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. 2020 Aug;8(15):e14515.
doi: 10.14814/phy2.14515.

Nephrotic syndrome-associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors

Amanda P Waller  1 , Shipra Agrawal  1   2 , Katelyn J Wolfgang  1 , Jiro Kino  1 , Melinda A Chanley  1 , William E Smoyer  1   2 , Bryce A Kerlin  1   2 ; Pediatric Nephrology Research Consortium (PNRC)
Collaborators, Affiliations

Nephrotic syndrome-associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors

Amanda P Waller et al. Physiol Rep. 2020 Aug.

Abstract

Background: Thrombosis is a potentially life-threatening nephrotic syndrome (NS) complication. We have previously demonstrated that hypercoagulopathy is proportional to NS severity in rat models and that pioglitazone (Pio) reduces proteinuria both independently and in combination with methylprednisolone (MP), a glucocorticoid (GC). However, the effect of these treatments on NS-associated hypercoagulopathy remains unknown. We thus sought to determine the ability of Pio and GC to alleviate NS-associated hypercoagulopathy.

Methods: Puromycin aminonucleoside-induced rat NS was treated with sham, Low- or High-dose MP, Pio, or combination (Pio + Low-MP) and plasma was collected at day 11. Plasma samples were collected from children with steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) upon presentation and after 7 weeks of GC therapy. Plasma endogenous thrombin potential (ETP), antithrombin (AT) activity, and albumin (Alb) were measured using thrombin generation, amidolytic, and colorimetric assays, respectively.

Results: In a rat model of NS, both High-MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (p < .05). Proteinuria (p = .005) and hypoalbuminemia (p < .001) were correlated with ETP. In childhood NS, while ETP was not different at presentation, GC therapy improved proteinuria, hypoalbuminemia, and ETP in children with SSNS (p < .001) but not SRNS (p = .330).

Conclusions: Both Pio and GC diminish proteinuria and significantly alleviate hypercoagulopathy. Both Pio and MP improved hypercoagulopathy in rats, and successful GC therapy (SSNS) also improved hypercoagulopathy in childhood NS. These data suggest that even a partial reduction in proteinuria may reduce NS-associated thrombotic risk.

Keywords: Hypercoagulopathy; Methylprednisolone; Nephrotic Syndrome; Nuclear Receptors; Pioglitazone; Thrombosis.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Both Methylprednisolone and Pioglitazone Alleviate Proteinuria and Hypoalbuminemia. Mean ± SE of (a) proteinuria and (b) plasma albumin concentration in a PAN‐induced rodent model of NS, with/without concomitant methylprednisolone (MP) and/or pioglitazone (Pio) treatment (n = 8‐13/group). *p < .05 ***p < .001
Figure 2
Figure 2
Hypercoagulopathy Improves in Parallel with Nephrosis Following Treatment. (a) Representative thrombin generation graphs from one individual rat per group, and graph of mean ± SE for (b) endogenous thrombin potential (ETP), in a PAN‐induced rodent model of NS, with/without concomitant methylprednisolone (MP) and/or pioglitazone (Pio) treatment (n = 8‐13/group). (c‐d) Linear regression analysis correlating disease severity (proteinuria (c) and hypoalbuminemia (d)) with ETP. *p < .05 ***p < .001
Figure 3
Figure 3
Qualitative Antithrombin Deficit and Treatment Response. Mean ± SE of plasma AT concentration (a‐c) and AT activity (d‐f) in PAN‐NS, with/without methylprednisolone (MP) and/or pioglitazone (Pio) treatment, and linear regression analysis with proteinuria (b, e) and hypoalbuminemia (c, f) (n = 8‐13/group). There was no correlation between ETP and AT activity or AT concentration (p = .066 and p = .186, respectively; data not shown). *p < .05
Figure 4
Figure 4
Prothrombin Antigen and Activity are not Altered by PAN‐Induced Nephrosis or Treatment. Mean ± SE of plasma prothrombin concentration (a) and activity (b) in PAN‐induced rodent model of NS, with/without concomitant methylprednisolone (MP) and/or pioglitazone (Pio) treatment (n = 8‐13/group)
Figure 5
Figure 5
PAN Nephrotic Rats Are Responsive to High‐Dose Methylprednisolone Treatment. Mean ± SE of (a) proteinuria and (b) plasma albumin concentration in a PAN‐induced nephrotic rats (n = 4‐8/group). Varying disease severity was induced in male Wistar rats by a single intravenous (IV) or intraperitoneal (IP) injection of 75 or 100 mg/kg PAN. Steroid sensitivity was confirmed after 11 days of treatment with either high‐dose methylprednisolone (+MP; 15 mg/kg) or sham saline (‐). *p < .05 ***p < .001
Figure 6
Figure 6
High‐Dose Methylprednisolone Corrects Thrombin Generation in More Severe PAN‐Induced Nephrosis. Mean ± SE of Endogenous Thrombin Potential (a) in rats made nephrotic with a single intravenous (IV) or intraperitoneal (IP) injection of 75 or 100 mg/kg PAN injection, and then treated with high‐dose methylprednisolone (+MP; 15 mg/kg) or sham saline (‐) for 11 days (n = 4‐8/group). ETP was significantly correlated with proteinuria and hypoalbuminemia (b, c). *p < .05
Figure 7
Figure 7
Qualitative Antithrombin Deficit Persists in More Severe PAN‐Induced Nephrosis. Mean ± SE of plasma antithrombin (AT) antigen (a‐c) & AT activity (d‐f) in a PAN‐induced rodent model of NS treated with high‐dose methylprednisolone (+MP; 15 mg/kg) or sham saline (‐) for 11 days (n = 4‐8/group). Plasma AT antigen and activity were significantly correlated with disease severity. *p < .05 **p < .01
Figure 8
Figure 8
Hypercoagulopathy Responses Persist Across a Broad Range of Disease Severity. Linear regression analysis of disease severity (proteinuria (a, c) and hypoalbuminemia (b, d)) and coagulation markers (ETP (a, b), AT activity (c, d)), in all PAN‐NS rats combined (n = 95). (e, f) Linear regression analysis of the relationship between ETP and AT activity (e) and AT concentration (f) in the combined rat groups (n = 95)
Figure 9
Figure 9
Healthy Rats Treated with Methylprednisolone and Pioglitazone Alone or in Combination Exhibit Elevated Endogenous Thrombin Potential and Altered Coagulation Parameters. Mean ± SE of (a) proteinuria, (b) plasma albumin concentration, (c) Representative thrombin generation graphs from one individual rat per group, (d) mean ± SE Endogenous Thrombin Potential, (e) antithrombin antigen (f) and activity, (g) prothrombin antigen (h) and activity, in healthy Wistar rats treated with saline (Control), high‐dose methylprednisolone (MP; 15 mg/kg), pioglitazone (Pio), or combination therapy (Pio + Low‐GC) (n = 4‐13/group). *p < .05 **p < .01
Figure 10
Figure 10
Hypercoagulopathy Improves in Children with Steroid‐Sensitive, but not Steroid‐Resistant Nephrotic Syndrome. Mean ± SE of proteinuria (a), plasma albumin (b), and ETP (c) in childhood steroid‐sensitive NS (SSNS; n = 24) and steroid‐resistant NS (SRNS; n = 14) at disease presentation (“Pre”) and following glucocorticoid treatment (“Post”). (d) Representative thrombin generation graphs from one individual per group and a human pooled normal plasma (PNP) control. (e, f) Linear regression analysis correlating ETP with proteinuria and hypoalbuminemia, respectively. *p < .05 **p < .01 ***p < .001
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