NEFROLOGIA. Val. XIV. Núm. 3. 1994 EDITORIAL Cell biology of Diabetic Nephropathy E. N. Wardle, MD, MRCP Oxford. Reino Unido Introduction 40 % of patients with juvenile insulin dependent diabetes mellitus (IDDM) develop nephropathy within 10-20 years of diagnosis 1, 20 % of middle aged, noninsulin dependent diabetes mellitus (NIDM) patients a r e f o u n d t o have n e p h r o p a t h y o n l y 5-10 years after the initial diagnosis 2 . Generally the patients with early proteinuria are those who have had poor glucose control 3, a slightly higher blood pressure (135: 85 mm Hg) and more marked retinopathy. They are those who soon have an increase of their GFR above normal and so they have hyperfiltration. They tend also to have a higher cholesterol, a higher LDL and VLDL, and elevation of their plasma fibrinogen 4. After 5-10 years of diabetes we might detect microalbuminuria, which means an albumin excretion rate of 20-200 micrograms/minute in an overnight urine, or 30-300 mg/24 hrs in a complete collection. Then after another 5-10 years they will develop albumin stick positive proteinuria. The clinical sequence of the development of diabetic nephropathy was described in detail by Castiglioni and Savazzi 5. When biopsies are obtained from diabetic patients, the changes that are typical are ' the hyalinosis of the afferent and efferent arterioles of the glomeruli 2 , the thickening of the basement membranes of the glomerular capillaries 6 , and3 the increased volume of mesangial cells leading to an increase in volume of glomeruli. Once the expansion of the mesangium is more than 37 % of the glomerular volume, there is pressure on the capillaries with loss of filtration surface 7, so that thereafter there is functional renal impairment. Ultimately there is closure and obsolence of glomeruli, called glomerulosclerosis. Those patients with severe glomerular lesions have hypertension and reduced creatinine clearance, and there is renal interstitial fibrosis. There is evidente that this fibrosis develops concurrently with the increased fractional mesangial volume 8. Clomerular Capillary Hipertension and Hyperfiltration In rats that are made diabetic by means of streptorotocin and kept alive by injections of insulin there is a decreased resistance of the afferent arterioles, so that there will be increased pressure in the glomerular capillaries and there is hyper-filtration 9 , which is determined by the increase in plasma flow. The factors that determine hyperfiltration have been discussed by Bank 10 . Hyperglycaemia is a first consideration 11 but so is a high protein intake 12. A very high glucose (20 mM) inhibits cytosolic calcium signaling in cultured mesangial or vascular smooth muscle cells 11b.Hence there is vasodilatation. Sodium intake is also relevant. During hyperglycaemia induced osmotic flow, there is increased reabsorption of sodium in the proximal tubules 13 . In subjects who are using insulin, insulin is known to increase sodium reabsorption in the tubules 14. The pituitary of diabetic patients secretes large pulses of growth hormone, possibly as a result of raised glucose in the CSF, and growth hormone increases GFR and renal blood flow 15 by the intermediary of insulin growth factor IGF-1 16 , In any case there is increased formation of nitric oxide vasodilator in the afferent arterioles in diabetes 17. The hyperglycaemia also results in production of vasodilator prostaglandins in the afferent artérioles 18 . This production of prostaglandins seems to depend on polyol formation, because it is arrested 19. when an aldose reductase inhibitor is used Hyperglycaemia determines also an increased production of thromboxanes at the vascular pole of diabetic kidneys and thus an increased tone in the efferent arterioles 10 . Indeed one knows that urinary thromboxane excretion is increased 10, and that proteinuria is ameliorated in animals given thromboxane synthetase inhibitor 20 Yet another consideration is that hyperglycaemia leads to formation of excess diacylglycerol withi cells and thus there is activation 257 E. N. WARDLE of protein kinase C. Excess protein kinase C is known to mediate vascular permeability of the endothelium of blood vessels 21, In fact it mediates down-regulation of thromboxane receptors in diabetic glomeruli and mesangial cells 22, Angiotensin II receptors are likewise downregulated. In all this will explain the predominant vasodilatation in the afferent arterioles and so the hyperfiltration. The data in humans indicates some relation between early hyperfiltration and the development of diabetic renal disease. Thus in one study of children a GFR in excess of 125 ml/min 1.73 m2 conferred a predictive value for nephropathy of 53 % 23 . Yet one must note that in that study half of the hyperfiltering subjects did not develop nephropathy during the period of follow-up. Thus hyperfiltration is not a highly sensitive predictive parameter. N I D D M patients also show hyperfiltration 2 4 . Itis common in Pima Indians 25. It has been suggested that the patients who progress to diabetic nephropathy are those with a family predisposition to hypertension 26. Hypertension will raise intracapillary pressures and worsen proteinuria. In some populations genetic susceptibility to essential hypertension can be linked to increased erythrocyte sodium-lithium counter-transport. There is a similar link with diabetic nephropathy 27. However one must emphasize that this does not apply in all populations that have been studied 28 . There has to be a metabolic component to the hypertrophy that stems from the hyperglycaemia. Thus it has now been demonstrated that in the kidneys of diabetic rats there is an increase of glucosylceramide, a glycosphingolipid precursor. Furthermore an inhibitor of glucosyl-ceramide prevents the renal hypertrophy 39 . Vascular Changes in Diabetes Hyalinosis of blood vessels is recognised as an adverse feature. It means that the vessel walls are permeable to molecules as large as fibrinogen. It implies that glomerulosclerosis will develop 40. When it occurs leakiness of the postglomerular peritubular capiIlaries leads to protein accumulation in the interstit i u m of the kidneys 4 1 T h a t will lead to renal . interstitial fibrosis. The process is probably facilitated by iron that comes from transferrin in the urine of diabetics 42. What causes increased endothelial cell permeability in diabetes? Firstly metabolism of glucose to diacylglycerol that mediates the formation of excess protein kinase C has been mentioned 21. Secondly a role for conversion of glucose to sorbito1 via aldose reductase enzyme is probable, because the inhibitor sorbinil can reduce vascular permeability 43 . Thirdly there is a definite role for lipid peroxides, which arise when there is free radical formation linked to nonenzymic glucosylation of proteins 4 4 - 4 5 . The freeradicals arise from auto-oxidation reactions of sugars and sugar adducts to proteins and by auto-oxidation of unsaturated lipids adjacent to altered membrane prot e i n s . Monocytes of diabetics often produce superoxide anions and thus hydrogen peroxide 4 6 . Polymorphonuclear leucocytes in serum that is high in cholesterol also form superoxide anions 47. It is usual for the low density lipoproteins of diabetics to become oxidised 48 . Apart from that one can gauge free radical activity in diabetic rats by their expiration of pentane 4 9 , and one canmeasure plasma lipid peroxides and lipid peroxidation of kidneys in diabetic rats 50, In human studies severa1 groups have now related lipid peroxides in plasma 51 (measured by thethiobarbituric acid reaction) to endothelial cell damage as shown by a raised plasma von Willebrand factor. The patients with raised plasma malonyldialdehyde (indicative of lipid peroxides) and raised plasma von Willebrand factor were also those who had microalbuminuria 52-54. Furthermore patients with raised plas. ma MDA were those with tubular damage as shown by increased excretion of NAG, N-acetyl-glucosaminidase, in their urine 54. When sought in the correct Hypertrophy of the Kidneys Soon after the onset of diabetes the size of the kidneys increase. All the glomeruli and their nephrons hypertrophy 29. In the rat this hypertrophy precedes the increase of GFR 3 0 . The finding of increasedprotein kinase C activity in the glomeruli is relevant to growth 31. Both hyperglycaemia and the growth factors work via pkC activation. It has been shown that the action of EGF, epidermal growth factor, works through the intermediary of increased protein kinase C activity 32. EGF is produced in the distal tubules and ascending loops of Henle 33, There is increased excretion of EGF in the urine of streptozotocin diabetic rats 3 4 , The effects of growth hormone are known to be mediated by IGF-1 and this is anabolic and it is involved in hypertrophy of diabetic kidneys 35. In short term studies the somatostatin analogue, octreotide, which suppresses growth hormone secretion, stops the early renal hypertrophy of diabetic rats 3 6 . Yet in the longterm it does not 37. Hyperglycaemia stimulates production of TGFbeta in cultured proximal tubules 38 .It is therefore possible that it could contribute to hypertrophy of the tubules, albeit how TGFbeta behaves depends on the phase of development. 258 E. N. WARDLE manner, marked evidence of tubular damage will be found in many diabetics 55,56. Oxidised LDL has altered properties 57 that are pertinent to atherogenesis 58. Oxidised LDL is chemotactic for monocytes and it is taken up by macrophages in arterial walls to cause their cholesterol enrichment and foam cell formation. Oxidised LDL causes platelet aggregation. It stops the action of nitric oxide and so it can promote vasoconstriction in small arterioles. Oxidised LDL can be cytotoxic for endothelial cells. In lesser doses it causes the expression of «tissue factor» thromboplastin on endothelial cells 59, so that there is a thrombotic tendency in small arterioles. It also prevents activation of protein C and so it thwarts protective fibrinolysis. The severe atheroma that many diabetics develop has implications for kidney function. For example platelet aggregates forming in the aorta will be swept into the glomeruli to cause lesions like focal segmental sclerosis 60. Also many diabetics do have renal artery stenoses. Recognition of this fact is necessary before the prescription of ACE inhibitors. Non-enzymatic glucosylation of proteins produces ACEP, advanced glycosylation end-products 45. They accumulate in the tissues of diabetics 61 and will undoubtedly play a role in nephropathy. Mesangial cells express AGEP receptors 6 2 and when stimulated by AGEP they form basement membrane proteins 63. AGEP have oxidising potential 64, and when they accumulate in blood, as they do in terminal renal failu6 5 they encourage procoagulant change on the endothelial cells 66, both directly and via release of cytokines like ll-1 and TNFa 67. Glycosaminoglycans and Collagen There is poor synthesis of heparan sulphate proteoglycans by the renal glomeruli in diabetes mellitus 68. There is a genetic factor involved in different strains of rats 71, and it has also been suggested that the varying liability to diabetic vascular disease in humans 69 m i g h t also d e p e n d on some factor like this. Hyperglycaemia also stops proteoglycan synthesis by mesangial cells 7 0 . When there is poor diabetic control there is inhibition of the N-acetyl heparan deacetylase enzyme that is required for heparan sulphate proteoglycan synthesis. Furthermore there is loss of heparan sulphates in the urine at the onset of diabetes in rats 72. When biopsies from human diabetic nephropathy were examined, a marked reduction in reactivity to anti-heparan sulphate proteoglycan antibodies was observed, but one has to acknowledge that these were cases of quite advanced disease 73. The importance of the heparan sulphates is that the 260 negative charges of their sulphate groupings on endothelial cells, and in the basement membranes of the glomeruli and on the mesangial cells, repel negatively charged albumin molecules, so that their filtration is prevented. So it would seem that loss of heparan sulphates at such an early stage in diabetes 74 could account for the onset of micro-albuminuria. Indeed Gambaro et al. 75 have show.n that, when streptozotocin diabetic rats are given injections of either low MW heparin or dermatan sulphate glycosaminoglycan, there was inhibition of mesangial cell expansion and thickening of the glomerular basement membranes was reduced. The heparan sulphates of the basement membrane are essential for the integrated binding of the other components like the type IV collagen and laminin. Another factor that contributes to proteinuria must also be the poor synthesis in diabetes 76 of the negatively charged sialoproteins that line the slit pores between the epithelial cells. The basement membrane width expands by about 30 % during the first 5 years of diabetes and by the time of clinical nephropathy its width has doubled 6 . Not only is there loss of negative charges 73 but the closely woven structure must be disorganised, perhaps by the addition of glucoadducts in the process of non-enzymic glucosylation 61,77-79, surely by the effect of ACE products causing collagen browning 80 , and surely as a result of the loss of heparan sulphate proteoglycans 81. When in an experimental situation a m i n o g u a n i d i n e is used to decrease AGE products 8 2 , the proteinuria is reduced 83 . A high ambient glucose (30 mM) increases the synthesis of type IV collagen by cultured endothelial, mesangial and epithelial cells 84-85. Undoubtedly the ability of high glucose to drive protein kinase C may explain this 86. However one should also be aware that lipid peroxidation enhances the synthesis of type I V collagen Also it can be shown in vivo that thromboxanes play a role, because when thromboxane synthase inhibitors are used basement membrane thickening and mesangial matrix expansion is reduced 87,88. The basic fact that high ambient glucose c a u s e s increases messenger RNA for type IV collagen 89,90 has now been verified by many groups. Likewise proximal tubules that are exposed to glucose will synthesise type IV collagen of tubular basement membranes 91, It is reported that sorbinil prevents the biosynthesis 91. Clearly the altered structure of the GBM explains the albuminuria and the loss of charge selectivity that can now be measured by study of the clearance of IgG/lgG4 92,93. As can be shown by dextran clearances, there is initially no increase of pore size at a time when there is substantial loss of albumin and IgG, E. N. WARDLE We know from histology that glomerulosclerosis (mesangial sclerosis) will ultimately develop and that follows increasing deposition of fibronectin and type IV collagen. High glucose leads to loss of proteoglycans 111 and it promotes formation of fibronectin and collagen 9 7 - 9 8 , albeit one set of studies showed that over a long time high glucose suppresses collagen The Altered Mesangia of Glomeruli of Diabetics production 111. That might be due to ascorbic acid deThe feature that is so typical of diabetes is the early pletion, since that is well recognised in diabetes. Certainly it seems that hyperlipidaemia will mediamesangial cell expansion that is followed years later by mesangial sclerosis 5 , 7 . Thus the picture differs t e glomerulosclerosis, as in other situations 112. from the glomerulonephritides in which one often exImmunohistochemical studies of the localisation of apolipoproteins in glomeruli has shown that fixation pects to see mesangial cell proliferation that is cauof apolipoprotein B with apoE gives rise to more glosed by growth factors 96. By now one can list various merulosclerosis and interstitial scarring 1 1 3 . experimental observations that explain why the mesangial cells do not proliferate. 1) A high ambient What more does one need to know? Since mice glucose (20 mM) inhibits mesangial cell proliferation, transgenic for bovine growth hormone1 1 4 develop although it does promote fibronectin synthesis 9 7 . 2) mesangial cell proliferation by 4 weeks, mesangial Although a high glucose increases protein kinase C in sclerosis by 20 weeks and glomerulosclerosis by 36 mesangial cells 9 8 and thus formation of extracellular weeks, what are the mediators of the response? matrix fibronectin, laminin and type IV collagen, Presumably PDGF autocrine production in glomeruli is involved at the early stage 110. Since TGFbeta is the high glucose also inhibits cytosolic calcium signa9 9 . Hence the cells will tend to be relaxed and Iling mediator of glomerulosclerosis in most situations, one has to assume that this is also the case in diabespread out rather than contractile. 3) Non-enzymatic tic nephropathy 110, Indeed it does seem that high gluglucosylation and formation of ACE products causes mesangial expansion and inhibition of mesangial cell cose stimulates autocrine production of TGFbeta by mesangial cells 1 0 4 . p r o l i f e r a t i o n 4) On account of the aldose reductase content of the mesangia, exposure to high glucose Furthermore the release of TGFbeta stops mesanw i l l result in formation of polyols 101 and there will be gial cell proliferation but increases the deposition of mesangial matrix 115. a reduction of the myoinositol of the cells 102. 5) When there is a high glucose, prostaglandin production by mesangial CelIs is increased 103 Prostaglandins Final Synopsis inhibit cell oroliferation 9 6 . Potential high glucose induced mesangial cell proliferation is inhibced also by By now it has been shown in rats with diabetic nephropathy and in humans that TGFbeta values are transforming growth factor beta 1 0 4 . 6) Highglucose can inhibit the cell proliferative effect of ICF-1 105 . 7) elevated in the glomeruli 115 . Thisis a clear indication Low density lipoproteins at a concentration of only that TGFbeta mediates glomerulosclerosis. 10 g/ml stimulate proliferation of mesangial cells and Secondly Cohen and Ziyadeh 117 have examined yet at a level of 1 0 0 - 5 0 0 g/ml, as would be the case the effects of glycosylated proteins on glomerular mesangial cells. They have shown that glycosylated in any hyperlipidaemia, there is inhibition 106. Such proteins (i) stop proliferation of mesangial cells, and LDL will stimulate superoxide production by mesangial cells and thus may become oxidised 107 . 8) (ii) stimulate the mesangial cells to transcribe the geOxidised LDL reduces release of growth factors from nes for type IV collagen. So growth of mesangial cells is inhibited, as was recorded by Crowley et macrophage like cells 1 0 8 . Oxidised LDL bind very well to mesangial cells and inhibit their proliferation al.100, and type IV collagen production occurs in dia109. betic glomeruli. One should note that when glycated at concentrations as low as 10-25 g/ml Although these observations make good biochemiproteins are taken up by mesangial cells, there is incal sense, they are mainly based on in vitro studies. tracellular production of hydrogen peroxide 64,118 When RNA messengers are looked at in the early staWe do know that lipid peroxidation is a stimulus to ges of diabetes in Sprague-Dawley rats those for collagen production 6 6 , In fact Cohen and Ziyadeh TNFalpha, basic fibroblast growth factor and PDGF-B used glycated serum proteins that had no cross-links chain and for transforming growth factor beta are inand showed no AGE fluorescente, whereas Crowley creased 110. Indeed their levels are reduced by insulin et al. 1 0 0 specifically used fluorescent cross-linked AGE products. Thus either product can influente the therapy 110. Nevertheless rats are not as hyperlipaemic as man might be. genes for collagen. 262 but pore size is increased later. It is relevant to note that glycated albumin readily leaks through the glomeruli and it is not reabsorbed by the proximal tubules 94. In any case.there is often proximal tubule dysfunction as shown by lysozymuria 95. CELL BIOLOGY OF DIABETIC NEPHROPATHY Thirdly there is more information on the contraversial topic of whether or not nitric oxide production by endothelium is increased o decreased in diabetes. The answer is that NO production can be increased or decreased depending on the vascular bed. Nitric oxide production is increased in the afferent arteriales of diabetic glomeruli and thus this is a factor that explains vasodilatation and hyperfiltration. On the other hand it has been shown that carbamvlcholine induced cyclic GMP is decreased within isolated diabetic glomeruli in parallel with hyperglycaemia induced increases of protein kinase C 119. Furthermore in that situation thromboxane formation is increased88 and this is another factor 120 that contributes to production of glomerular extraceIlular matrix proteins. Hence hyperglycaemia causes non-enzymatic glycosylation of proteins and they reduce mesangial cell proliferation but cause mesangial expansion and type IV collagen production. Also hyperglycaemia induced protein kinase C, and hence thromboxanes, cause matrix deposition. References 1 . Anderson AR, Christiansen JS, Anderson JK, Kreiner S and Deckert T: Diabetic nephropathy in type I diabetes: an epidem i o l o g i c a l study. Diabetologia 25:496-451, 1983. 2. Tung P and Levin SR: Nephropathy in non-insulin dependent diabetes mellitus. AmerJ Med 85 (suppl 5A):1-9, 1988. 3. Feld-Rasmussen B, Mathieson ER, Jensen T, Lauritzen T and D e c k e r t T: Effect of improved metabolic control on loss of kidney function in type I diabetic patients: an update of the Steno studies. Diabetologia 34:164-70, 1991. 4. Wardle EN, Piercy JS and Anderson J: Chemical parameters of diabetic vascular disease. Postgrad Med J 49:1-9, 1973. 5 . Castiglioni A and Savazzi GM: Physiopathology and clinical aspects of diabetic nephropathy. Nephron 50:151-1463, 1988. Walker JD, Close CF, Jones SL, Raftery M, Keen H et al: G l o m e r u l a r structure in type I insulin dependent diabetic pat i e n t s with normo and microalbuminuria. Kidney Int 41 :7418, 1992. 7 . St e f f e s MW, Osterby R , C h a v e r s B and M a u e r , S M : Mesangial expansion as a central mechanism for loss of kidn e y function in diabetic patients. Diabetes 38:1077-81, 1989. Lane PH, Steffes MW, Fioretto P and Mauer SM: Renal interst i t i a l expansion in insulin dependent diabetes mellitus. K i d n e y Int 43:661-7, 1993. H a s i m o t o Y, Ideura T, Yoshimura A and Koshikawa S: Autoregulation of renal blood flow in streptozotocin induced diabetic rats. Diabetes 38:1109-1113, 1989. B a n k N: Mechanisms of diabetic hyperfiltration. Kidney Int 40:792-807 1991. W i s e m a n J, M a n g i l i R , A l b e r e t t o M a n d V i b e r t i G C : G l o m e r u l a r response mechanisms to glycaemic changes in insulin dependent diabetes. Kidney Int 31 :1012-28, 1987. M e n e P, Publiese G, Pricci F, DiMario U, Cinotti GA and Pugliese F: High glucose inhibits citosolic calcium signaling in cultured ras mensagial cells. 1 2 . Wiseman MJ, Bugnetti E, Dodds R, Keen H and Viberti GC: Changes in renal function in response to protein restricted diet in type I diabetic patients. Diabetes 30:154-9, 1987. 13. Woods LL, Mizelle HL and Hall JE: Control of renal hemodyn a m i c s in hyperglycemia: possible role of tubuloglomerular f e e d b a c k . AmerJ Physiology 257:F65-F73, 1987. 1 4 Skott P, Hother-Nielsen 0 and Brun NE: Effects of insulin on k i d n e y function and sodium excretion in diabetic subjects. Diabetologia 32:694-699, 1989. 15 Töushoff B, Nowack R, Kurilenko S, Blum WF, Seyberth HW, Mehls 0 and Ritz E: Growth hormone induced glomer u l a r hyperfiltration is dependent on vasodilating prostanoids. Amer J Kidney Dis 21 :145-151, 1993. 1 6 . H i r s c h b e r g RR and Kople JD: Increase in renal blood flow and GFR during growth hormone treatment may be mediated b y insulin growth factor 1. Amer J Nephrology 8:249-253, 1988. 17. B a n k N and Aynedjian HS: Role of endothelium dependent relaxing factor (nitric oxide) in diabetic renal hyperfiltration. Kidney Int 43:1306-12, 1993. 18. Craven PA, Caines MA and DeRubertis FR: Sequential alterations in glomerular prostaglandin and thromboxane synthesis in diabetic rats: relation to hyperfiltration of early diabetes. Metabolism 36:95- 103, 1987. 1 9 Craven PA and DeRubertis FR: Sorhinil suppresses glomerul a r prostaglandin production and reduces hyperfiltration in the streptozotocin diabetic rat. Clin Res 36:517a, 1988. 2 0 Craven PA and DeRubertis FR: Suppression of urinary albumin excretion in diabetic rats by 4 (imidazolyl) acetophenone, a selective inhibitor of thromboxane synthesis. J Lab Clin Med 116:469-478, 1990. 21. Lynch JJ, Ferro TJ, Blumenstock FA, Brockenauer AM and M a l i k AB: Increased endothelial albumin permeability mediated by protein kinase C activation. J Clin Invest 85:19911998, 1990. 22. Studer RK, Craven PA and DeRubertis FR: Activation of prot e i n kinase C reduces thromboxane receptors in glomeruli and mesangial cells. Kidney Int 44:58-64, 1993. 23. Rudberg S, Persson B and Dahlquist G: Increased glomerular filtration rate as a predictor of diabetic nephropathy. Kidney Int 41:822-828, 1992. 24. Vora JP, Dolben J, Dean JJ, Thomas D, Williams JD et al: R e n a l hemodynamics in newly presenting non-insulin dependent diabetes mellitus. Kidney Int 41:829-35, 1992. 25. Myers BD, Nelson GR, Williams GW, Bennett PH, Hardy SA et al: Glomerular function in Pima Indians with non-insulin d e p e n d e n t diabetes mellitus of recent onset. J Clin Invest 88:524-530, 1991. 26. Krolewski AS, Canessa M, Warran JH, Laffel L, Christlieb AR et al: Predisposition to hypertension and susceptibility to ren a l disease in insulin dependent diabetes mellitus. New England J Med 318:140-5, 1988. 2 7 . Trevisan R, Nosadini R, Fioretto P, Semplicini A, Donadon V et al: Clustering of risk factors in hypertensive insulin depend e n t diabetes with high sodium-lithium countertransport. Kidney Int 41:855-861, 1992. 28. Jensen JS, Mathiesen ER, Norgaard K, Hommel E, BorchJ o h n s e n KI et al: Increased blood pressure and sodium/lit h i u m countertransport are not inherited in diabetic nephropathy. Diabetologia 33:619-24, 1990. 29. Seyer-Hansen K: Renal hypertrophy in experimental diabetes mellitus. Kidney Int 23:643-646, 1983. 30. Cortes P, Dumler F, Goldman J and Levin NW: Relation betw e e n renal function and metabolic alterations in early strept o z o t o c i n induced diabetes in rats. D i a b e t e s 36:80-87, 1987. 263 E. N. WARDLE 31. Craven PA and DeRubertis FR: Protein kinase C is activated in the glomeruli of streptozotocin diabetic rats. J Clin Invest, 83:1667-75, 1989. 32. Albert i P, Bardella L and Comolli R: Ribosomal protein S6 kinase and protein kinase C activation by EGF after renal isc h a e m i a . Nephron 64:296-302, 1993. 3 3 . Hammerman MR, O'Shea M and Miller SB: Role of growth factors in regulation of renal growth. Ann Review Physiol 55:305-21, 1993. 34. Guh JY, Lai YH, Shin SJ, Chuang LY and Tsai JH: Epidermal growth factor in renal hypertrophy in streptozotocin diabetic rats. Nephron 59:641-7, 1991. 3 5 . Muchaneta-Kubara EC and El Nahas AM: Role of insulin growth factor I in expemental diabetic ne Nephrol Dial Transplant 7(7):663 abst., 1992. 36. Flyvberg A, Frystyk J, Thorlacius-Ussing O and Orskov H: S o m a t o s t a t i n analogue administration prevents increase in k i d n e y somatomedicin C and initial renal growth in diabetic and uninephrectomised rats. Diabetologia 32:261-5, 1989. 37. Muntzel M, Hannedouche T, Niesor R, Nole LH, S o u b e r b i e l l e JC, Lacour B and Drüeke T: Long term effects of a somatostatin analogue on renal haemodynamics and hyp e r t r o p h y in diabetic rats. Clinical Science 83:575-581, 1992. 38. Rocco MV, Chen Y, Goldfarb S and Ziyadeh FN: Elevated glucose stimulates TGFbeta gene expression and bioactivity in proximal tubules. Kidney Int 41 :107-114, 1992. 39. Zador IZ, Deshmukh GD, Kunkel R, Johnson K, Radin NS and Shayman JA: A role for glycosphingolipid accumulation in the renal hypertrophy of streptozotocin induced diabetes mellitus. J Clin Invest 91:797-803, 1993. 40. Bader R, Bader H and Grund KE: Structure and function of the kidney in diabetic glomerulosclerosis. Correlations betw e e n morphologic and functional parameters. PathoI Res Pract 167:204-216, 1980. 41. Pinter GG and Atkins JL: Role of post-glomerular microvess e l s in the pathophysiology of diabetes mellitus. D i a b e t e s 40:791-795, 1991. 4 2 . Howard RL, Buddington B and'Alfrey C: Urinary albumin, t r a n s f e r r i n and iron excretion in diabetic patients. Kidney /nt 40:923-6, 1991. 43. Cunha-Vaz JG, Mota CC, Leite EC, Abreu JR and Ruas MA: Effects of sorbinil on the blood retina1 barrier in early diabetic retinopathy. Diabetes 35:574-8, 1986. 44. Baynes JW: Role of oxidative stress in development of complications in diabetes. Diabetes 40: 405-413, 1991. 45. Bucala R and Cerami A: Advanced glycosylation: chemistry, b i o l o g y and implications for diabetes and ageing. A d v Pharmacology 23:1-34, 1992. 4 6 . Noritake M, Katsura Y, Shinomiya N, Kanatani M and Uwabe Y: Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Clin Exp Immuno/ogy 88:269-274, 1992. 4 7 . Ludwig P, Hunninghake DG and Hoidal JR: Increased leuc o c y t e o x i d a t i v e m e t a b o l i s m in hyperlipoproteinaemia. Lancet ii:348-50, 1987. 48. Lyons TJ: Oxidised low density Iipoproteins: a role in the pat h o g e n e s i s of atherosclerosis in diabetes. Diabetic Medicine 8:41 1-9, 1991. 49. Pitkanen OM, Martin JM, Hallman M, Akerblom HK, Sariola H and Andersson S: Free radical activity during development of diabetes in the rat. Life Science X):335-9, 1992. 5 0 . Tomino Y, Wang LN, Fukui M, Yyaguchi Y and Koide H: G lomeru lar nonenzymatic glycosylation and I ipid peroxide are increased in the early phase of streptozotocin induced diabetic rats prior to histologic changes. Nephron 59:632-636, 1991. 51. Jennings P, McLaren M, Scott NA, Saniabadi AR and Belch J: The relationship of oxidative stress to thrombotic tendency in type 1 diabetic patients with retinopathy. Diabetic Medicine 8:860-5, 1991. 52. Collier A, Rumley A, Rumley AG et al: Free radical activity a n d hemostatic factors in NIDDM patients with and without microalbuminuria. Diabetes 41:909-913, 1992. 5 3 . S t e h o u w e r CDA, Nauta J, Zeldenrust GC, Hackeng W, Donker AJM and Ottolander GJ: Urinary albumin excretion, c a r d i o v a s c u l a r disease and endothelial dysfunction in non i n s u l i n dependent diabetes mellitus. Lancet 340:319-23, 1992. 54. Yaqoob M, Patrick AW and McLelland P: Oxidant injury and t u b u l a r damage precedes albuminuria in diabetic nephropathy. Nephrol Dial Transplant 7(7):698, 1992. . .. 5 5 . Ikenaga Hi Swukl H. M-m N f Itoh H and Saruta T: Enzymuria in non-insulin dependent diabetic patients: signs of tubular d y s f u n c t i o n . Clinical Science 84:469-475, 1993. 56. Catalano C, Winocour PH, Gillespie S, Gibb I and Albert¡ K: Effect of posture and acute glycaemic control on the excretion of retinol binding protein in normoalbuminuric insulin dependent diabetic patients. Clinical Science 84:461-7, 1993. 57. Esterbauer H, Gebicki J, Publ H and Jurgens G: The role of lip i d peroxidation and antioxidants in oxidative modification of LDL. Free Radica/ Biol Med 13:341-390, 1992. 58. Steinberg D, Parthasarathy D, Carew TE et al: Modifications o f LDL that increase its atherogenicity. New En gl Med 320:915-923, 1989. 59. Weis JR, Pitas RE, Wilson BD and Rodgers GM: Oxidised L D L increases cultured human endothelial cell tissue factor activity and reduces protein C activation. FASER / 5:2459-63. 1991. 6 0 . Wardle EN: Diabetic nephropathy. Nephron 45:177-181, 1987. 61. Brownlee M: Glycosylation products as toxic mediators of d i a b e t i c complications. Annual Review Med 42:159-66, 1991. 6 2 . Skolnik EY, Yang Z, Makita Z, Radoff S, Kistein M et al: Human and rat mesangial cell receptors for glucose modified proteins. J Experimental Medicine 174:931-g, 1991. 63. Doi T, Vlassara H, Kirstein M, Yamada Y, Striker GE et al: R e c e p t o r specific increase in extracellular matrix production in mouse mesangial cells by advanced glycosylation end products is mediated by PDGF. Proc Natl Atad Sci 89:287377, 1992. 6 4 . Monhoisse JC, Gillery P, Maquart FX and Borel JP: P r o d u c t i o n of superoxide anion by glycated proteins. A d v Exp Med Bio/ 264:551-4, 1990. 65. Makita Z, Radoff S, Rayfield EJ et al: Advanced glycosylation e n d products in patients with diabetic nephropathy. New EngIl Med 325:836-842, 1991. 66. Stern DM, Esposito C and Gerlach H: Endothelium and regul a t i o n of coagulation. Diabetes Care 14(suppl 1 ):160-166, 1991. 67. Hasegawa G, Nakano K, Sawada M et al: Possible role of tumor necrosis factor and interleukin 1 in the development of d i a b e t i c nephropathy. Kidney In-t 40:1007-1 2, 1991. 68. Cohen MP and Surma ML: 35-sulfate incorporation into glomerular basement membrane glycosaminoglycans is decreased in experimental diabetes. / Lab Clin Med 98:715-722, 1981. 69. Deckert T, Feldt-Rasmussen B, Burch-Johnsen K, Jensen T a n d Kofoed-Enevoldsen A: Albuminuria reflects widespread v a s c u l a r damage. Diabetojogia 32:219-226, 1989. 70. Mason RM, Thomas G and Davies M: Proteoglycan synthesis by human mesangial cells is depressed by hyperglycaemic glucose concentrations. Biochem Soc Trans 20:965, 1992. 71. Kofoed-Enevoldsen A and Eriksson UJ: Inhibition of N-acetyl heparan deacetylase in diabetic rats. Diabetes 40:1449-52, 1991. 264 CELL BIOLOGY OF DIABETIC NEPHROPATHY 72. Reddi AS: Glomerular and urinary glycosaminoglycans in diabetic rats. Clin Chim Acta 189:21 I-220, 1990. 73. Makino H, Ikeda S, Haramoto T and Ota Z: Heparan sulfate proteoglycans are lost in patients with diabetic nephropathy. N e p h r o n 61:415-21, 1992. 74. Reddi AS, Ramamurthi R, Miller M, Dhuper S and Laskar N: E n a l a p r i l improves albuminuria by prevent loss of heparan s u l f a t e in diabetic rats. Biochem Med Metab Biol45:l 19131,199l. 75. Gambaro G, Cavazzana AO, Luzi P, Piccoli Z, Borsatti A et a l : Glycosaminoglycans prevent morphological renal alterat i o n s and albuminuria in diabetic rats. Kidney Int 42:285291,1992. 76 . Cárdenas A, S c h a d e c k C , B e r n a r d A a n d Lauweys R: D e p l e t i o n of sialic acid in glomeruli of uninephrectomised rats. Biochem Med Metab Biol 46:416-21, 1991. 7 7 . Review: Non-enzymic glycosylation of proteins. N u t r i t i o n Reviews 47:281-2, 1989. 7 8 . LePape A, Guitton JD and Mull JP: Modification of glomerular basement membrane cross-links in experimental diabetic rats. Biochem Biophys Res Commun 1OO:1 214-21, 1981. 7 9 . Coperland KR, Yatscoff RW, Thliveris JA, Mehta A and P e n n e r B: Non-enzymatic glycation and altered renal structure and function in the diabetic rat. Kidney Int 32:664-670. 8 0 . Monnier VM, Sell DR, Abdul Karim KW and Emancipator S W : Collagen browning and crosslinking are increased in chronic experimental hyperglycemia relevant to diabetes and ageing. Diabetes 37:867-72, 1988. 81. Makino H, Yamasaki Y, Haramoto T, Shikata K, Hironska K, Ota Z and Kanware YS: Ultrastructural changes of extracellular matrices in the nephron revealed by high resolution scann i n g and immunoelectron microscopy. Lab Invest 68:49155, 1993. 82. Edelstein D and Brownlee M: Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine. Diabetes 41:26-g, 1992. 83. Itakura M, Yoskikawa H and Bannal C: Aminoguanidine dec r e a s e s urinary albumin and high MW proteins in diabetic rats. Life Science 49:889-897, 1991. 84. Danne T, Spiro MJ and Spiro RG: Effect of high glucose on type IV collagen production by cultured glomerular epithelial, endothelial and mesangial cells. Diabetes 42:170-7, 1993. 8 5 . Kreisberg JI: Hyperglycemia and micro-angiopathy. Direct r e g u l a t i o n by glucose of microvascular cells. Lab Invest 67:416-426, 1992. 8 6 . Wardle EN: Glomerulosclerosis and lipid peroxidation. N e p h r o n 64:487, 1993. 87. Ledbetter S, Copeland EJ, Noonan D, Vogeli G and Hassell JR: Altered steady state messenger RNA levels of basement m e m b r a n e proteins in diabetic mouse kidneys and thromboxane synthase inhibition. Diabetes 39:196-203, 1990. 88. Craven PA, Melham M and DeRubertis FR: Thromboxane in t h e pathogenesis of glomerular injury in diabetes. Kid /nt 42:937-946, 1992. 8 9 . Cagliero E, Maiello M, Boeri D, Roy S and Lorenzi M: Increased expression of basement membrane components by human endothelial cells cultured in high glucose concentration. / Clin Invest 82:735-8, 1988. 90. Ihm CG, Lee G, Nast CC, Artishevsky AA, Guillermo R et al: E a r l y increased renal procollagen al (IV) mRNA levels in s t r e p t o z o t o c i n induced diabetes. Ridney Int 41 :768-77, 1992. 9 1 . Ziyadeh FN: Renal tubular basement membrane and collagen type IV in diabetes mellitus. Kidney Int 43:l 14-120, 1993. 92. Pietravalle P, Morano S, Castina G et al: Charge selectivity of p r o t e i n u r i a in type 1 diabetes explored by IgG subclass clearance. Diabetes 40:1685-90, 1991. 9 3 . Bangstad HJ, Kofoed-Enevoldsen A, Dahl-Jorgensen K and Hanssen KF: Glomerular charge selectivity and the influente of improved blood glucose control in type I diabetic patients w i t h microalbuminuria. Diabetologia 35:1165-69, 1992. 94. Kowluru A, Kowluru RA, Solomon S and Martínez L: Protein g l y c a t i o n effects upon protein recognition by the proximal tubule. Life Science 50:281-6, 1992. 95. Wardle EN and Farr M: Lysozymuria in diabetes. Brit Medj i:624-5, 1976. 96. Wardle EN: Cytokine growth factors and glomerulonephritis, N e p h r o n 57:257-261, 1991. 97. Nahman NS, Leonhart KL, Cosio FG and Hebert CL: Effects of high glucose on cellular proliferation and fibronectin pro. ductlon by cultured human m e s a n g r a l cells. Kldncy Int 41:396-402, 1992. 98. Kreisberg JI and Ayo SH: The glomerular mesangium in diabetes mellitus. Kidney Int 43:109-l 13, 1993. 99. Mené P, Pugliese G, Pricci F, Di Mario U, Cinotti GA and Pugliese F: High glucose inhibits cytosolic calcium signaling in cultured rat mesangial cells. K i d n e y Int 43:585-591, 1993. 1 0 0 . Crowley ST, Brownlee M, Edelstein D and Schlondorff D: E f f e c t s of non-enzymatic glycosylation mesangial matrix on proliferation of mesangial cells. Diabetes 40:540-7, 1991. 1 0 1 . Beyer-Mears A, Ku L and Cohen MP: Glomerular polyol acc u m u l a t i o n in diabetes and its prevention by sorbinil. Diabetes 33:604-7, 1984. 1 0 2 . Haneda M, Kikkcawa R, Arimura T, Ebata K et al: Glucose i n h i b i t s myo-inositol and reduces myo-inositol content in c u l t u r e d rat glomerular mesangial cells. Metabolism 39:4045, 1990. 103. Williams B and Schrier RW: Glucose induced protein kinase C activity regulates arachidonic acid release and prostaglandin production by cultured rat glomerular mesangial cells. J Am Soc Nephrol2:302, 1991. 1 0 4 . Wolf G, Sharma K, Chen V, Ericksen M and Ziyadeh FN: High glucose induced mesangial cell proliferation is reversed b y autocrine transforming growth factor beta. Kidney Int 42 :647-56, 1992. 105. Kikkawa R, Hameda M, Togawa M, Koga D, Kajiwara N and Shigata Y: Differential modulation of mitogenic and metabolic actions of IGF-1 in rat glomerular mesangial cells in high glucose culture. Diabetologia 36:276-281, 1993. 1 0 6 . Wasserman J, Santiago A and Ritici V: Interactions of low d e n s i t y Iipoproteins with rat mesangial cells. Kidney Int 35:1168-74, 1989. 1 0 7 . Shapiro RJ and Xie Y: Mesangial cells modify LDL by a lipoxygenase dependent pathway. J Amer Soc Nephrol 2:778 abst., 1991. 108. Chisholm GM, Irwin KC and Penn MS: Lipoprotein oxidation a n d Iipoprotein induced cell injury in diabetes. Diabetes 41 (suppl 2):61-66, 1992. 109. Coritsidis G, Rifici V, Gupta S and Schlondorff D: Preferential binding of oxidised LDL to rat glomeruli in vivo a n d cultured mesangial cells in vitro. Kidney Int 39:858-866, 1991. 110. Nakamura T, Fukui M, Ebihara I, Osada S, Nagaoka I, T o m i n o Y and Koide H: Messenger RNA expression of g r o w t h factors in glomeruli from diabetic rats. D i a b e t e s 42:450-56, 1993. 111. Silbiger S, Crowley S, Shan Z, Brownlee M, Satriano J and Schlondorff D: Non-enzymatic glycation of mesangial matrix and prolonged exposure of mesangial matrix to elevated gluc o s e reduces collagen synthesis and proteoglycan charge. Kidney Int 43:853-864, 1993. 112. Rovin BH and Tan LC: LDL stimulates mesangial fibronectin p r o d u c t i o n and chemoattractant expression. Kidney Int 43:218-225, 1993. 265 E. N. WARDLE 113. Sato H, Suzuki S and Kobayashi H: Immunohistological localization of apolipoproteins in glomeruli in renal diseases, spec i f i c a l l y apoB and apoE. Clin Nephrology 36:127-134, 1991. 114. Doi T, Striker LJ, Cibson CC et al: Glomerular lesions in mic e transgenic for growth hormone and IGF-1. Relation between glomerular size and mesangial sclerosis. Amer J Pathol 137:541-552, 1990. 115. Ziyadeh FN, Chen Y, Davila A, Goldfarb S and Wolf G: Self limited stimulation of mesangial cell growth in high glucose: autocrine activation of TGFP reduces proliferation but increases mesangial matrix. J Amer Soc Nephrology 2:304 abst., 1991. 116. Yamamoto T, Nakamura T, Noble N, Ruoslahti E and Border W: Expression of TGFbeta is elevated in human and experimental diabetic nephropathy. Proc Natl Atad Sci USA 90:1814-18, 1993. 117. Cohen MP y Ziyadeh FN: Amadori glucose adducts modulat e mesangial cell growth and collagen gene expression. Kidney Int 45:475-489, 1994. 118. Agarwal M and Daniels BS: Increased uptake of glycated alb u m i n by glomerular mesangial and epithelial cells in situ is a c c o m p a n i e d by augmented local production of hydrogen peroxide. Clinical Res 40:179 abst., 1992. 119. Craven PA, Studer RK and DeRubertis FR: Impaired nitric o x i d e dependent cyclic GMP generation in glomeruli from d i a b e t i c rats evidence for protein kinase C mediated supp r e s s i o n of cholinergi response. J Clin Invest 93:311-320, 1994. 120. Kohan DE: Eicosanois: potential regulators of extracellular lin ed 120:4-6, 1992. 266