal proteins and the progression of renal insufficiency. Interstitial &agr;-SMA proved to be a predictor of progressive diabetic nephropathy (R² for 1/serum Cr slope=0.608, P=0.00001). This predictive parameters; tubular atrophy (R²=0.477, P=0.00004) and interstitial fibrosis (R²=0.28, P=0.001). Conclusion. We have demonstrated in this study the neoexpression of cytoskeletal proteins within diabetic kidneys. This has allowed the identification of new predicting histological markers for the progression of diabetic nephropathy.     

Transforming growth factor-beta(1) in the kidney and urine of patients with glomerular disease and proteinuria.

BACKGROUND: Transforming growth factor-beta(1) (TGF-beta(1)) is the major fibrogenic growth factor implicated in the pathogenesis of renal scarring. Proteinuria is a poor prognostic feature for various types of glomerular disease and its toxic action may be related to the activation of tubular epithelial cells towards increased production of cytokines and chemoattractant peptides. In this work we studied the site of synthesis and expression profile of TGF-beta(1) in the renal tissue of patients with heavy proteinuria and examined the relation of this expression with the urinary excretion of TGF-beta(1). METHODS: Twenty-five patients with heavy proteinuria (8.4+/-3.0 g/24 h) were included in the study. All patients underwent a diagnostic kidney biopsy and were commenced on immunosuppressive therapy with corticosteroids and cyclosporin. The sites of synthesis and expression profile of TGF-beta(1) mRNA and protein in the kidney were examined by in situ hybridization and immunohistochemistry. Urinary and plasma TGF-beta(1) levels were determined by ELISA before the initiation of treatment and 6 months later and compared with those of normal subjects and of patients with IgA nephropathy and normal urinary protein excretion. RESULTS: The site of synthesis and expression of TGF-beta(1) in the renal tissue of patients with heavy proteinuria was mainly localized within the cytoplasm of tubular epithelial cells. Interstitial expression was also present but glomerular TGF-beta(1) expression was found only in patients with mesangial proliferation. Urinary TGF-beta(1) excretion was significantly higher in nephrotic patients compared with normal subjects and with patients with IgA nephropathy and normal urinary protein excretion (783+/-280 vs 310+/-140 and 375+/-90 ng/24 h, respectively; P<0.01). In patients with remission of proteinuria after immunosuppressive therapy, urinary TGF-beta(1) excretion was significantly reduced (from 749+/-290 to 495+/-130 ng/24 h; P<0.01), while in patients with persistent nephrotic syndrome, it remained elevated. CONCLUSIONS: The localization of TGF-beta(1) mRNA and protein within tubular epithelial cells, along with its increased urinary excretion in patients with nephrotic syndrome, suggest the activation of these cells by filtered protein towards increased TGF-beta(1) production.     

The experimental use of cocaine in human subjects

Continuation of the practice of experimental administration of cocaine to cocaine-addicted volunteers has been recommended by some investigators. The author's view is that the risk of experimental use of cocaine outweighs its benefits and that this practice should not be pursued. The author describes the damaging effects of cocaine on the cardiovascular system, particularly its ability to induce myocardial band necrosis, and the unique reinforcing properties of the drug. The legal and ethical issues raised by the experimental use of cocaine are also discussed.     

Efficacy and safety of transcranial magnetic stimulation in the acute treatment of major depression: a multisite randomized controlled trial.

BACKGROUND: We tested whether transcranial magnetic stimulation (TMS) over the left dorsolateral prefrontal cortex (DLPFC) is effective and safe in the acute treatment of major depression. METHODS: In a double-blind, multisite study, 301 medication-free patients with major depression who had not benefited from prior treatment were randomized to active (n = 155) or sham TMS (n = 146) conditions. Sessions were conducted five times per week with TMS at 10 pulses/sec, 120% of motor threshold, 3000 pulses/session, for 4-6 weeks. Primary outcome was the symptom score change as assessed at week 4 with the Montgomery-Asberg Depression Rating Scale (MADRS). Secondary outcomes included changes on the 17- and 24-item Hamilton Depression Rating Scale (HAMD) and response and remission rates with the MADRS and HAMD. RESULTS: Active TMS was significantly superior to sham TMS on the MADRS at week 4 (with a post hoc correction for inequality in symptom severity between groups at baseline), as well as on the HAMD17 and HAMD24 scales at weeks 4 and 6. Response rates were significantly higher with active TMS on all three scales at weeks 4 and 6. Remission rates were approximately twofold higher with active TMS at week 6 and significant on the MADRS and HAMD24 scales (but not the HAMD17 scale). Active TMS was well tolerated with a low dropout rate for adverse events (4.5%) that were generally mild and limited to transient scalp discomfort or pain. CONCLUSIONS: Transcranial magnetic stimulation was effective in treating major depression with minimal side effects reported. It offers clinicians a novel alternative for the treatment of this disorder.     

Haploinsufficiency of Pkd2 is associated with increased tubular cell proliferation and interstitial fibrosis in two murine Pkd2 models.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human kidney disease and is caused by germline mutations in PKD1 (85%) or PKD2 (15%). It has been estimated that around 1% of tubular cells give rise to cysts, and cell hyperproliferation has been noted to be a cardinal feature of cystic epithelium. Nevertheless, it is uncertain whether the increase in proliferative index observed is an early or late feature of the cystic ADPKD kidney. METHODS: Two Pkd2 mouse mutants (WS25 and WS183) have been recently generated as orthologous models of PKD2. To determine the effect of Pkd2 dosage on cell proliferation, cyst formation and renal fibrosis, we studied renal tissue from Pkd2(WS25/WS25) and Pkd2(+/-) mice by histological analysis. We also examined the proliferative index in archival nephrectomy tissue obtained from patients with ADPKD and normal controls. RESULTS: The proliferative index of non-cystic tubules in Pkd2 mutant mice as assessed by proliferating cell nuclear antigen and Ki67-positive nuclei was between 1-2%, values 5-10 times higher than control tissue. Similarly, the proliferative index of non-cystic tubules in human ADPKD kidneys was 40 times higher than corresponding controls. In Pkd2 mutant mice, significant correlations were found between the fibrosis score and the mean cyst area as well as with the proliferative index. Of significance, proliferating tubular cells were uniformly positive for polycystin-2 expression in Pkd2(+/-) kidney. CONCLUSION: These results suggest that an increase in cell proliferation is an early event preceding cyst formation and can result from haploinsufficiency at Pkd2. The possible pathogenic link between tubular cell proliferation, interstitial fibrosis and cyst formation is discussed.     

Suitability and durability of multiple internal thoracic artery coronary artery bypasses.

OBJECTIVE: The authors evaluate operative and extended outcomes of coronary artery bypass surgery using the bilateral internal thoracic arteries (ITAs) as bypass grafts. The authors conclude that the procedure is viable and of long-term benefit to most patients. SUMMARY BACKGROUND DATA: Multiple ITA grafting was met with early enthusiasm by the surgical profession, but skepticism and controversy arose with reports of increased operative morbidity, insufficient graft blood flow, a high incidence of failure of the right ITA, and uncertainty about durability and long-term benefits. METHODS: To assess the actual incidence and impact of these complications and long-term results, the authors prospectively studied 500 consecutive patients with multiple ITA bypasses, constituting the closely observed and carefully documented experience of one surgeon over an 11-year period. RESULTS: Operative mortality in the series of 500 patients was 1.8%, perioperative myocardial infarction (new Q wave) rate was 0.6%, and deep sternal wound infection occurred in 1%. Six patients (1.2%) had strokes, and nine patients (1.8%) were returned to the operating room to control bleeding. One hundred ninety-eight patients who had abnormal stress test results before surgery were retested within 3 months of surgery. Ninety-four percent of these were normal, 3% were nondiagnostic, and 3% were abnormal. After a mean follow-up of 7.1 years (mode, 7.2 years), 87.5% of patients in the sample were alive, and 93.2% of this group have experienced continuing good clinical results (New York Heart Association class I or II). Eighty-nine patients who underwent an angiogram had 90.8% patency rates of ITA bypasses and 84.5% patency of vein grafts. Only two patients required repeat operations. CONCLUSIONS: The operative results did not support the contention that the coronary artery bypass using ITA procedure produces higher than acceptable mortality and morbidity rates. Multiple ITA bypasses can be performed without excessive morbidity, with low reoperation rates and long-term outcomes that should encourage skeptics to reconsider the procedure's clinical value.     

Increases in renal epsilon-(gamma-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy: pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, epsilon-(gamma-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney epsilon-(gamma-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p < 0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p < 0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular epsilon-(gamma-glutamyl) lysine (+361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular epsilon-(gamma-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca(2+) and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.     

Gene conversion is a likely cause of mutation in PKD1

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.      

Consanguinity-associated kidney diseases in Lebanon: an epidemiological study

Consanguineous marriages are common in many countries of the Middle East including Lebanon. Their impact on the repartition of kidney diseases and on the risk for familial nephritis is not known. We surveyed all of the dialysis centers in Lebanon. Nine hundred and twenty-five (925) patients and their private physicians were asked to answer a questionnaire. More than half of the hemodialysis (HD) patients had an unknown etiology of their kidney disease. Diabetes, polycystic kidney disease (PKD), chronic pyelonephritis and nephrosclerosis (NS) were the most commonly documented diagnoses. Consanguinity was present in 26% of the total HD population. More consanguineous patients with unknown renal etiology were diagnosed with their kidney diseases and initiated on dialysis before the age of 30 when compared with their non-consanguineous counter-parts (45% versus 33%, P<0.02 and 42% versus 27%, P<0.01), respectively. Similarly, consanguineous polycystic patients were diagnosed and started earlier on dialysis when compared with the non-consanguineous population (34% versus 12%, P<0.05 and 28% versus 8%, P<0.05), respectively suggesting a different disease pattern. Furthermore, the risk for family history of kidney disease was noticeable in the non-consanguineous population and significantly higher among the consanguineous patients (12% versus 18%, P<0.04). Consanguinity-associated kidney diseases affected all religious communities, in particular the Muslim and the Druze (36 and 39%), respectively versus 17% of the Christian community. Certain geographical areas were more involved than others such as the North, South and the Bekaa with the highest percentage (40%) in the latter. Socio-economical level was not a contributing factor. We conclude that the documentation of the underlying etiology in end-stage renal diseases (ESRD) seems to be deficient. Furthermore, consanguinity is prevalent in the Lebanese dialysis patients population, in particular the Muslim and the Druze communities. Consanguinity-associated kidney diseases pattern seems to differ from that of the general HD population by disease diagnosis and initiation at a younger age and a significantly higher risk for familial renal disease. It is a cultural phenomenon prevalent predominantly in the rural areas. We recommend a multi-approach including educational, informative and probably legislative strategy in order to limit and hopefully discourage consanguineous marriages.