Diabetic nephropathy in humans requires activation of multiple molecular programs to become manifest. The current pathophysiological understanding of diabetic nephropathy is derived largely from analysis of animal and cellular models. Several pathways were identified by these approaches and were confirmed to play critical roles in the evolution of diabetic nephropathy in humans (27
). However, despite pathogenic changes in a variety of different signaling pathways, current murine models of diabetic nephropathy fail to completely replicate progressive human diabetic nephropathy (glomerulosclerosis, tubulointerstitital fibrosis, and decline in GFR) (10
). This discrepancy could be due to partial, incomplete, or temporary activation of critical pathogenic responses and/or distinctive protective responses that counteract or prevent nephropathy in mice that are not manifest in humans. To identify those human-specific pathogenic or murine protective pathways, we used a comparative transcriptomic approach to study diabetic nephropathy in both species.
In human diabetes, as assessed by microarray analysis and confirmed by RT-PCR and immunohistochemistry, the Jak/Stat signaling pathway appeared to be one of the top regulated pathways and was consistently altered in both glomeruli and tubulointerstitium from patients with diabetic nephropathy. Marrero's group previously has identified Jak/Stat activation as potentially pathogenic in mediating angiotenisin II signaling, in inducing transforming growth factor-β expression, and in stimulating extracellular matrix protein production in cultured mesangial cells and animal models of diabetic nephropathy (14
). However, to our knowledge the current report is the first study to demonstrate enhanced Jak/Stat expression in human diabetic nephropathy. While multiple members of the Jak/Stat family showed increased mRNA expression in microarrays and RT-PCR expression studies, we focused on the expression patterns of Jak-2, since it is a critical upstream regulator of many Jak/Stat signaling events and was already implicated in processes that enhance fibrosis and epithelial mesenchymal transition in diabetic nephropathy (11
Our findings suggest an interesting compartmental and temporal association of enhanced Jak-2 expression. Glomerular Jak-2 mRNA levels increase several-fold in diabetic patients with early diabetic nephropathy and then decline in later stages as tubulointerstitial Jak-2 increases along with progressive tubulointerstitial fibrosis and reduction in kidney function. Thus, enhanced Jak-2 expression temporally corresponds to the evolution of human diabetic nephropathy, with glomerulopathy followed by tubulointerstitial fibrosis. The impressive inverse correlation between tubulointerstitial with eGFR suggests a potential causal relationship between enhanced Jak/Stat expression and progressive tubulointerstitial fibrosis and renal failure. While some of this apparent correlation could be due to population differences between the early diabetic nephropathy group, who are all Pima Indians, and the mostly European progressive diabetic nephropathy group, a strong correlation between tubulointerstitial Jak-2 expression and eGFR remained in the progressive diabetic nephropathy group alone, suggesting that this association was not due to population differences.
Jak-2 mRNA induction in diabetic nephropathy was confirmed by immunochemistry and appears to be diabetic nephropathy specific, as there was no induction of Jak-2 mRNA in glomeruli or tubulointerstitum in lupus nephritis, IgA nephropathy, or hypertensive nephrosclerosis. Jak-2 protein expression was increased in both glomeruli and proximal tubular cells in humans with diabetic nephropathy, but there was variable expression in other nephropathies with tubular damage and proteinuria, consistent with the notion that Jak-2 activation is not generic to all progressive renal diseases. This observation confirmed the results obtained by Affymetrix GeneChip compared with those for control patients (living donors, n = 21), where Jak-2 mRNA showed no consistent regulation in patients with hypertensive nephropathy (n = 20), IgA nephropathy (n = 27), and lupus nephritis (n = 32), with fold changes between 0.93 and 1.10 in the glomerular and tubulointerstitial compartments.
Similarly, there was no correlation between gene expression of Jak-2 or other Jak/Stat members in diabetic nephropathy samples with blood pressure, duration of diabetes, glycosylated hemoglobin, or BMI (with the sole exception of Jak-3 and blood pressure), suggesting that the correlation with eGFR was specific and not due to some confounding factor. Nonetheless, a significant correlation between Jak-2 and eGFR does not necessarily imply a causal connection between the two parameters, nor does it imply directionality if a causal relationship exists. It is imperative to follow up these studies with prospective analysis of patients with diabetic nephropathy to determine whether glomerular or tubulointerstitial Jak-2 expression truly predicts progression of diabetic nephropathy in humans. Additionally, tissue-specific Jak-2–overexpression studies in animal models of diabetic nephropathy will be important.
The absence of enhanced Jak-2 expression in the db/db
C57BLKS and STZ-induced diabetic DBA/2J mouse diabetic nephropathy models is interesting since these models, frequently used for studies of diabetic nephropathy (10
), develop high levels of albuminuria and undergo extensive early glomerular changes of diabetic nephropathy (22
) but rarely progress to severe glomerulosclerosis and do not develop significant tubulointerstitial fibrosis and progressive kidney failure (10
). No increases in Jak-2 mRNA or protein expression were found in either glomerular or tubulointerstitial compartments, suggesting one possible reason for the lack of progressive diabetic nephropathy induction in these mouse models. It is certainly likely that additional responses critical for progression of human diabetic nephropathy are missing in conventional murine models of diabetic nephropathy and that protective responses in mouse models may be absent from humans with progressive diabetic nephropathy. Thus, the strategy of uncovering divergent responses between human progressive diabetic nephropathy and murine models of early diabetic nephropathy may reveal other pathways that are essential for the pathogenesis of diabetic nephropathy and that are potential targets for therapeutic or prevention strategies.
Since Jak/Stat pathways are activated by growth factors, cytokines, or other upstream signals, and because Jak-2 protein activation is via autophosphorylation, enhanced Jak-2 expression should result in Stat tyrosine phosphorylation and activation only with Jak-2 activation. Thus, it was important to test whether Jak-2 overexpression alone could induce Stat phosphorylation and other downstream responses. While in vivo confirmation of such a response necessarily awaits the generation and testing of tissue-specific Jak-2 transgenic mice, we have confirmed that Jak-2 overexpression alone, without additional growth factors or cytokines, substantially increases Stat-3 phosphorylation on tyrosine 705. This observation confirms activation of Jak-2/Stat-3 signaling simply by overexpressing Jak-2 in mouse mesangial cells. Interestingly, high glucose had only a modest and statistically insignificant effect on Stat-3 phosphorylation, implying that high glucose alone is not sufficient to trigger Jak-2 signaling in our system. However, high glucose had an additive effect on Jak-2 overexpression on Stat-3 phosphorylation. The molecular mechanism by which Jak-2 overexpression induces Stat-3 phosphorylation remains to be determined.
Enhanced production of ROS has been described as a potential major activator of Jak/Stat signaling (33
) and can occur independently of the addition of exogenous cytokines (34
). This phenomenon appears to be relatively general since ROS mediate induction of Jak-2 activation in tissues such as cardiac myocytes and vascular smooth muscle cells (34
) as well as kidney cells (12
). We did not test whether ROS could further augment the signaling to Stat-3 in our Jak-2–overexpressing cells but we did show that Jak-2 overexpression induced ROS production in mesangial cells, especially those cultured in high glucose medium. This observation suggests the presence of an amplification loop in diabetic glomerular and proximal tubular cells in which there is both enhanced Jak-2 expression, as shown in this study, as well as enhanced ROS production, due to metabolic alterations resulting from enhanced glucose flux (26
). These two independent processes can each enhance the other, leading to progressive downstream signaling from both pathways and inexorable fibrosis and kidney failure.
Enhanced Jak-2 expression in both glomerular and proximal tubule cells may stimulate a host of downstream processes that lead to the glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure that affects up to 40% of diabetic patients. The absence of this response may protect mice from these late, but deadly, aspects of progressive diabetic nephropathy. Further exploration of Jak/Stat pathways in diabetic nephropathy as well as additional systems analysis of the differences between mice and humans may elucidate targets for therapies that could help humans with diabetic nephropathy be more like mice.