Leon G. Fine

Gene transfer into the rat renal glomerulus via a mesangial cell vector: site-specific delivery, in situ amplification, and sustained expression of an exogenous gene in vivo.

To evaluate the pathophysiological function of specific molecules in the renal glomerulus, selective, sustained, and modifiable expression of such molecules will be required. Towards achieving this end, we devised a gene transfer system using the glomerular mesangial cell as a vector for gene delivery. A reporter gene which encodes bacterial beta-galactosidase was introduced into cultured rat mesangial cells, and the stable transfectants were transferred into the rat kidney via the renal artery, leading to selective entrapment within the glomeruli. In the normal kidney, the reporter cells populated into 57 +/- 13% of glomeruli site specifically, and the expression of beta-galactosidase was sustained for 4 wk and declined thereafter. Within the glomerulus, some of the reporter cells remained in the glomerular capillaries, while others repopulated the mesangial area and, in part, extended their cytoplasmic processes toward the surrounding capillaries. When the cells were transferred into glomeruli subjected to transient mesangiolysis induced by monoclonal antibody 1-22-3, in situ expression of beta-galactosidase was amplified 7-12-fold, and the enhanced level of expression continued for up to 8 wk. The mesangial cell vector system thus achieves site-specific delivery of an exogenous gene into the glomerulus and is amenable to in situ amplification and sustained expression by preconditioning of the target site.

Intrarenal oxygenation in chronic renal failure.

1 In chronic renal failure (CRF), renal impairment correlates with tubulointerstitial fibrosis characterized by inflammation, interstitial expansion with accumulation of extracellular matrix (ECM), tubular atrophy and vascular obliteration. Tubulointerstitial injury subsequent to glomerular sclerosis may be induced by proteinuria, leakage of glomerular filtrate or injury to the post‐glomerular peritubular capillaries (hypoxia). 2 In vivo data in animal models suggest that CRF is associated with hypoxia, with the decline in renal Po2 preceding ECM accumulation. 3 Chronic renal failure is characterized by loss of microvascular profiles but, in the absence of microvascular obliteration, hypoxia can occur by a variety of complementary mechanisms, including anaemia, decreased capillary flow, increased vasoconstriction, increased metabolic demand and increased diffusion distances due to ECM deposition. 4 Hypoxia regulates a wide array of genes, including many fibrogenic factors. Hypoxia‐inducible factors (HIF) are the major, but not the sole, transcriptional regulators in the hypoxic response. 5 In CRF, hypoxia may play a role in the sustained inflammatory response. 6 In vitro studies in tubulointerstitial cells suggest that hypoxia can induce profibrogenic changes in proximal tubular epithelial cells and interstitial fibroblasts consistent with changes observed in CRF in vivo. The effect of hypoxia on renal microvascular cells warrants investigation. 7 Hypoxia may play a role in the recruitment, retention and differentiation of circulating progenitor cells to the kidney contributing to the disease process and may also affect intrinsic stem cell populations. 8 Chronic hypoxia in CRF fails to induce a sustained angiogenic response. 9 Therapeutic manipulation of the hypoxic response may be of benefit in slowing progression of CRF. Potential therapies include correction of anaemia, inhibition of the renin–angiotensin system, administration of exogenous pro‐angiogenic factors to protect the microvasculature, activation of HIF and hypoxia‐mediated targeting of engineered progenitor cells.

Tubular-Derived Growth Factors and Cytokines in the Pathogenesis of Tubulointerstitial Fibrosis: Implications for Human Renal Disease Progression

Detailed histomorphometric analysis of human biopsy tissue over the last 30 years has convincingly demonstrated that preservation of the tubulointerstitial compartment of the kidney is the major determinant of renal outcome in a variety of human renal diseases. Nevertheless, the pathophysiology of tubulointerstitial disease remains obscure. In particular, the primary role of tubular injury has not been explored adequately. There is now accumulating evidence that apart from their many transport functions, tubular cells also secrete an array of cytokines, including chemotactic cytokines, polypeptide growth factors, and vasoactive peptides. Three paracrine growth systems acting at different levels in the kidney are described as examples of potential interactions between tubular and interstitial cells in health and disease. We hypothesize that while glomerular injury may precede tubular injury, it is tubular injury that sets into motion the irreversible process of tubulointerstitial fibrosis characteristic of progressive human renal disease, leading to secondary loss of glomerular function.

Angiotensin II Blockade Augments Renal Cortical Microvascular pO2 Indicating a Novel, Potentially Renoprotective Action

Background: The existence of tubulointerstitial damage in most cases of progressive human glomerular disease suggests that this compartment of the kidney is likely to be targeted by renoprotective agents which slow the progression of disease. Angiotensin-converting enzyme (ACE) inhibitors have become the cornerstone of renal protection. Since we have proposed that perturbation of the interstitial capillary circulation with consequent chronic hypoxia could be critical to the progressive nature of many renal diseases, we developed a dynamic method of measuring renal cortical pO2 and sought to determine whether agents which block the renal effects of angiotensin II (AII) could affect interstitial microvascular oxygenation in the normal rat kidney. Methods: Instrumented, anaesthetised adult male Sprague-Dawley rats were studied. Cortical microvascular pO2 was measured on the surface of the exposed kidney using protoporphyrin phosphorescence. Blood pressure and renal artery blood flow (Doppler flowmetry) were measured concurrently over a 180-min experimental period. Animals received non-hypotensive doses of enalaprilat (100 µg/kg i.v.) or candesartan (40 µg/kg i.v.) either at the beginning of the experimental period or after an initial decline in cortical microvascular pO2. Results: After a 30-min stabilisation period there was a slow decline in pO2 from 48.6 ± 4.1 to 38.5 ± 6.9 mm Hg in control animals over the 180-min experimental period. Administration of the ACE inhibitor, enalaprilat at the beginning of the experimental period, completely abrogated this decline and protected pO2 levels throughout this period with no effect on blood pressure or renal blood flow. In separate experiments, administration of enalaprilat after microvascular pO2 had fallen by 5 mm Hg, resulted in a rise in RBF and pO2 within 15 min with pO2 remaining elevated for up to 60 min post-injection. The angiotensin II AT1 receptor antagonist, candesartan, had a similar effect to enalaprilat, inducing a rapid and sustained elevation in cortical pO2. Conclusions: These studies indicate that blockade of AII raises pO2 in the interstitial microvascular compartment of the normal rat kidney. This effect may contribute to the renoprotective action of ACE inhibitors and AII receptor antagonists in slowing the progression of chronic renal diseases.

Mechanisms of tubulo-interstitial injury in progressive renal diseases

Abstract. A vast amount of evidence, based upon human renal biopsy material, indicates that the presence of tubular atrophy and interstitial fibrosis is a better indicator of outcome of renal function than is the extent of glomerular sclerosis. The pathophysiological basis for this surprising fact has not been adequately addressed. In this review we point out that the systemic hypertension which accompanies most forms of chronic renal disease could impact adversely upon the vasodilated interstitial vascular compartment which, together with a component of primary capillary injury related to the disease process, could cause progressive obliteration of particular capillaries. This would initiate a process of chronic tubular ischaemia ultimately leading to tubular atrophy. Since tubular cells have been shown to produce an array of cytokines and growth factors which modulate fibroblast proliferation, extracellular matrix production and chemo‐attracts for infiltrating cells, it is further proposed that it is the tubular injury which initiates the deleterious cascade of events. Tubular injury may be aggravated by the filtration of potentially ‘noxious’ molecules through the diseased glomerulus and by infiltrating cells. As the vascular bed into which glomerular blood flow empties is progressively obliterated, glomerular function declines and renal failure advances in relation to the degree of tubulo‐interstitial fibrosis.

Functional Profile of the Isolated Uremic Nephron: ROLE OF COMPENSATORY HYPERTROPHY IN THE CONTROL OF FLUID REABSORPTION BY THE PROXIMAL STRAIGHT TUBULE

An in vitro approach to the study of single nephron function in uremia has been employed in evaluating the control of fluid reabsorption by the renal superficial proximal straight tubule (PST). Isolated segments of PSTs from the remnant kidneys of uremic rabbits (stage III) were perfused in vitro and their rate of fluid reabsorption compared with normal PSTs and with PSTs derived from the remnant kidneys of nonuremic rabbits (stage II). All segments were exposed to a peritubular bathing medium of both normal and uremic rabbit serum thereby permitting a differentiation to be made between adaptations in function which are intrinsic to the tubular epithelium and those which are dependent upon a uremic milieu.Compared with normal and stage II PSTs, there was significant hypertrophy of the stage III tubules as evidenced by an increase in length and internal diameter, and a twofold increase in the dry weight per unit length. Fluid reabsorption per unit length of tubule was 70% greater in stage III than in normal and stage II PSTs, and was closely correlated with the increase in dry weight. Substitutions between normal and uremic rabbit serum in the peritubular bathing medium did not affect fluid reabsorption significantly in any of the three groups of PSTs. Perfusion of the tubules with an ultrafiltrate of normal vs. uremic serum likewise failed to influence the rate of net fluid reabsorption. It has previously been observed that net fluid secretion may occur in nonperfused or stop-flow perfused normal rabbit PSTs exposed to human uremic serum. Additional studies were thus performed on normal and stage III PSTs to evaluate whether net secretion occurs in the presence of rabbit uremic serum. No evidence for net secretion was found. These studies demonstrate that fluid reabsorption is greatly increased in the superficial PST of the uremic remnant kidney and that this functional adaptation is closely correlated with compensatory hypertrophy of the segment. Humoral factors in the peritubular environment do not appear to be important mediators of the enhanced fluid reabsorption.

Progressive renal disease : Fibroblasts, extracellular matrix, and integrins

Progressive renal disease is characterized by expansion of the tubulo-interstitium and accumulation of extracellular matrix within this tissue compartment. Interstitial fibroblasts are the primary producers of the interstitial matrix, and in the evolution of tubulo-interstitial fibrosis these cells undergo changes, namely increased proliferation, differentiation to myofibroblasts, and altered extracellular matrix metabolism, all of which, in other cell types, have been shown to be regulated by the major family of extracellular matrix receptors, the integrins. In the normal kidney, interstitial fibroblasts express α1, α4, α5, and β1 integrins, and fibrosis is associated with increased expression of α1, α2, α5, αv, and β1 integrins. In particular, α5, β1, and αv are suggested to be linked with the fibrotic process. In vitro, renal fibroblasts express a similar range of integrins, and ligation of selected receptors is associated with specific functions. Ligation of α6 stimulates proliferation, while α5 promotes expression of myofibroblastic phenotype, and β1 integrin has been implicated in cell contraction. Recent studies suggest that renal fibroblasts also express the non-integrin matrix receptors, discoidin domain receptors, and that changes in activation of these receptors may be associated with fibrogenic events. Thus the current, albeit limited, data suggest an important role for receptors for extracellular matrix molecules in the pathogenesis of progressive renal fibrosis.

Physiology and Cell Biology Update: Polypeptide Growth Factors and Their Relation to Renal Disease

A number of polypeptide growth factors have been shown to act on different types of renal cells, and many are produced by the kidney itself. It is unclear whether the compensatory hypertrophy that follows the destruction of nephrons is under the control of growth factors, but they undoubtedly participate in various forms of progressive renal injury, including chronic glomerular and tubulointerstitial diseases via autocrine, paracrine, and possibly even endocrine routes. As new developments occur in this rapidly changing field, it is hoped that this knowledge can be used to ameliorate the damage, halt the progression, or enhance the recovery from a disease process in the kidney and in other tissues.

How to evaluate and improve the quality and credibility of an outcomes database: validation and feedback study on the UK Cardiac Surgery Experience

Abstract Objectives: To assess the quality and completeness of a database of clinical outcomes after cardiac surgery and to determine whether a process of validation, monitoring, and feedback could improve the quality of the database. Design: Stratified sampling of retrospective data followed by prospective re-sampling of database after intervention of monitoring, validation, and feedback. Setting: Ten tertiary care cardiac surgery centres in the United Kingdom. Intervention: Validation of data derived from a stratified sample of case notes (recording of deaths cross checked with mortuary records), monitoring of completeness and accuracy of data entry, feedback to local data managers and lead surgeons. Main outcome measures: Average percentage missing data, average κ coefficient, and reliability score by centre for 17 variables required for assignment of risk scores. Actual minus risk adjusted mortality in each centre. Results: The database was incomplete, with a mean (SE) of 24.96% (0.09%) of essential data elements missing, whereas only 1.18% (0.06%) were missing in the patient records (P<0.0001). Intervention was associated with (a) significantly less missing data (9.33% (0.08%) P<0.0001); (b) marginal improvement in reliability of data and mean (SE) overall centre reliability score (0.53 (0.15) v 0.44 (0.17)); and (c) improved accuracy of assigned Parsonnet risk scores (κ 0.84 v 0.70). Mortality scores (actual minus risk adjusted mortality) for all participating centres fell within two standard deviations of the mean score. Conclusion: A short period of independent validation, monitoring, and feedback improved the quality of an outcomes database and improved the process of risk adjustment, but with substantial room for further improvement. Wider application of this approach should increase the credibility of similar databases before their public release. What is already know in this topic Release of healthcare outcomes into the public domain has altered referral patterns and has led to improvement in some centres and elimination of others The tacit assumption is that such outcomes data are accurate and can be relied on by the public and by healthcare providers to guide improvements What this study adds Sampling of a published national cardiac surgery database in England revealed it to be both incomplete and unreliable in its ability to yield accurate, risk adjusted outcomes data An independent short process of monitoring, validation, and feedback improved the quality of the database Such databases probably require an ongoing process of monitoring in order to allow data of adequate quality to be generated for the purpose of improving healthcare outcomes

Regulation of Vasopressin Action by Prostaglandins: EVIDENCE FOR PROSTAGLANDIN SYNTHESIS IN THE RABBIT CORTICAL COLLECTING TUBULE

The present studies examined whether vasopressin increases prostaglandin biosynthesis in isolated rabbit cortical collecting tubules (CCT) and whether endogenous prostaglandin biosynthesis plays a role in modulating the response of this nephron segment to vasopressin. Three groups of studies were performed. In the first group, CCT and proximal straight tubules (PST) were incubated with [(3)H]arachidonic acid, and metabolites were separated and identified using silica gel thin-layer chromatography. CCT were capable of producing all of the major prostaglandins (PG) (PGE(2) > thromboxane B(2)[TxB(2)] > PGF(2alpha) > PGI(2)). PST produced significantly lesser quantities of these lipids. In the second group, radiolabeled arachidonic acid was incorporated into the phospholipid pool of both CCT and PST, vasopressin was added to the incubation medium, and metabolities were separated and identified as above. Vasopressin stimulated the release of all of the major prostaglandins in CCT but had no effect on PST. PGE release into the incubation medium, as assessed by a radioreceptor assay, increased 108%, and a vasopressin analogue, 1-desamino-8-d-arginine vasopressin, had a quantitatively similar effect. In the third group, a submaximal dose of vasopressin was administered to isolated, perfused CCT studied in the presence and absence of indomethacin to assess whether endogenous prostaglandins play a role in modulating the antidiuretic response to vasopressin. Studies were performed in rabbits on a normal diet and in desoxycorticosterone acetate (DOCA)- or KCl-loaded animals. In the state of mineralocorticoid excess, basal prostaglandin synthesis was 63% lower, and vasopressin-stimulated prostaglandin synthesis 76% lower, than the synthesis observed in rabbits on a normal diet. Cyclooxygenase inhibition exposed a significant hydroosmotic response to a submaximal dose of vasopressin in CCT from DOCA- or KCl-loaded animals. With arachidonic acid in the bath, the same dose of vasopressin failed to elicit a hydroosmotic response in CCT from rabbits on a normal diet even in the presence of a cyclooxygenase inhibitor. However, removal of exogenous arachidonic acid, with a consequently lower rate of prostaglandin synthesis, allowed the cyclooxygenase inhibitor to enhance the hydroosmotic response to vasopressin in these tubules.We conclude from these studies that the rabbit CCT has the capacity to synthesize all of the major prostaglandins and that the rate of synthesis of these lipids is enhanced by vasopessin. Prostaglandin synthesis by the CCT is postulated to modulate the antidiuretic action of vasopressin via a closed feedback loop. The effectiveness of this feedback regulation is dependent upon the mineralocorticoid status of the animal, which determines the level of basal and vasopressin-stimulated prostaglandin synthesis by the CCT.