Cultured skin fibroblasts as an in vitro model to assess phenotypic features in subjects with diabetic nephropathy

Related Article, p. 465 Diabetic nephropathy (DN) develops in about 25% of all patients with Type 1 diabetes and is the strongest predictor of cardiovascular disease and premature death in these patients.1Batlle D Lurbe A LaPointe M Agrawal R The Na+/H+ antiporter, type I diabetes, and hypertension.Kidney Curr Surv World Lit. 1995; 4: 1-3Google Scholar, 2Viberti GC Hill RD Jarrett RJ Argyropoulos A Mahmud U Keen H Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus.Lancet. 1982; 1: 1430-1432Google Scholar, 3Lurbe A Redon J Pascual JM Tacons J Alverez V Batlle D Altered blood pressure during sleep in normotensive subjects with type 1 diabetes.Hypertension. 1993; 21: 227-235Google Scholar, 4Caramori ML Fioretto P Mauer M The need for early predictors of diabetic nephropathy risk: is albumin excretion rate sufficient?.Diabetes. 2000; : 1399-1408Google Scholar, 5Seaquist ER Goetz FC Rich S Barbosa J Familial clustering of diabetic kidney disease. Evidence for genetic susceptibility to diabetic nephropathy.N Engl J Med. 1989; 320: 1161-1165Google Scholar, 6Krolewski AS Warram JH Christlieb AR Busick EJ Kahn CR The changing natural history of nephropathy in type I diabetes.Am J Med. 1985; 78: 785-794Google Scholar, 7Krolewski AS Canessa M Warram JH Laffel LM Christlieb AR Knowler WC Rand LI Predisposition to hypertension and susceptibility to renal disease in insulin-dependent diabetes mellitus.N Engl J Med. 1988; 318: 140-145Google Scholar, 8Borch-Johnsen K Norgaard K Hommel E Mathiesen ER Jensen JS Deckert T Parving HH Is diabetic nephropathy an inherited complication?.Kidney Int. 1992; 41: 719-722Google Scholar Understanding the cellular pathophysiology associated with development of DN is therefore of paramount importance. The risk for DN in Type 1 patients with diabetes appears to have a genetic component in addition to the metabolic abnormality associated with hyperglycemia.1Batlle D Lurbe A LaPointe M Agrawal R The Na+/H+ antiporter, type I diabetes, and hypertension.Kidney Curr Surv World Lit. 1995; 4: 1-3Google Scholar, 2Viberti GC Hill RD Jarrett RJ Argyropoulos A Mahmud U Keen H Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus.Lancet. 1982; 1: 1430-1432Google Scholar, 3Lurbe A Redon J Pascual JM Tacons J Alverez V Batlle D Altered blood pressure during sleep in normotensive subjects with type 1 diabetes.Hypertension. 1993; 21: 227-235Google Scholar, 4Caramori ML Fioretto P Mauer M The need for early predictors of diabetic nephropathy risk: is albumin excretion rate sufficient?.Diabetes. 2000; : 1399-1408Google Scholar, 5Seaquist ER Goetz FC Rich S Barbosa J Familial clustering of diabetic kidney disease. Evidence for genetic susceptibility to diabetic nephropathy.N Engl J Med. 1989; 320: 1161-1165Google Scholar, 6Krolewski AS Warram JH Christlieb AR Busick EJ Kahn CR The changing natural history of nephropathy in type I diabetes.Am J Med. 1985; 78: 785-794Google Scholar, 7Krolewski AS Canessa M Warram JH Laffel LM Christlieb AR Knowler WC Rand LI Predisposition to hypertension and susceptibility to renal disease in insulin-dependent diabetes mellitus.N Engl J Med. 1988; 318: 140-145Google Scholar, 8Borch-Johnsen K Norgaard K Hommel E Mathiesen ER Jensen JS Deckert T Parving HH Is diabetic nephropathy an inherited complication?.Kidney Int. 1992; 41: 719-722Google Scholar This is evident from the findings that DN develops in some Type 1 patients despite good glycemic control and a substantial proportion of patients with poor glycemic control do not develop DN.5Seaquist ER Goetz FC Rich S Barbosa J Familial clustering of diabetic kidney disease. Evidence for genetic susceptibility to diabetic nephropathy.N Engl J Med. 1989; 320: 1161-1165Google Scholar, 6Krolewski AS Warram JH Christlieb AR Busick EJ Kahn CR The changing natural history of nephropathy in type I diabetes.Am J Med. 1985; 78: 785-794Google Scholar, 7Krolewski AS Canessa M Warram JH Laffel LM Christlieb AR Knowler WC Rand LI Predisposition to hypertension and susceptibility to renal disease in insulin-dependent diabetes mellitus.N Engl J Med. 1988; 318: 140-145Google Scholar, 8Borch-Johnsen K Norgaard K Hommel E Mathiesen ER Jensen JS Deckert T Parving HH Is diabetic nephropathy an inherited complication?.Kidney Int. 1992; 41: 719-722Google Scholar Direct study of the cellular mechanisms responsible for altered mesangial expansion and the determinants of the development of overt nephropathy in predisposed subjects is limited by the availability of kidney biopsies and adequate sampling of cells. As a surrogate model, cultured skin fibroblasts have been used as an in vitro system for studying the cellular mechanisms associated with development of DN.9Lurbe A Fioretto P Mauer M LaPointe MS Batlle D Growth phenotype of cultured skin fibroblasts from patients with insulin-dependent diabetes mellitus with and without nephropathy and its association to overactivity of the Na+/H+ antiporter.Kidney Int. 1996; 50: 1684-1693Google Scholar, 10Davies JE Ng LL Kofoed-Enevoldsen A Li LK Earle KA Trevisan R Viberti G Intracellular pH and Na+/H+ antiport activity of cultured skin fibroblasts from diabetics.Kidney Int. 1992; 42: 1184-1190Google Scholar, 11Trevisan R Li L Messent J TariQ T Earle K Walker J Viberti G Na+/H+ antiport activity and cell growth in cultured skin fibroblasts of IDDM patients with nephropathy.Diabetes. 1992; 41: 1239-1246Google Scholar Cultured skin fibroblasts from diabetic patients with DN exhibit distinctive phenotypic differences compared with cells from patients with normoalbuminuria and normal control subjects. For example, we9Lurbe A Fioretto P Mauer M LaPointe MS Batlle D Growth phenotype of cultured skin fibroblasts from patients with insulin-dependent diabetes mellitus with and without nephropathy and its association to overactivity of the Na+/H+ antiporter.Kidney Int. 1996; 50: 1684-1693Google Scholar and others11Trevisan R Li L Messent J TariQ T Earle K Walker J Viberti G Na+/H+ antiport activity and cell growth in cultured skin fibroblasts of IDDM patients with nephropathy.Diabetes. 1992; 41: 1239-1246Google Scholar showed that cultured skin fibroblasts from patients with Type 1 diabetes with DN have increased DNA synthesis compared with those from patients with Type 1 diabetes with normoalbuminuria and from nondiabetic control subjects. Increased DNA synthesis in fibroblasts from patients with Type 1 diabetes with DN may involve alterations in cell cycle regulatory proteins that are active in the G1 phase of the cell cycle.12Danesh F Ye M Salmi S LaPointe M Batlle D Temporal profile of serum induced S phase entry and pRB phosphorylation in human skin fibroblasts.Kidney Int. 1999; 56: 1282-1285Google Scholar, 13Danesh F Hannan E Ye M LaPointe M Molitch M Batlle D Cyclin dependent kinase inhibitor p16 is decreased in cultured skin fibroblasts from type 1 diabetic patients with nephropathy (DN). A marker of diabetic nephropathy?.J Am Soc Nephrol. 2000; 11 (abstr): 638AGoogle Scholar The hyperplastic phenotype of skin fibroblasts from patients with Type 1 diabetes with DN is also associated with an increased activity in Na+-H+ exchange (NHE), which in itself could potentiate cell growth.9Lurbe A Fioretto P Mauer M LaPointe MS Batlle D Growth phenotype of cultured skin fibroblasts from patients with insulin-dependent diabetes mellitus with and without nephropathy and its association to overactivity of the Na+/H+ antiporter.Kidney Int. 1996; 50: 1684-1693Google Scholar It is interesting that in cultured skin fibroblasts from patients with Type 2 diabetes with DN, the Vmax for NHE is increased as well.14Trevisan R Cipollina MR Duner E Trevisan M Nosadini R Abnormal Na+/H+ antiport activity in cultured fibroblasts from NIDDM patients with hypertension and microalbuminuria.Diabetologia. 1996; 39: 717-724Google Scholar This finding suggests a common denominator for the development of nephropathy in Type 1 and Type 2 diabetes. In this respect, NHE overactivity may be a marker of nephropathy for both types of diabetics. Using the skin fibroblast model, Podestá et al15Podestaá F Meregalli G Ghelardi R Del Giudice R Asnaghi V Maestroni A Zerbini G Low Ca2+ pump activity in diabetic nephropathy.Am J Kidney Dis. 2001; 38: 465-472Abstract Full Text Full Text PDF Scopus (9) Google Scholar confirm in this issue of the Journal that cells from patients with Type 1 diabetes with DN exhibit elevated Vmax of the Na+-H+ antiporter. They further report that Ca2+-pump-mediated Ca2+ efflux was lower in cells from patients with DN than in patients with normoalbuminuria and nondiabetic subjects. Neither cytosolic Ca2+ concentrations nor intracellular pH (pHi) were altered, despite the alterations in the aforementioned ion transporters. Measurable alterations in pHi and Ca2+ may not be present as other transporters, such as the Cl− /HCO3− and the Na+-Ca2+ exchangers, may compensate16Godinich M LaPointe M Batlle D Free cytosolic Ca2+ regulation via Na+/Ca2+ exchange in vascular smooth muscle cells.Ann N Y Acad Sci. 1991; 639: 556-561Google Scholar, 17Redon J Batlle D Regulation of intracellular pH in the spontaneously hypertensive rat. Role of bicarbonate-dependent transporters.Hypertension. 1994; 23: 503-512Google Scholar, 18Stakisaitis D LaPointe MS Batlle D Mechanisms of chloride transport in thymic lymphocytes.Am J Physiol Renal Physiol. 2001; 280: F314-F324Google Scholar and thereby prevent alterations in pHi and cytosolic Ca2+, respectively. Unlike those used in previous studies,9Lurbe A Fioretto P Mauer M LaPointe MS Batlle D Growth phenotype of cultured skin fibroblasts from patients with insulin-dependent diabetes mellitus with and without nephropathy and its association to overactivity of the Na+/H+ antiporter.Kidney Int. 1996; 50: 1684-1693Google Scholar, 10Davies JE Ng LL Kofoed-Enevoldsen A Li LK Earle KA Trevisan R Viberti G Intracellular pH and Na+/H+ antiport activity of cultured skin fibroblasts from diabetics.Kidney Int. 1992; 42: 1184-1190Google Scholar, 11Trevisan R Li L Messent J TariQ T Earle K Walker J Viberti G Na+/H+ antiport activity and cell growth in cultured skin fibroblasts of IDDM patients with nephropathy.Diabetes. 1992; 41: 1239-1246Google Scholar, 19Ng LL Davies JE Siczkowski M Sweeney FP Quinn PA Krolewski B Krolewski AS Abnormal Na+/H1 antiporter phenotype and turnover of immortalized lymphoblasts from type 1 diabetic patients with nephropathy.J Clin Invest. 1994; 93: 2750-2757Google Scholar the cells used in the study by Podestaá et al15Podestaá F Meregalli G Ghelardi R Del Giudice R Asnaghi V Maestroni A Zerbini G Low Ca2+ pump activity in diabetic nephropathy.Am J Kidney Dis. 2001; 38: 465-472Abstract Full Text Full Text PDF Scopus (9) Google Scholar were grown in normal glucose media (5 mmol/L) and assayed in the sixth or seventh passage. It has been shown that a high glucose media exaggerates the difference in NHE activity in immortalized lymphoblasts from patients with Type 1 diabetes with DN and from controls.20Davies JE Siczkowski M Sweeney FP Quinn PA Krolewski B Krolewski AS Ng LL Glucose-induced changes in turnover of Na+/H+ exchanger of immortalized lymphoblasts from type I diabetic patients with nephropathy.Diabetes. 1995; 44: 382-388Google Scholar Thus, the use of normal glucose in this study seems to rule out the possibility that cells from patients with DN are simply more sensitive to a high glucose media than are cells from patients with normoalbuminuria and from normal controls. Restriction of the number of times the cells are passaged is also important because the phenotype could be lost after several passages. For instance, cultured vascular smooth muscle from SHR and WKY rats show differences in NHE activity and proliferation rates that were only present in early passaged cells (before the sixth passage). Differences were not evident when later passaged cells were used.21Berk BC Vallega G Muslin AJ Gordon HM Canessa M Alexander RW Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.J Clin Invest. 1989; 83: 822-829Google Scholar, 22LaPointe M Ye M Moe O Alpern R Batlle D Na+/H+ antiporter (NHE-1 isoform) in cultured vascular smooth muscle from the spontaneously hypertensive rat.Kidney Int. 1995; 47: 78-87Google Scholar Moreover, skin fibroblasts from patients with Type 1 diabetes with DN have been shown to undergo premature senescence.23Vracko R Benditt EP Restricted replicative lifespan of diabetic fibroblasts in vitro: Its relation to microangiopathy.Fed Proc. 1975; 34: 68-70Google Scholar, 24Goldstein S Moerman EJ Soeldner JS Gleason RE Barnett DM Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts.J Clin Invest. 1979; 63: 358-370Google Scholar, 25Morocutti A Earle KA Sethi M Piras G Pal K Richards D Rodemann P Viberti G Premature senescence of skin fibroblasts from insulin-dependent diabetic patients with kidney disease.Kidney Int. 1996; 50: 250-256Google Scholar The requirement for limiting the number of cell passages, however, greatly restricts the number of cells available for study. Coupled with the requirement for pairing the subjects based on age, sex, disease duration, and other factors, these types of studies using cultured human skin fibroblasts are demanding and should be commended. The subjects used in the 3 study groups were well matched for age, duration of disease, body mass index, male/female ratio, and daily insulin dose. There were, however, important clinical features that differed significantly between the subject groups. HbA1c values were significantly increased in patients with DN, indicating poorer glycemic control. This is a common feature of diabetic patients with DN, and it is hard to control for this feature. Adjustment for glycated hemoglobin and plasma lipids, however, did not alter the findings of the study. Another striking and potentially confounding in vivo variable was that 16 of the 20 patients with DN also suffered from hypertension. Although the authors found no direct correlation between Ca2+ efflux and/or NHE activity and mean blood pressure, this does not exclude that the findings of altered activities of NHE and the Ca2+-pump in patients with DN could, nevertheless, reflect a predisposition to hypertension. Determination of this would require additional studies using patients with DN who had not developed hypertension or cells from nondiabetic hypertensive subjects as controls. The remaining question is: What is the underlying molecular mechanism by which seemingly diverse aspects in the cell biology of cultured skin fibroblasts become altered? The cause of the increased NHE activity in patients with Type 1 diabetes with DN is not known, although studies indicate that it is not due to polymorphisms of NHE-126Doria A Warram JH Krolewski AS Genetic susceptibility to nephropathy in insulin-dependent diabetes: From epidemiology to molecular genetics.Diabetes Metab Rev. 1995; 11: 287-314Google Scholar or to increased protein expression.27Siczkowski M Davies JE Sweeney FP Kofoed-Enevoldsen A Ng LL Na+/H+ exchanger isoform-1 abundance in skin fibroblasts of type I diabetic patients with nephropathy.Metabolism. 1995; 44: 791-795Google Scholar This would suggest that the increase in NHE activity is possibly due to a posttranslational modification, possibly via an alteration in Ca2+-dependent second messenger systems. Reduced Ca2+ pump activity in skin fibroblasts from patients with DN is apt to impact not only on cytosolic Ca2+, but also on pHi. The Ca2+ pump acts as a Ca2+-H+ exchanger and provides not only a pathway for cellular Ca2+ exit, but also a pathway for cell acidification.28Daugirdas JT Arrieta J Ye M Flores G Batlle DC Intracellular acidification associated with changes in free cytosolic calcium: Evidence for Ca2+/H+ exchange via a plasma membrane Ca2+ ATPase in vascular smooth muscle cells.J Clin Invest. 1995; 95: 1480-1489Google Scholar Thus, a decrease in Ca2+ pump activity would be anticipated to cause increased cytosolic Ca2+ levels and decreased H+ levels (ie, an increase in cell pH). A higher pH inside the cells would tend to inhibit NHE activity. Thus, a decrease in Ca2+ pump activity cannot explain an increase in the Vmax of the Na+-H+ antiporter via alterations in pHi. On the other hand, a decrease in Ca2+ pump activity could cause an increase in cytosolic Ca2+ that could then activate Ca2+-dependent protein kinases or calmodulin binding and thus increase the Vmax for NHE. The finding that there was an inverse correlation between the Vmax for NHE and Ca2+ pump activity in the study by Podestaá et al15Podestaá F Meregalli G Ghelardi R Del Giudice R Asnaghi V Maestroni A Zerbini G Low Ca2+ pump activity in diabetic nephropathy.Am J Kidney Dis. 2001; 38: 465-472Abstract Full Text Full Text PDF Scopus (9) Google Scholar supports this possibility. Such a correlation, however, does not imply a cause and effect. Both features could be the result of upstream signaling alterations or be totally unrelated. In evaluating a study such as this, one has to consider whether the findings were caused by inherent genetic differences between the patient groups or whether the differences observed were triggered by in vivo environmental factors such as metabolic control. In this and other studies, the cultured cells were grown and assayed under identical environmental conditions, which strongly suggests the presence of a genetic difference between patients susceptible to DN and those who are protected. The possibility cannot be ruled out that a permanent change in gene expression through modifications of the genome has occurred in vivo. In any case, it is remarkable that cultured skin fibroblasts display phenotypic features that are potentially useful for identifying diabetic subjects susceptible to the development of diabetic nephropathy. Low Ca2+ pump activity in diabetic nephropathyAmerican Journal of Kidney DiseasesVol. 38Issue 3PreviewElevated cell Na+-H+ exchange (NHE) activity characterizes diabetic nephropathy (DN), but the mechanisms of this abnormality are unclear. Recent evidence suggests that NHE and the Ca2+ pump share similar regulatory pathways, but whether abnormalities in Ca2+ metabolism characterize DN is not known. We investigated Ca2+ efflux rates, NHE activity, cytosolic Ca2+ ([Ca2+ ]i) concentrations, and intracellular pH (pHi) in human skin fibroblasts from 20 patients with type 1 (insulin-dependent) diabetes and nephropathy; 20 patients with diabetes with normoalbuminuria matched for age, sex, and duration of diabetes; and 10 individuals without diabetes. Full-Text PDF