- Email: [email protected]
Suicide of the nephron
HYPOTHESIS
Hypothesis
Suicide of the nephron
C Nicholas Hales There are various causes of renal disease. However, progressive renal disease is closely linked to the degree and duration of proteinuria. At first sight, this seems a perverse response in which a compromised organ unleashes a coordinated series of reactions that exacerbate the damage already done. Although the nephron has mechanisms whereby it can compensate for damage both by hypertrophy and hyperfunction after renal injury or ablation, these changes seem to provide only a temporary compensation. I and my colleagues found altered renal telomere shortening in the male rat linked to increased or decreased proteinuria and longevity, which suggests a mechanism whereby this compensatory process may be limited. I hypothesise that when the damaging or hypertrophic processes shorten renal telomeres to a critical length, the cells senesce with loss of function. I also suggest that the complex series of responses triggered in a protein-leaking nephron is normally a beneficial and limited process. It leads to the replacement by fibrosis of a malfunctioning unit in an otherwise healthy organ that has substantial spare capacity. The response only becomes life threatening when there is widespread nephron damage, the acceleration of which results in the ablation of all nephron function. The processes activated by proteinuria are complex and coordinated; they include the synthesis of endothelin-1 with production of monocyte chemoattractant protein 1 and an immunoregulatory cytokine chemotactic for monocytes and memory T cells. These chemokines may be expected to induce the proliferation of fibroblasts, increase the synthesis of extracellular matrix, and cause inflammation. There is also an increase in the proinflammatory glycoprotein osteopontin. The transcription factor nuclear factor B, which switches on inflammatory genes, is also increased. Components of the complement system when filtered may also cause interstitial injury accompanied by the formation of oxygen free radicals. Inflammatory cells thereby recruited secrete transforming growth-factor , which induces fibroblasts to proliferate and secrete matrix components that cause interstitial scarring and fibrosis. Tubular cells themselves seem to be transformed into fibroblasts or hypertrophy with the production of type-IV collagen. Angiotensin II plays a key part in this process. In addition, loss of selectivity of protein filtration may allow the proteinbound cytokines to reach the proximal tubule, which, at the low pH of the tubular fluid, dissociate causing further inflammation. There is clear evidence that the reduction of proteinuria slows or prevents this series of damaging events.1–3
Limited capacity of the kidney to compensate Despite these processes, the nephron clearly has mechanisms whereby it can compensate for renal injury or ablation.4 After partial nephrectomy in the rat there is compensatory hypertrophy and hyperfunction. Albumin or proteinuric urine stimulates proximal tubular-cell division.5 Notwithstanding these compensatory responses in the rat, glomerular sclerosis develops and progresses and the animal eventually dies of renal failure. Similarly in
human beings even when an initiating process of renal injury has remitted or been controlled by treatment the glomerular filtration rate may continue to decline.4 The question then arises as to why the process of compensatory hypertrophy is so limited. A possible explanation lies in kidney telomeres.6 Telomeres are the DNA-protein complexes found at the ends of chromosomes. In the absence of the enzyme telomerase these structures shorten at each cell division. After a critical degree of shortening has occurred, to a length of 1–4 kb, cells senesce and finally die through apoptosis.7 Human beings and rats who are growth retarded during fetal life are born with smaller kidneys and a reduced number of nephrons.8,9 We retarded the growth of male rats by feeding their dams a reducedprotein diet during fetal or early postnatal life. Rats fetally growth retarded but suckled by normally-fed dams after birth reached normal growth by the time they were weaned onto a conventional diet. At 3 months of age their kidneys weighed the same as controls. However, they died younger than controls and also than rats growth retarded only postnatally (by suckling a dam fed a reduced-protein diet). The latter, despite being weaned onto a normal diet remained smaller than controls and had an extended life span. Because male rats usually die of kidney disease10 we hypothesised that animals that caught up in growth and kidney weight had had an accelerated shortening of kidney telomeres leading to renal-cell senescence and early renal failure. We found that at the age of 13 months the animals that had caught up in growth had shorter kidney telomeres than those growth retarded postnatally.6 Subsequently we have found that the rats that caught up in growth had more proteinuria than those postnatally growth retarded, which is consistent with them having earlier renal failure (unpublished findings).
Hypothesis Lancet 2001; 357: 136–37 Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QR, UK (e-mail: [email protected])
136
I propose that renal deficiency and disease, including renal injury or ablation, lead to proteinuria. Proteinuria by virtue of the processes listed above, plus the telomereshortening limit on hyperplasia, leads to renal-cell senescence and loss of renal function. Furthermore, the existence of the complex, apparently highly integrated,
THE LANCET • Vol 357 • January 13, 2001
For personal use only. Not to be reproduced without permission of The Lancet.
HYPOTHESIS
response leading to renal failure and death of the organism is unlikely to be a biological accident. As a hypothesis to explain this apparently self-destructive behaviour I propose that the process, which is damaging in severely compromised kidneys, is of advantage in other more common situations of limited nephron damage or loss of function. I suggest that it represents the mechanism for elimination of failing nephrons. This would usually be advantageous since the failing nephron would sense its own incompetence by the arrival of too much or unusual proteins at its tubular apical surface. This arrival would in turn, by a time-dependent and dose-dependent effect, trigger the nephron to eventually self ablate by a massive and coordinated process in which it is replaced by fibrous tissue. In the intervening stage there is evidence that both glomerular11 and tubular5 cells proliferate but the limit to this process, I suggest, is imposed by telomere shortening. This shortening, originally thought to be due to the consequence of cell division in the absence of telomerase, is now known to occur even more rapidly in the presence of oxidative damage.12 Thus the shortening that we have observed with ageing in the rat6 and that has been reported in the human renal cortex13 could occur by either or both of these mechanisms. Exposure of cultured human mesangial cells to oxygen radicals increased age-dependent telomere shortening14 consistent with a combination of mechanisms being involved. The ultimate demise of the nephron presumably occurs when the rate of apoptosis exceeds that of proliferation.15 The incompetent nephron then ceases to be a liability in terms of renal malfunction, it no longer filters blood, and its function is subsumed by the remaining great mass of normal nephrons. The whole process only becomes life threatening when most of the nephrons are compromised, leading to escalating renal failure. In this respect what I propose fits into the pattern shown by other responses to injury or infection, in which a beneficial response is mounted to a limited injury but a life-threatening response may arise from a major event. Examples of these responses are disseminated intravascular coagulation16 and septicaemic shock.17 Again one can speculate whether these reactions represent the stupidity or sense of the biological systems. Survival of the species may be best served by the rapid elimination of seriously damaged individual members of the species. However, in human societies, in which great importance is attached to the survival of the individual, even at the cost of disability, the therapeutic logic is to inhibit or attenuate the otherwise overwhelming defence mechanism.
Testing the hypothesis The suggestion that telomere shortening is a consequence of renal-cell, particularly tubular-cell, hyperplasia, and that such shortening limits the extent to which hyperplasia can occur, can be tested both in vivo and in vitro. In-vivo surgical ablation leads to compensatory hypertrophy and hyperplasia of the remaining renal tissue. If enough tissue is removed renal failure ultimately results. The hypothesis predicts that this failure will be accompanied by shortening of telomeres into a range believed to be critical for cell survival. In-vitro cultured renal tubules respond to the presence of albumin or proteinuric urine by growth5— the prediction is that this growth will be accompanied by shortening of telomeres. Dependent upon how long the cells can be maintained in culture, telomere shortening should become critical for cell replication and survival.
THE LANCET • Vol 357 • January 13, 2001
The proposal that oxidative damage is a major component of the mechanism of telomere shortening may again be tested both in vivo and in vitro. In vivo the effects of renal tissue ablation should be attenuated by the administration of various antioxidants. The same should be true in vitro. The overexpression in renal tubular cells of components of cellular defence mechanisms against oxidative damage may again be done transgenically in vivo or by transfection in vitro. One or more of these components should reduce telomere shortening. The test of the overall hypothesis—that on a small scale the processes described are beneficial to renal function and the organism’s survival—really requires a more complete description of the pathway and mechanisms involved. The strategy will be to knock out one or more components of the pathway. Animals will then undergo small-scale injuries to the kidney of the type that would lead to limited renal scarring. In the absence of the nephron-ablation mechanism the prediction is that the limited duration and extent of tubular dysfunction would persist, leading, for example, to a more longlasting proteinuria. It is undoubtedly true that better understanding of this sequence of events at the molecular level will lead to new and improved methods of intervention to prevent, stop, or delay progressive renal disease. I am grateful to Robert Unwin for helpful discussions.
References 1 2 3 4 5
6
7 8
9
10
11
12
13 14
15
16 17
Remuzzi G, Bertani T. Pathophysiology of progressive nephropathies. N Engl J Med 1998; 339: 1446–56. de Jong PE, Navis G, de Zeeuw D. Renoprotective therapy: titration against urinary protein excretion. Lancet 1999; 354: 352–53. Remuzzi G. Nephropathic nature of proteinuria. Curr Opin Nephrol Hypertens 1999; 8: 655–63. Brenner BM. Nephron adaptation to renal injury or ablation. Am J Physiol 1985; 249: F324–37. Burton CJ, Bevington A, Harris KPG, Walls J. Growth of promiximal tubular cells in the presence of albumin and proteinuric urine. Exp Nephrol 1994; 2: 345–50. Jennings BJ, Ozanne SE, Dorling MW, Hales CN. Early growth determines longevity in male rats and may be related to telomere shortening in the kidney. FEBS Lett 1999; 448: 4–8. de Lange T. Telomeres and senescence: ending the debate. Science 1998; 279: 334–35. Hincliffe SA, Lynch MRJ, Sargent PH, Howard CV, van Velzen D. The effect of intrauterine growth retardation on the development of renal nephrons. Br J Obstet Gynaecol 1992; 99: 296–301. Merlet-Benichou C, Gilbert T, Muffat-Joly M, Lelievre-Pegorier M, Leroy B. Intrauterine growth retardation leads to a permanent nephron deficit in the rat. Pediatr Nephrol 1994; 8: 175–80. Iwasaki K, Gleiser CA, Masoro EJ, McMahan CA, Seo E, Yu BP. The influence of dietary-protein source on longevity and age-related disease processes of Fischer rats. J Gerontol 1988; 43: B5–B12. Rodriguez-Lopez AM, Flores O, Arevalo MA, Lopez-Nova JM. Glomerular cell proliferation and apoptosis in uninephrectomised spontaneously hypertensive rats. Kidney Int 1998; 68: 536–40. Oikawa S, Kawanishi S. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Lett 1999; 453: 365–68. Melk A, Ramassar V, Helms L, et al. Telomere shortening in kidneys with age. J Am Soc Nephrol 2000; 11: 444–53. Inui K, Yoshimura A, Watanabe S, et al. Telomere shortening by ageing of cultured human mesangial cells is accentuated by continuous exposure to oxygen radicals. J Am Soc Nephrol 1999; 10: 548–49A. Thomas ME, Brunskill NJ, Harris KPG, et al. Proteinuria induces tubular cell turnover: a potential mechanism for tubular atrophy. Kidney Int 1999; 55: 890–98. Levi M, ten Cate H. Disseminated intravascular coagulation. N Engl J Med 1999; 341: 586–92. Karima R. Matsumoto S, Higashi H, Matsushima K. The molecular pathogenesis of endotoxic shock and organ failure. Mol Med Today 1999; 5: 123–32.
137
For personal use only. Not to be reproduced without permission of The Lancet.
Hypothesis
Suicide of the nephron
C Nicholas Hales There are various causes of renal disease. However, progressive renal disease is closely linked to the degree and duration of proteinuria. At first sight, this seems a perverse response in which a compromised organ unleashes a coordinated series of reactions that exacerbate the damage already done. Although the nephron has mechanisms whereby it can compensate for damage both by hypertrophy and hyperfunction after renal injury or ablation, these changes seem to provide only a temporary compensation. I and my colleagues found altered renal telomere shortening in the male rat linked to increased or decreased proteinuria and longevity, which suggests a mechanism whereby this compensatory process may be limited. I hypothesise that when the damaging or hypertrophic processes shorten renal telomeres to a critical length, the cells senesce with loss of function. I also suggest that the complex series of responses triggered in a protein-leaking nephron is normally a beneficial and limited process. It leads to the replacement by fibrosis of a malfunctioning unit in an otherwise healthy organ that has substantial spare capacity. The response only becomes life threatening when there is widespread nephron damage, the acceleration of which results in the ablation of all nephron function. The processes activated by proteinuria are complex and coordinated; they include the synthesis of endothelin-1 with production of monocyte chemoattractant protein 1 and an immunoregulatory cytokine chemotactic for monocytes and memory T cells. These chemokines may be expected to induce the proliferation of fibroblasts, increase the synthesis of extracellular matrix, and cause inflammation. There is also an increase in the proinflammatory glycoprotein osteopontin. The transcription factor nuclear factor B, which switches on inflammatory genes, is also increased. Components of the complement system when filtered may also cause interstitial injury accompanied by the formation of oxygen free radicals. Inflammatory cells thereby recruited secrete transforming growth-factor , which induces fibroblasts to proliferate and secrete matrix components that cause interstitial scarring and fibrosis. Tubular cells themselves seem to be transformed into fibroblasts or hypertrophy with the production of type-IV collagen. Angiotensin II plays a key part in this process. In addition, loss of selectivity of protein filtration may allow the proteinbound cytokines to reach the proximal tubule, which, at the low pH of the tubular fluid, dissociate causing further inflammation. There is clear evidence that the reduction of proteinuria slows or prevents this series of damaging events.1–3
Limited capacity of the kidney to compensate Despite these processes, the nephron clearly has mechanisms whereby it can compensate for renal injury or ablation.4 After partial nephrectomy in the rat there is compensatory hypertrophy and hyperfunction. Albumin or proteinuric urine stimulates proximal tubular-cell division.5 Notwithstanding these compensatory responses in the rat, glomerular sclerosis develops and progresses and the animal eventually dies of renal failure. Similarly in
human beings even when an initiating process of renal injury has remitted or been controlled by treatment the glomerular filtration rate may continue to decline.4 The question then arises as to why the process of compensatory hypertrophy is so limited. A possible explanation lies in kidney telomeres.6 Telomeres are the DNA-protein complexes found at the ends of chromosomes. In the absence of the enzyme telomerase these structures shorten at each cell division. After a critical degree of shortening has occurred, to a length of 1–4 kb, cells senesce and finally die through apoptosis.7 Human beings and rats who are growth retarded during fetal life are born with smaller kidneys and a reduced number of nephrons.8,9 We retarded the growth of male rats by feeding their dams a reducedprotein diet during fetal or early postnatal life. Rats fetally growth retarded but suckled by normally-fed dams after birth reached normal growth by the time they were weaned onto a conventional diet. At 3 months of age their kidneys weighed the same as controls. However, they died younger than controls and also than rats growth retarded only postnatally (by suckling a dam fed a reduced-protein diet). The latter, despite being weaned onto a normal diet remained smaller than controls and had an extended life span. Because male rats usually die of kidney disease10 we hypothesised that animals that caught up in growth and kidney weight had had an accelerated shortening of kidney telomeres leading to renal-cell senescence and early renal failure. We found that at the age of 13 months the animals that had caught up in growth had shorter kidney telomeres than those growth retarded postnatally.6 Subsequently we have found that the rats that caught up in growth had more proteinuria than those postnatally growth retarded, which is consistent with them having earlier renal failure (unpublished findings).
Hypothesis Lancet 2001; 357: 136–37 Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QR, UK (e-mail: [email protected])
136
I propose that renal deficiency and disease, including renal injury or ablation, lead to proteinuria. Proteinuria by virtue of the processes listed above, plus the telomereshortening limit on hyperplasia, leads to renal-cell senescence and loss of renal function. Furthermore, the existence of the complex, apparently highly integrated,
THE LANCET • Vol 357 • January 13, 2001
For personal use only. Not to be reproduced without permission of The Lancet.
HYPOTHESIS
response leading to renal failure and death of the organism is unlikely to be a biological accident. As a hypothesis to explain this apparently self-destructive behaviour I propose that the process, which is damaging in severely compromised kidneys, is of advantage in other more common situations of limited nephron damage or loss of function. I suggest that it represents the mechanism for elimination of failing nephrons. This would usually be advantageous since the failing nephron would sense its own incompetence by the arrival of too much or unusual proteins at its tubular apical surface. This arrival would in turn, by a time-dependent and dose-dependent effect, trigger the nephron to eventually self ablate by a massive and coordinated process in which it is replaced by fibrous tissue. In the intervening stage there is evidence that both glomerular11 and tubular5 cells proliferate but the limit to this process, I suggest, is imposed by telomere shortening. This shortening, originally thought to be due to the consequence of cell division in the absence of telomerase, is now known to occur even more rapidly in the presence of oxidative damage.12 Thus the shortening that we have observed with ageing in the rat6 and that has been reported in the human renal cortex13 could occur by either or both of these mechanisms. Exposure of cultured human mesangial cells to oxygen radicals increased age-dependent telomere shortening14 consistent with a combination of mechanisms being involved. The ultimate demise of the nephron presumably occurs when the rate of apoptosis exceeds that of proliferation.15 The incompetent nephron then ceases to be a liability in terms of renal malfunction, it no longer filters blood, and its function is subsumed by the remaining great mass of normal nephrons. The whole process only becomes life threatening when most of the nephrons are compromised, leading to escalating renal failure. In this respect what I propose fits into the pattern shown by other responses to injury or infection, in which a beneficial response is mounted to a limited injury but a life-threatening response may arise from a major event. Examples of these responses are disseminated intravascular coagulation16 and septicaemic shock.17 Again one can speculate whether these reactions represent the stupidity or sense of the biological systems. Survival of the species may be best served by the rapid elimination of seriously damaged individual members of the species. However, in human societies, in which great importance is attached to the survival of the individual, even at the cost of disability, the therapeutic logic is to inhibit or attenuate the otherwise overwhelming defence mechanism.
Testing the hypothesis The suggestion that telomere shortening is a consequence of renal-cell, particularly tubular-cell, hyperplasia, and that such shortening limits the extent to which hyperplasia can occur, can be tested both in vivo and in vitro. In-vivo surgical ablation leads to compensatory hypertrophy and hyperplasia of the remaining renal tissue. If enough tissue is removed renal failure ultimately results. The hypothesis predicts that this failure will be accompanied by shortening of telomeres into a range believed to be critical for cell survival. In-vitro cultured renal tubules respond to the presence of albumin or proteinuric urine by growth5— the prediction is that this growth will be accompanied by shortening of telomeres. Dependent upon how long the cells can be maintained in culture, telomere shortening should become critical for cell replication and survival.
THE LANCET • Vol 357 • January 13, 2001
The proposal that oxidative damage is a major component of the mechanism of telomere shortening may again be tested both in vivo and in vitro. In vivo the effects of renal tissue ablation should be attenuated by the administration of various antioxidants. The same should be true in vitro. The overexpression in renal tubular cells of components of cellular defence mechanisms against oxidative damage may again be done transgenically in vivo or by transfection in vitro. One or more of these components should reduce telomere shortening. The test of the overall hypothesis—that on a small scale the processes described are beneficial to renal function and the organism’s survival—really requires a more complete description of the pathway and mechanisms involved. The strategy will be to knock out one or more components of the pathway. Animals will then undergo small-scale injuries to the kidney of the type that would lead to limited renal scarring. In the absence of the nephron-ablation mechanism the prediction is that the limited duration and extent of tubular dysfunction would persist, leading, for example, to a more longlasting proteinuria. It is undoubtedly true that better understanding of this sequence of events at the molecular level will lead to new and improved methods of intervention to prevent, stop, or delay progressive renal disease. I am grateful to Robert Unwin for helpful discussions.
References 1 2 3 4 5
6
7 8
9
10
11
12
13 14
15
16 17
Remuzzi G, Bertani T. Pathophysiology of progressive nephropathies. N Engl J Med 1998; 339: 1446–56. de Jong PE, Navis G, de Zeeuw D. Renoprotective therapy: titration against urinary protein excretion. Lancet 1999; 354: 352–53. Remuzzi G. Nephropathic nature of proteinuria. Curr Opin Nephrol Hypertens 1999; 8: 655–63. Brenner BM. Nephron adaptation to renal injury or ablation. Am J Physiol 1985; 249: F324–37. Burton CJ, Bevington A, Harris KPG, Walls J. Growth of promiximal tubular cells in the presence of albumin and proteinuric urine. Exp Nephrol 1994; 2: 345–50. Jennings BJ, Ozanne SE, Dorling MW, Hales CN. Early growth determines longevity in male rats and may be related to telomere shortening in the kidney. FEBS Lett 1999; 448: 4–8. de Lange T. Telomeres and senescence: ending the debate. Science 1998; 279: 334–35. Hincliffe SA, Lynch MRJ, Sargent PH, Howard CV, van Velzen D. The effect of intrauterine growth retardation on the development of renal nephrons. Br J Obstet Gynaecol 1992; 99: 296–301. Merlet-Benichou C, Gilbert T, Muffat-Joly M, Lelievre-Pegorier M, Leroy B. Intrauterine growth retardation leads to a permanent nephron deficit in the rat. Pediatr Nephrol 1994; 8: 175–80. Iwasaki K, Gleiser CA, Masoro EJ, McMahan CA, Seo E, Yu BP. The influence of dietary-protein source on longevity and age-related disease processes of Fischer rats. J Gerontol 1988; 43: B5–B12. Rodriguez-Lopez AM, Flores O, Arevalo MA, Lopez-Nova JM. Glomerular cell proliferation and apoptosis in uninephrectomised spontaneously hypertensive rats. Kidney Int 1998; 68: 536–40. Oikawa S, Kawanishi S. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Lett 1999; 453: 365–68. Melk A, Ramassar V, Helms L, et al. Telomere shortening in kidneys with age. J Am Soc Nephrol 2000; 11: 444–53. Inui K, Yoshimura A, Watanabe S, et al. Telomere shortening by ageing of cultured human mesangial cells is accentuated by continuous exposure to oxygen radicals. J Am Soc Nephrol 1999; 10: 548–49A. Thomas ME, Brunskill NJ, Harris KPG, et al. Proteinuria induces tubular cell turnover: a potential mechanism for tubular atrophy. Kidney Int 1999; 55: 890–98. Levi M, ten Cate H. Disseminated intravascular coagulation. N Engl J Med 1999; 341: 586–92. Karima R. Matsumoto S, Higashi H, Matsushima K. The molecular pathogenesis of endotoxic shock and organ failure. Mol Med Today 1999; 5: 123–32.
137
For personal use only. Not to be reproduced without permission of The Lancet.