Jill T. Norman

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.

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.

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.

Hypoxia-induced changes in extracellular matrix metabolism in renal cells

The mechanisms underlying the progressive fibrosis that characterises end-stage renal disease in vivo remain to be established but hypoxia, as a result of microvascular injury and loss, has been suggested to play an important role. In support of this hypothesis, in vitro studies show that hypoxia (1% O2) induces a fibrogenic phenotype in human renal tubular endothelia, interstitial fibroblasts and microvascular endothelial cells, simultaneously increasing extracellular matrix (ECM) production and decreasing turnover via effectors on matrix-degrading enzymes and their inhibitors. The effects of hypoxia on ECM metabolism are independent of hypoxia-induced growth factors and are mediated by a haem-protein sensor and activation of both protein kinase C- and tyrosine kinase-mediated signal transduction pathways. De novo gene transcription is regulated by both hypoxia-inducible factor-1-dependent and -independent mechanisms. Further understanding of the molecular mechanisms by which decreased oxygen alters expression of genes involved in ECM metabolism in renal cells may provide new insights into the pathogenesis of fibrosis and identify novel avenues for intervention.

αv integrins play an important role in myofibroblast differentiation

Transforming growth factor‐β1 is a potent mediator of the differentiation of fibroblasts into myofibroblasts, which is characterized by the appearance of the cytoskeletal protein α‐smooth muscle actin. The aim of this study was to investigate the role of integrin extracellular matrix receptors in transforming growth factor‐β1–induced myofibroblast differentiation. We show that blockade of the αv and/or β1 integrins prevents the transforming growth factor‐β1–induced myofibroblast differentiation, seen by the increased expression of α‐smooth muscle actin and enhanced collagen gel contraction in three human fibroblast cell lines (from the mouth, skin, and kidney). Further, blockade of αv specific integrins αvβ5 and αvβ3 suppressed myofibroblast differentiation in fibroblasts from the mouth and skin; however, in the kidney cells, the prevention of differentiation was seen only with blockade of αvβ5 integrin but not αvβ3. A possible reason for this result may be different degrees of responsiveness to transforming growth factor‐β1 treatment seen from different anatomical origins of the cell lines. These data indicate a novel role for αv integrins in the differentiation of human fibroblasts from the mouth, skin, and kidney into myofibroblasts and suggest that there is a common differentiation pathway.

The Role of Angiotensin II in Renal Growth

Angiotensin II (AII) has many of the features of the archetypical growth factors and appears to be a growth regulator in the kidney. AII binds to specific cell surface receptors present on a number of different renal cell types including mesangial, vascular smooth muscle, tubular and interstitial cells, and activates many of the intracellular signalling pathways associated with cell growth. In vitro AII can potentiate the mitogenic effect of other growth factors such as EGF. AII induces hypertrophy of vascular smooth muscle cells but the role of AII in the growth of other renal cell types has not been systematically studied.

Renal growth responses to acute and chronic injury: routes to therapeutic intervention

Knowledge of the control of cell growth and extracellular matrix deposition has assumed center stage in the understanding of how the diseased kidney responds to injury. After acute tubular injury, there may be reversible, partial depolarization of renal cells or cell necrosis. The latter requires a regenerative response, which could be under the control of growth factors such as epidermal growth factor (EGF). Up-regulation of EGF receptors on viable cells provides the cell with an enhanced growth response despite a reduction in EGF production by the kidney. Acute glomerular injury involves a highly complex network of cytokines and growth inhibitors, the most important of which appear to be platelet-derived growth factor as a mitogen and transforming growth factor beta as an activator of extracellular matrix deposition. The long-term growth responses of the kidney to injury, reflected by chronic renal diseases, include tubular hypertrophy in those nephrons which are less affected by the primary disease. Tubular cell enlargement appears to proceed along a pathway that is different from the growth in cell size which precedes cell division, at least as indicated by a fundamentally different pattern of early gene expression. This pattern is not suggestive of a classical growth factor-initiated process. Other chronic changes that seem to correlate well with the progression of human disease are tubular atrophy and interstitial fibrosis. Growth factors produced by tubular cells may cause proliferation and matrix deposition by adjacent interstitial fibroblasts. A scheme is proposed in which low-grade ischemic injury to tubular cells, secondary to microvascular injury, leads to tubular atrophy, the release of growth factors, interstitial fibrosis, and the obliteration of peritubular capillaries. This would aggravate primary glomerular injury by compromising the vascular outflow from the glomerulus and would account for the long-recognized association between tubulo-interstitial injury and the progression of a variety of renal diseases. The use of growth factors to stimulate specific growth responses, antibodies, or inhibitory molecules to inhibit scarring generated by cytokines and the potential for genetic manipulation of the kidney provide future avenues for manipulating the growth response of the diseased kidney.

Restoring the renal microvasculature to treat chronic kidney disease

Chronic kidney disease is characterized by progressive loss of the renal microvasculature, which leads to local areas of hypoxia and induction of profibrotic responses, scarring and deterioration of renal function. Revascularization alone might be sufficient to restore kidney function and regenerate the structure of the diseased kidney. For revascularization to be successful, however, the underlying disease process needs to be halted or alleviated and there must remain a sufficient number of surviving nephron units that can serve as a scaffold for kidney regeneration. This Perspectives article describes how revascularization might be achieved using vascular growth factors or adoptive transfer of endothelial progenitor cells and provides a brief outline of the studies performed to date. An overview of how therapeutic strategies targeting the microvasculature could be enhanced in the future is also presented.

Connective tissue growth factor causes EMT-like cell fate changes in vivo and in vitro

Summary Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of chronic fibrotic diseases. However, the mechanism by which paracrine effects of CTGF control the cell fate of neighboring epithelial cells is not known. In this study, we investigated the paracrine effects of CTGF overexpressed in fibroblasts of Col1a2-CTGF transgenic mice on epithelial cells of skin and lung. The skin and lungs of Col1a2-CTGF transgenic mice were examined for phenotypic markers of epithelial activation and differentiation and stimulation of signal transduction pathways. In addition to an expansion of the dermal compartment in Col1a2-CTGF transgenic mice, the epidermis was characterized by focal hyperplasia, and basal cells stained positive for &agr;SMA, Snail, S100A4 and Sox9, indicating that these cells had undergone a change in their genetic program. Activation of phosphorylated p38 and phosphorylated Erk1/2 was observed in the granular and cornified layers of the skin. Lung fibrosis was associated with a marked increase in cells co-expressing epithelial and mesenchymal markers in the lesional and unaffected lung tissue of Col1a2-CTGF mice. In epithelial cells treated with TGF&bgr;, CTGF-specific siRNA-mediated knockdown suppressed Snail, Sox9, S100A4 protein levels and restored E-cadherin levels. Both adenoviral expression of CTGF in epithelial cells and treatment with recombinant CTGF induced EMT-like morphological changes and expression of &agr;-SMA. Our in vivo and in vitro data supports the notion that CTGF expression in mesenchymal cells in the skin and lungs can cause changes in the differentiation program of adjacent epithelial cells. We speculate that these changes might contribute to fibrogenesis.